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"I've never had an orgasm with my partner of four years. Any tips?"
I have been with my partner for nearly four years now, and have never had an orgasm with him. The last time I had one with a guy was about seven years ago.
When I'm on my own I can orgasm quite easily. I think I have got so used to it on my own, it seems impossible to achieve it with my partner.
He seems to want to masturbate more often than have sex, I'm not sure if this is down to him thinking he's not satisfying me, or if it's just a guy thing.
I do enjoy the sex, but feel I need to relax and enjoy it more. Can you give me any tips?
It's a myth that couples orgasm effortlessly together. In real life, less than half of all women regularly orgasm with their guys (the stats for lesbians is higher, surprise, surprise).
So don't feel bad about it - it's not unusual to orgasm easiest when you're solo and masturbating. To orgasm with him you need several things to be happening.
First up, he needs to use the technique that does it for you. Secondly, you need to feel safe enough emotionally to let go.
Thinking your partner would prefer to masturbate rather than have sex with you isn't pleasant and it's certainly not the right head space to orgasm in. Time for an honest conversation.
Tell him your fear that he doesn't really enjoy sex with you (because this is essentially what you are saying). Tell him what he needs to do to you to orgasm with him.
If your masturbation technique isn't couple friendly, you need to teach yourself to orgasm using one that you can teach him. Especially make sure he knows there's absolutely nothing wrong with you and given the statistics, other girlfriends who claimed to orgasm with him were probably faking.


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Located in the heart of downtown La Jolla, commonly known as the Village, sits a spectacular ocean front Charming La Jolla Condo on Coast Boulevard; 909 Coast Boulevard #9. With over 1200 square feet, two bedrooms and two baths, this is an extremely rare opportunity to enjoy luxurious seaside living in such a prime, sought after location.
Just steps from the Cove, this magnificent single story Charming La Jolla Condo on Coast Boulevard, located on the top floor, has been recently renovated to include designer finishes and high-end amenities. The bright and open layout flows seamlessly together, offering comfortable, yet elegant living. And with an attached balcony just off the living room, you can easily enjoy the ocean breeze!
For those who love to cook, the well-equipped kitchen, complete with granite counter-tops and stainless steel appliances, offers everything you need to make cooking stress-free! Just down the hall, the spacious master suite, boasts a walk in closet and beautiful en-suite bathroom with walk in shower. A second bedroom provides the perfect space for an office or guest room.
Not only does this Charming La Jolla Condo on Coast Boulevard come with everything you need, the complex includes underground parking, BBQ areas, and a community pool!
Conveniently located right in the heart of La Jolla Village, there are plenty of activities to keep you occupied! Take a short stroll up the street to famous Prospect Avenue, where you can enjoy everything from world class shopping and five star restaurants to unique boutiques and delicious cafes! Don't forget to explore the legendary La Valencia Hotel, or walk through the many exhibits at the Museum of Contemporary Art!
The prime location is perfect for outdoor enthusiasts who wish to enjoy the year-round Mediterranean climate. Whether you head down to the breathtaking La Jolla Cove, a premier destination for kayaking, snorkeling and diving or walk out on to the sea wall at the Children's Pool, you are sure to feel at peace in some of Southern California's most picturesque attractions!
Don't miss out on your opportunity to enjoy the La Jolla lifestyle in a luxurious & Charming La Jolla Condo on Coast Boulevard just steps from the sand!

subset: commoncrawl


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Our society has become an ideal business environment where everything works: from basic infrastructure to high ethical standards. Investments in education and an open, equal society have made Finns independent and creative problem solvers who can boost productivity with new exciting new ideas.
Finns like to make sensible use of their time. They follow timetables and other plans faithfully and expect the same from others. They expect well-defined targets and processes, while taking authority and responsibilities seriously. Finns are careful and gather background information in advance, but they make decisions quickly once they have the necessary facts.
There are thousands of lakes in Finland, and Finland has thousands of examples of world class innovation, ranging from high tech solutions to everyday inventions.
Regardless of their position in the company, many people are on a first-name basis. Finnish companies generally have a flat organizational structure and informal work relationships are commonplace. A healthy sense of humor is also an asset, and if you have the opportunity to take a sauna, try it out, your Finnish hosts will be very impressed.
Equality is at the very heart of Finnish society. Finland has had a woman as the President of the Republic and the President of the Supreme Court. A woman professor or an executive is nothing exceptional.
The best way to experience Finnish culture is to become a part of it. In Finland, you won't just stand and watch – you're invited to join in. Come say hi and find your inner Finn!


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West Virginia's Eastern Panhandle boasts numerous recreational, historic and cultural activities within a short drive of The Woods.
For the adventurous, whitewater rafting and kayaking, professional auto racing and thoroughbred racing are close by. In addition, The Woods is bordered on three sides by the 23,000-acre Sleepy Creek Wildlife Management Area, which contains more than 75 miles of hiking trails and a 205-acre lake for fishing and canoeing.
History buffs enjoy Harpers Ferry National Park, historic Shepherdstown (oldest town in W. Va.), Antietam Battlefield and Berkeley Springs (a favorite retreat of George Washington). Each summer, the Contemporary American Theater Festival comes to nearby Shepherdstown, offering new, cutting-edge plays from America's best playwrights.
Nearby outlet centers are favored by Woods' guests. Great buys can be found in women's apparel, menswear, luggage, glass, collectibles and much more. Specialty shopping is abundant in the Eastern Panhandle, where visitors may find one-of-a-kind items created by local artists or vintage treasures from a variety of antique shops.
"Most men are so occupied with the cares and labors of life that
its finer fruits cannot be plucked by them."
Henry David Thoreau from "Walden"
P.O. Box 5
Mountain Lake Road
Hedgesville, WV 25427
(800) 248-2222


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- Website
- http://www.utahstateaggies.com/
- Address
- 7400 Old Main Hill, Logan UT, 84322-7400
- 7400
- Phone
- (435) 797-1850
- Fax
- (435) 797-2615
|Jamie Sweeten
|Administration - Executive Assistant
|Brian Evans
|Student Athlete Services - Sr. Associate AD
|Maggie McInerney
|Business Services - Associate AD
|Paul Finn II
|Development - Assist. AD, Annual Giving/Tickets
|Kyle Cottam
|Media Relations - Assistant AD for Communications
|Reed Capener
|Administration - Assist. AD Event Operations
|Jovon Bouknight
|Football - Coach
|Julius Brown
|Football - Coach
|Stacy Collins
|Football - Coach
|Steven Farmer
|Football - Coach
|Frank Maile
|Football - Coach
|Kendrick Shaver
|Football - Coach
|Mark Tommerdahl
|Football - Coach
|Luke Wells
|Football - Coach
|David Yost
|Football - Coach
|Timothy Duryea
|Men's Basketball - Head Coach
|Jerry Finkbeiner
|Women's Basketball - Head Coach
|Dean Johansen
|Golf - Head Coach
|Nadalie Walsh
|Gymnastics - Head Coach
|Heather Cairns
|Soccer - Head Coach
|Steven Johnson
|Softball - Head Coach
|David Scholz
|Strength & Conditioning - Director of Football Strength and Conditioning
|James Wilson
|Men's Tennis - Head Coach
|Gregory Gensel
|Track & Field - Head Coach
|Grayson DuBose
|Volleyball - Head Coach
|Jessica Achatz
|Athletic Trainers - Assistant
|Laura McLane
|Athletic Trainers - Assistant
|Amber Rae Childers
|Student Athlete Services - Assist. AD
|William Garren
|Video - Assist. AD for Video Operations
|Stephanie Gonzales
|Business Services - Travel Coordinator
|Michael Bair
|Equipment - Director of Equipment Operations
|Jana Doggett
|Administration - Deputy Athletics Director
|DJ Eckman
|Administration - Event Operations Coordinator
|John Hartwell
|Administration - Vice President and Director of Athletics
|Andrew Dickinson
|Development - Assistant AD for Major Gifts
|Michael Williams
|Athletic Trainers - Associate AD, Head Trainer
|Karen Yindeepit
|Athletic Trainers - Insurance Coordinator
|Kalli Egbert
|Business Servces - Estes Center Administration Assistant
|Ronda Christoffersen
|Business Servces - Manager
|Shay Jeppsen
|Business Servces - Spectrum Administration Assistant
|Jake Garlock
|Compliance - Associate AD
|Michelle Bowman
|Compliance - Coordinator
|James Moore
|Development - Assistant AD for Special Projects
|Jeremy Winn
|Development - Associate AD - Major Gifts
|KC Smurthwaite
|Development - Major Gifts Officer
|Bobbi Chatterton
|Development - Manager of Athletic Development Operations
|James Bell
|Equipment - Inventory Control
|Steve Wiley
|Equipment - Inventory Control
|Brandy Saunders
|Football - Administrative Assistant
|Waqa Damuni
|Football - Director for Football Operations
|Jared Russell
|Football - Director of Player Personal
|Matthew Wells
|Football - Head Coach
|Erik Skinner
|Golf - Assistant Coach
|Christopher Bogantes
|Gymnastics - Assistant Coach
|Andrea Dolinsky-Webb
|Gymnastics - Assistant Coach
|Katherine Prchlik
|Marketing - Assistant
|Ryan McLane
|Marketing - Assistant AD
|Wade Denniston
|Media Relations - Assistant Media Relations Coordinator
|Megan McCuistion
|Media Relations - Assistant Media Relations Coordinator
|Doug Hoffman
|Media Relations - Associate AD/Director for Media Relations
|Spencer Nelson
|Men's Basketball - Assistant Coach
|Louis Wilson
|Men's Basketball - Assistant Coach
|Tarvish Felton
|Men's Basketball - Associate Head Coach
|jesse Parker
|Men's Basketball - Director of Basketball Operations
|Kyle Taylor
|Men's Basketball - Video Coordinator / Director of Player Development
|Ben Mullis
|Men's Tennis - Assistant
|Molli Sorenson
|Soccer - Assistant Coach
|Trevor Wachsman
|Soccer - Associate Head Coach
|Nathan Miller
|Softball - Assistant Coach
|Windy Thees
|Softball - Assistant Coach
|Nicole Troumbley
|Spirit Squad - Director
|Joseph Bergles
|Strength & Conditioning - Assistant Strength and Conditioning Coach
|Brandon Howard
|Strength & Conditioning - Assistant Strength and Conditioning Coach
|Alan Bishop
|Strength & Conditioning - Director of Olympic Sports Strength and Conditioning
|Steph Ingalls
|Student Athlete Services - Academic Coordinator
|Kimi Jennings
|Student Athlete Services - Academic Sport Coordinator
|Andrea Patton
|Student Athlete Services - Learning Specialist
|Sarah Griggs
|Track & Field - Assistant Distance Coach
|Jack Mullaney
|Track & Field - Director of Operations
|Arthur Gulden
|Track & Field - Distance Coach
|Erik Rasmussen
|Track & Field - Jumps and Multi Coach
|Jeramie Murray
|Track & Field - Sprints & Hurdles Coach
|Matthew Ingebritsen
|Track & Field - Assistant Head Coach / Throws
|Jacob Boxx
|Video - Multi Media Coordinator
|Daniel Magalogo
|Video - Program Technician
|Taubi Palmer
|Volleyball - Assistant Coach
|Sammi Stuart
|Volleyball - Assistant Coach
|JC Carter
|Women's Basketball - Assistant Coach
|Benjamin Finkbeiner
|Women's Basketball - Assistant Coach
|Kelsie Kruger
|Women's Basketball - Assistant Coach
|Meghan Blevins
|Women's Tennis - Assistant
|Sean Mcinerney
|Women's Tennis - Head Coach
|Jessica Dickinson
|Development - Special Events Manager
|Tyler Knowles
|Marketing - Director of Marketing


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What News Sites Find as Important as Politics
Isn't it amazing what can be considered "real news" these days? People like Kim Kardashian and Justin Bieber seem to be taking the spotlight for most of the news that comes off the presses, when it should be directed towards things that matter, such as citizens that are revered in their community, or unbiased political updates. Yet we get more celebrity news rather than anything worth taking your time to read. Here are some headlines that were among the same group as more important news:
"Is Kardashian dating Bieber?"
Taken from CNN.com, a world renowned news site known for its credibility.
Keep in mind this was in the same category as "Trump at 41% in a new poll." which is also from CNN.com, that is real news, a story about how a potential presidential candidate is getting more and more approval ratings in society, and becoming a better candidate in the race for presidency. Compared to the Kardashian/Bieber article, it's the biggest thing in news since the moon landing in 1969.
"Snoring polar bear cub is the cutest thing you'll see today"
Okay, a cute baby animal video. We get it, it has a really small face and high pitched snore, but does that really deserve to be on the home page of CNN.com? It is within a quick glance away from a story about how an american soldier is facing court-martial after being imprisoned by the Taliban. Now there, there is real, interesting, worthwhile news. Not some cute polar bear, not some celebrity couple that may or may not be a thing, but an american soldier, a man who had fought for his country overseas, getting put in front of a grand jury for something he did overseas. That is more important than those unimportant articles about cute animals sleeping because it is national news that is to be seen as a guide to not do in the future. The man facing court-martial could very well make history, whereas the Kardashian/Bieber story is 1. a rumor and 2. just another stupid celebrity couple that will end faster than any other.
So there it is, the ugly truth. News sites see cute animal videos at the same level of importance as updates about the 2016 presidential candidates'.


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Northern Ireland victory over Faroe Islands is only thing that matters for fired-up Lafferty
Kyle just wants to qualify even if his goals dry up
He's scored five times in six games during the Euro 2016 qualifying campaign.
An impressive strike rate in anyone's book, unless you're David Healy, who hit a record 13 in 10 matches in the 2008 European Championship series.
Like most strikers, however, Kyle Lafferty's mind is still on the one that got away - namely the opportunity that went begging against Romania at Windsor Park back in June when he fired his shot straight at the goalkeeper in the closing minutes of the scoreless draw when a win would have put Northern Ireland top of Group F with four games to go.
It might have gone through his head on numerous occasions over the last two and a half months, but making up for the miss isn't Lafferty's motivation going into tomorrow night's qualifier against the Faroe Islands.
He has scored against the north Atlantic archipelago twice before in his international career, but a third isn't the be all and end all either.
All that matters to Lafferty is winning so that he and his team-mates can write their names into Northern Ireland footballing folklore.
"I think my mind has been stuck on that chance I missed against Romania in the last 10 minutes to be honest," the man from Kesh said.
"I've always said that I don't care if I don't score another goal in this campaign, as long as we cross the finish line. As long as we are in France next year that's the most important thing.
"I've scored five goals so far and I'm not going to say I'm happy with that.
"I'd love to go on and score another three, four or five, but if I don't I'm pretty happy with the tally that I have - as long as we are there and celebrating."
Fitness concerns following a pre-season knee injury had thrown Lafferty's involvement against the Faroes into doubt.
Having to play without him doesn't bear thinking about for Northern Ireland manager Michael O'Neill.
He needn't worry though, because the news from the man himself is that if another chance like the one against Romania comes his way, he is in a better shape to put it away this time.
"I'd say that I am fitter and sharper going into these games than I was going into the Romania one," he said.
"I have absolutely worked my socks off on the bike and on the pitch over the last five weeks when I've been injured.
"Getting the injury when I did was disappointing.
"I think I'd done 10 days of pre-season and my first day back with the whole group I did my knee.
"It's just one of those things and I think things happen for a reason. I've had to pick my head up and I am looking forward to these two games."
Looking forward is something of an understatement from the normally not so shy and retiring Lafferty.
As he faced the press, the 27-year-old hardly stopped smiling, such is his eager anticipation of what lies ahead in the next five days.
Like every player and fan, Lafferty is well aware that expectation has risen to a new level, with the European finals in France well within reach.
Rather than being scared off by that, Lafferty is embracing the situation and tomorrow night's match can't come soon enough for him as he looks to clinch a place in France.
"It excites me. I can't wait for Friday night," he said.
"I just can't wait to get out onto the pitch and get the three points that we need and look forward to Monday night.
"I said after the Romania game that we wished the Faroes game was coming up the next day because we have that feeling around the squad now that we just can't wait for the next game to come along because we are excited and want to cross that finish line.
"Everyone knows that we will cross it - it's just a matter of time.
"We can basically get there in the next week, be on the plane to France, if the results go our way and we win the games so it's going to be exciting and I'm excited to be here and hopefully help the team to get the six points that we need.
"If it turns out that we don't we still have two games to play and I am sure that we'll be in France next year."
Belfast Telegraph


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George and Amal Clooney New "Divorce" Story Is Bogus Bait-and-Switch (EXCLUSIVE)
George and Amal Clooney have NOT filed divorce papers after six months of marriage, despite a misleading In Touch cover story designed to trick readers into thinking the couple's marriage has ended. Gossip Cop can exclusively bust the story.
According to the magazine's cover, the Clooneys' "nightmare" union is over. "He can't take it anymore," declares the outlet, claiming he "storm[ed] out after their worst fight ever." Of course, readers may recall that In Touch ran a divorce cover story about the couple back in February, alleging George had "dumped" Amal after 143 days of marriage. A month before that, in January, In Touch claimed the couple was set for a "$200M Divorce" in another cover story.
In Touch sure loves to run cover stories claiming the Clooneys are getting divorced! But the tabloid has been repeatedly wrong (as Gossip Cop has pointed out), and this latest report is no different. There have been NO "divorce papers." The only actual "divorce papers" the tabloid uses in the story is the filing from Clooney's 1992 split from Talia Balsam. That's how desperate In Touch is.
The story itself is mostly rehashed filler about the alleged "problems" in the Clooneys' marriage, including their supposed fights about his career, the time they spend apart for work reasons, disagreements about whether to start a family, and so on. These rumors haven't been true no matter how many times the same tabloids repeat them.
Meanwhile, In Touch doesn't even have the courage to say the Clooneys are actually filing for divorce. Instead, buried in the story, is a source saying, "I wouldn't be surprised if he drew up divorce papers any day now." They are NOT getting divorced. A rep tells Gossip Cop the new report is "all fabricated." A source close to the situation additionally tells Gossip Cop it's "outrageous" for the publication to bait-and-switch using Clooney's divorce papers from more than two decades ago.


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And not a very good one I might add
Should we ask for something they would never do?
Isn't posting a question about if a request should be made in a place where the ones who would receive the request the same thing as making the request?
Could I think of a third rhetorical question?
Hahah, however I do agree, I think they should fix it first:)
Wouldn't it be nice to find lots of fixes in it?
No, it is ready barrings some bugs that are being ironed out. Its perhaps the best expansion for a game I have seen in years.
Isn't Crusade the fix?
Ok the 2.0 isnt great you can always find bugs, and make patches, but what is broken it is a good game.
There are many great features available to you once you register, including:
Sign Up Now!


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One of the biggest party weekends of the summer has finally arrived in the US, and to celebrate Memorial Day Weekend, the Revel resort in Atlantic City, New Jersey is bringing one of the hottest line-ups of the weekend to their HQ Beach Club and Nightclub!
Kicking it off this weekend at the Beach Club on Thursday are the EC Twins, with electro house superstar Wolfgang Gartner taking over HQ Nightclub that night. The Beach Club gets a versatile and massive line-up throughout the weekend, with Manufactured Supertars, Sultan + Ned Shepard, and Bambi on Friday, Armin van Buuren on Saturday, and Boris and Victor Calderone on Memorial Day Monday! HQ Nightclub is bringing Manufactured Superstars on Friday, R3hab on Saturday, and Laidback Luke with Mike Bugout on Memorial Day Monday!
Ticket links and event information can be found below – see you there!
HQ Beach Club
EC Twins
Manufactured Superstars, Sultan + Ned Shepard, Bambi
Armin van Buuren (SOLD OUT)
Boris + Victor Calderone
HQ Nightclub
Wolfgang Gartner
Manufactured Superstars
Laidback Luke w/ Mike Bugout


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A man walking on a sidewalk in Brooklyn was knocked over by the force of snow being plowed out of the street by a truck Wednesday, video shows. The snow also shattered the storefront of an auto dealership. Video courtesy JM Legend.
Published at 8:48 PM EST on Feb 6, 2014 | Updated at 8:50 PM EST on Feb 6, 2014


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Disposable Patient Gowns
Case of 50
Medline Disposable Patient Gowns
- Opening in front or in back.
- Gowns have attached ties.
- Tissue-Poly-Tissue 3-ply material.
- Gowns are 30 x 42".
Product Overview
Medline Disposable Patient Gowns
Disposable Patient Gowns
AllegroMedical.com presents The Disposable Patient Gowns. Opening in front or in back. Gowns have attached ties. Tissue-Poly-Tissue 3-ply material. Gowns are 30 x 42".
- Color: White
- Size: Regular/Large
- Dimension: 30" X 42"
- Fabric Type: Tissue/Poly/Tissue
- Gown Closure Style: Front/Back Opening With Belt
- Product Type: Patient Gown
- Sleeve Style: Sleeveless
- Snap Detail: Not Applicable
Place Your Order Here
Medline Disposable Patient Gowns
Disposable Patient Gowns Reviews Summary
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Part Number(s): 732 574412 01, 732 574412 02
Mfg Part Number(s): NON24245, NON24244
Scrip Part Number(s): 732 574412 02, 732 574412 01
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|Home | About | Journals | Submit | Contact Us | Français
Nuclear and cytoplasmic forms of the yeast exosome share 10 components, of which only Rrp44/Dis3 is believed to possess 3′ exonuclease activity. We report that expression only of Rrp44 lacking 3′-exonuclease activity (Rrp44-exo) supports growth in S288c-related strains (BY4741). In BY4741, rrp44-exo was synthetic-lethal with loss of the cytoplasmic 5′-exonuclease Xrn1, indicating block of mRNA turnover, but not with loss of the nuclear 3′-exonuclease Rrp6. The RNA processing phenotype of rrp44-exo was milder than that seen on Rrp44 depletion, indicating that Rrp44-exo retains important functions. Recombinant Rrp44 was shown to possess manganese-dependent endonuclease activity in vitro that was abolished by four point mutations in the putative metal binding residues of its N-terminal PIN domain. Rrp44 lacking both exonuclease and endonuclease activity failed to support growth in strains depleted of endogenous Rrp44. Strains expressing Rrp44-exo and Rrp44-endo–exo exhibited different RNA processing patterns in vivo suggesting Rrp44-dependent endonucleolytic cleavages in the 5′-ETS and ITS2 regions of the pre-rRNA. Finally, the N-terminal PIN domain was shown to be necessary and sufficient for association with the core exosome, indicating its dual function as a nuclease and structural element.
The exosome complex is implicated in many RNA processing and degradation activities. Ten 'core' exosome components are shared between the nuclear and cytoplasmic forms of the complex, and all of these are essential for cell viability (1). The nuclear exosome participates in many RNA degradation and surveillance pathways, as well as processing the precursors to the 5.8S rRNA and other stable RNA species (2). The cytoplasmic exosome functions in mRNA degradation, participating in general mRNA turnover and several activated decay and surveillance pathways (3).
Structural and functional analyses indicate that Rrp44 (Dis3) is the only catalytically active 3′–5′ exonuclease in the yeast exosome core (4–6), whereas the nuclear exosome is associated with a second active nuclease (Rrp6) (7). Rrp44 is related to Escherichia coli RNase R, a member of the RNase II (RNase B) family of hydrolytic exonucleases. As shown in Figure 1A, Rrp44 has an N-terminal PIN-domain (PilT N-terminus—derived from the name of an E. coli protein implicated in pilus formation), which is not shared with RNase R or RNase II. Recent studies reported endonuclease activity associated with the PIN-domains of human Smg6 and yeast Swt1, both of which are implicated in mRNA surveillance (8,9), and the PIN domain protein Nob1 was predicted to be a pre-rRNA endonuclease (10,11). The PIN domain in Rrp44 is followed by a putative RNA-binding, cold-shock domain (CSD), for which functional analyses have not yet been reported. The exonuclease activity resides in the RNB domain and this is abolished by the point mutation D551N (4,6). A C-terminal S1 RNA-binding domain is also important for substrate binding and for activity in vitro and in vivo, and this is impaired by the G916E mutation (6,12).
Strains lacking the exonuclease activity of Rrp44 are viable, whereas the integrity of the exosome complex is essential since depletion of any single core component is lethal (1,4–6). These observations raised several questions, including whether other nucleases in the nucleus or cytoplasm can functionally substitute for the exonuclease activity of Rrp44 and whether other nuclease activities are associated with the core exosome?
Growth and handling of Saccharomyces cerevisiae were by standard techniques. Strains were grown at 25°C or 30°C in YPD or synthetic dropout medium containing 0.67% nitrogen base (Difco) and either 2% glucose or 2% galactose. Yeast RNA extraction and northern hybridization were performed as described (13). Northern signals were generally visualized by autoradiography, with the exception of the lighter exposure in Figure 6B, which was generated by a Fuji FLA-5100 PhosphorImager. Oligonucleotide probes are listed in Table S1.
The construction of yeast expression plasmids for Rrp44 is described in detail in (6). Briefly, the RRP44 ORF is fused to a C-terminal tag containing a streptavidin-binding peptide (Strep-tag II), TEV cleavage site and two copies of the z-domain of protein A (szz-tag) and cloned into the XhoI restriction sites of either pRS316 (URA3) or pRS315 (LEU2) (14). For both yeast and E. coli expression plasmids (see below), point mutations were created using the QuikChange kit (Stratagene) and deletion of the PIN-domain was achieved by PCR using the oligos listed in Table S1.
The BY4741 wild-type (WT) strain was first supplemented with a plasmid carrying RRP44-szz and a URA3 selective marker. The genomic RRP44 ORF was then precisely deleted by homologous recombination with a PCR product generated from the pFA6A-kanMX6 deletion cassette (15). Following construction of rrp44Δ, a second plasmid (pRS315) (14) was introduced that carried LEU2 and expressed either WT or mutant Rrp44. The URA3 plasmid was then counter-selected on plates containing 5-fluoroorotic acid (5-FOA), which is converted to the toxic uracil analogue 5-fluoro-uracil by the action of Ura3. RRP44 was modified in W303 to provide control by the GAL promoter by integration of a PCR product generated from pYM-N27 (16). Oligonucleotides used in generation of PCR cassettes are described in Table S1.
E. coli strain BL21(DE3)pLysS (Stratagene) was transformed with plasmids encoding WT or mutant GST-Rrp44 [derived from GST-Dis3sc (17)] or GST alone (pGEX-4T1). Expression, cell lysis and single-step purification on glutathione sepharose was performed as previously described (6).
Ribonuclease assays were performed with 200 fmol recombinant GST-Rrp44 and 10 fmol 5′- or 3′-end-labeled A30 RNA or 150 fmol 5′-end-labeled 52-nt stem loop RNA substrate (5′-GGCCCCGGGC CCCGUAGAAA AUCUUAGUAA UCCUUCUUAC AUUGCCCGGG GC-3′) in 10 mM Tris–HCl pH 7.6, 75 mM NaCl, 2 mM DTT, 100 μg/ml BSA, 0.8 U/μl RNasin, 4.5% glycerol, 0.05% Nonidet P40, 0.5 mM MgCl2 and 0, 0.5 or 5 mM MnCl2. Prior to addition of the labeled RNA, 10 µl reactions were pre-incubated for 10 min at 30°C. After an additional 1–2 h at 30°C, reactions were mixed with one volume of RNA formamide buffer, heated for 10 min at 65°C and separated on a denaturing 12% polyacrylamide/8 M urea sequencing gel. Reaction products were visualized by autoradiography.
One step purifications of exosomes on IgG sepharose columns were performed as described (18). To isolate exosome complexes, Csl4-TAP strains were grown in YPD medium to OD600 0.7 at 25°C. Each preparation used 1 l of culture, producing 150 μl of the final TEV fraction. Exosomes were purified in buffer TMN150 (20 mM Tris–HCl pH 7.6, 150 mM NaCl, 0.1% NP-40, 5 mM MgCl2) or treated with 800 mM MgCl2 before TEV elution to dissociate endogenous Rrp44. Enzyme concentrations were normalized by immunoblotting using an anti-Rrp6 antibody (19) or anti-peptide antibodies raised against Rrp44 and Rrp43 (this study).
Equal amounts of GST-bait proteins (~1 pmol) were immobilized on glutathione sepharose beads and incubated with purified yeast exosomes (15 µl TEV eluate) for 1 h at 4°C in Buffer NB (20 mM Tris–HCl pH 7.6, 150 mM NaCl, 8.7% glycerol, 0.1% Nonidet P40 and 5 mM MgCl2). The beads were then washed five times with buffer NB. The retained proteins were separated on an 8% polyacrylamide/SDS gel and the presence of the core exosome was analyzed by immunoblotting with an antibody specific for Rrp43 (Figure 7D) and Rrp4 (data not shown). Inputs represent 20% of the starting material.
Yeast lysate from 27.5 ODs of cells was prepared in buffer TMN150 containing 1 mM dithioerythritol and complete EDTA-free protease inhibitors (Roche) and loaded on a linear 4-ml 10 to 30% (w/v) glycerol gradient. After centrifugation for 17 h at 45 000 rpm in a Beckman SW60 rotor, the gradient was harvested manually from the top. Proteins were isolated from the gradient fractions by acetone precipitation, separated on an 8% polyacrylamide/SDS gel and analyzed by immunoblotting.
To assess the effects of the loss of the enzymatic activity of Rrp44 in vivo, we modified strain BY4741 to express HA-tagged Rrp44 under control of the repressible GAL10 promoter (Table S1), allowing depletion of endogenous Rrp44. This strain was transformed with plasmids expressing either WT-Rrp44 or Rrp44 lacking exonuclease activity (Rrp44-exo, Figure 1A) under the control of the endogenous RRP44 promoter, or with the empty cloning vector (pRS316). To allow purification of exosome complexes to test for in vitro exonuclease activity, the plasmid-expressed proteins also carried C-terminal fusions with a tag containing the streptavidin-binding peptide, TEV cleavage site and two copies of the z-domain of protein A (szz-tag; see Materials and methods section and Table S1).
The growth of individual transformants was then analyzed under repressive conditions on SD -His/-Ura medium at 30°C (Figure 1B). Growth of the strain carrying the empty vector was progressively reduced after transfer to glucose. In contrast, growth was fully restored by expression of WT Rrp44. A reduced rate of growth was maintained by expression of the catalytically inactive Rrp44-exo (Figure 1B). After prolonged growth in glucose medium (24 h), the doubling time of strains expressing only Rrp44-exo was ~3 h when compared to the WT doubling time of ~2 h (data not shown). This indicates that the essential function of Rrp44 in vivo does not require its exonuclease activity.
Genetic depletion of Rrp44 or any other core exosome component results in characteristic defects in 3′-maturation of the 5.8S rRNA from the 7S pre-rRNA and in degradation of the excised 5′-external transcribed spacer region (5′-ETS) of the pre-rRNA (20–23). Exosome mutants were also reported to accumulate a 3′-truncated and polyadenylated fragment of the 5S rRNA (24).
Northern analysis of the GAL::rrp44 strain also expressing WT Rrp44 (Figure 1D, lanes 1 and 2) revealed similar levels of 3′ extended pre-5.8S rRNA and the 5′-ETS in galactose (GAL, lane 1) and glucose (GLU, lane 2) media. Expression of only Rrp44-exo resulted in the accumulation of truncated 5S rRNA (labeled as 5S*) and the 7S pre-rRNA plus truncated fragments (Figure 1D, lane 4). Accumulation of longer 3′-extended 5.8S species was greater in the Rrp44-depleted strain, whereas the shortened 6S pre-rRNA was reduced, indicating a greater inhibition of processing following depletion of Rrp44 than in the rrp44-exo strain. Accumulation of the full length 5′-ETS was greater in the Rrp44-depleted strain (Figure 1D, lane 6) than in the rrp44-exo strain (Figure 1D, lane 4), which also showed prominent 5′-ETS fragments that were absent from the Rrp44-depleted strain. These observations are consistent with residual degradation activity associated with Rrp44-exo.
Rrp44-depleted strains also exhibit defects at earlier steps in pre-rRNA maturation on the pathway of 18S rRNA synthesis (25). These effects are likely to be indirect, since 18S rRNA synthesis involves only endonuclease activities, and similar defects are seen in many strains with late-acting defects in the 25S and 5.8S rRNA synthesis pathway (26). Northern analyses of high molecular weight pre-rRNA precursors (35S, 27SA2 pre-rRNA and the aberrant 23S RNA) showed only very modest defects in the strain expressing Rrp44-exo (Figure S1, and see below).
The exonuclease activity of Rrp44 appears to play an important role in maturation of the 7S pre-rRNA, and in degradation of the excised 5′-ETS pre-rRNA region and truncated 5S rRNA. In contrast, the exonuclease activity is less required for the, presumably indirect, role of Rrp44 in early pre-rRNA processing steps. However, strains lacking the exonuclease activity of Rrp44 are viable and showed less RNA processing defects than the Rrp44-depleted strain on the pre-rRNA substrates.
Constructs expressed under the GAL promoter are never fully repressed, and so a low level of Rrp44 will always be expressed in a GAL::rrp44 strain. To avoid this problem, the RRP44 ORF was precisely deleted in strain BY4741 supplemented with a plasmid carrying RRP44 and a URA3 selective marker. Following construction of rrp44Δ, a second plasmid was introduced that carried LEU2 and expressed either Rrp44 or Rrp44-exo. Mitotic segregants that had lost the URA3 plasmid containing wild type RRP44 were then isolated on plates containing 5-FOA, which selectively kills cells expressing Ura3. The rrp44Δ strains showed an increased doubling time when complemented by expression of Rrp44-exo (3 h when compared to 2 h for WT-Rrp44, Figure 2), similar to the results obtained with GAL::rrp44 strains (Figure 1B).
'Since Rrp44 is essential for viability, whereas its exonuclease activity is largely dispensable, it seemed likely that it was partially redundant with the activities of one or more other nucleases. An obvious possibility was the nuclear, exosome-associated exonuclease Rrp6. Indeed, the rrp44-exo mutation was reported to result in synthetic lethality with an rrp6Δ (4). However, this was not observed in our strains (Figures 2 and and3).3). To test for synthetic lethality between rrp44-exo and rrp6Δ, the RRP6 ORF was first deleted in the rrp44Δ strain complemented with a plasmid carrying RRP44 and a URA3 selective marker (see above). A second plasmid (pRS315) carrying LEU2 and expressing either Rrp44 or Rrp44-exo was subsequently introduced and the URA3 plasmid was then counter-selected on 5-FOA containing SD -His/-Leu plates. The growth of individual transformants was then analyzed on SD -His/-Leu medium at 25°C (Figure 2). The loss of Rrp44-exonuclease activity reduced the growth of RRP6 and rrp6Δ by a similar proportion, relative to the same strains expressing Rrp44 (Figure 2).
Northern analysis was further used to compare the RNA processing phenotypes of rrp44Δ rrp6Δ double mutant strains complemented by plasmids expressing either WT Rrp44 or Rrp44-exo (Figure 3). Yeast strains carrying rrp6Δ exhibit a distinctive accumulation of the 3′-extended precursor 5.8S + 30 (27) (Figure 3A, lane 3), which is found as a short (S) and a 5′-extended long (L) form. Expression of only Rrp44-exo in the rrp6Δ strain leads to accumulation of a longer form of this precursor (Figure 3A, lane 4). To confirm the identity of the extended 5.8S species, primer extension was performed using a probe located across the 5.8S-ITS2 boundary (data not shown). This revealed identical 5′ ends, showing that the species detected in the rrp6Δ rrp44-exo double mutant is 3′ extended by ~10 nt relative to the 5.8S + 30 RNA seen in rrp6Δ single mutant strains.
Loss of Rrp6 has little effect on degradation of the excised 5′-ETS (Figure 3A, lane 3) and the phenotype of the rrp6Δ rrp44-exo strain (Figure 3A, lane 4) resembled that of rrp44-exo alone (Figure 3A, lane 2). Both 3′ extended and truncated forms of the box C + D snoRNA snR13 were previously observed in strains with defects in nuclear RNA surveillance (28,29). These forms of snR13 were also detected in both the rrp6Δ and rrp44-exo single mutants (Figure 3A, lanes 3 and 2), with stronger accumulation in the double mutant strain (Figure 3A, lane 4).
High molecular weight RNA was also analyzed (Figure 3B). The rrp6Δ mutation alone resulted in increased levels of the 27SA2 pre-rRNA, the first committed intermediate on the pathway of 5.8S and 25S rRNA synthesis. The aberrant 23S, 21S and 17S RNAs were also accumulated (Figure 3B, lane 3); these are known targets for degradation by Rrp6 and the TRAMP5 polyadenylation complex (24,25,30). In contrast, rrp44-exo alone reduced the level of 27SA2 but had little effect on 23S (Figure 3B, lane 2). The double mutant showed an intermediate phenotype.
Since the exonuclease activity of Rrp44 is dispensable for growth, even in the absence of Rrp6, we considered the possibility that the essential role of the other, apparently non-catalytic components of the core exosome might lie in restraining and controlling an otherwise over-promiscuous exonuclease activity of Rrp44. To test this model, the core exosome component Rrp41 was placed under the control of a GAL promoter in the rrp44Δ strain expressing either intact Rrp44 or Rrp44-exo. However, following transfer to glucose medium, the GAL::rrp41 strains showed similar growth inhibition upon expression of Rrp44 or Rrp44-exo (data not shown). Northern analyses (Figure 3A and B, lanes 5–8) also indicated that the loss of Rrp44 exonuclease activity did not suppress or give synergistic defects when combined with depletion of Rrp41. We conclude that the phenotype of Rrp41 depletion is neither dependent on, nor attenuated by, the exonuclease activity of Rrp44.
Since the exonuclease activity of Rrp44 was apparently not strongly redundant with Rrp6, we tested for synthetic lethal interactions with other exonucleases.
The non-essential, 5′–3′-exonuclease Xrn1 (31) plays a major role in cytoplasmic mRNA turnover and is synthetically lethal with the cytoplasmic cofactors for the exosome, due to synergistic inhibition of mRNA degradation (32,33). Deletion of XRN1 was synthetically lethal with rrp44-exo (Figure 4A). We conclude that loss of the 3′-exonuclease activity of Rrp44 is synthetic-lethal with loss of the cytoplasmic 5′–3′ exonuclease Xrn1, probably due to synergistic inhibition of mRNA degradation. In contrast, loss of the PIN-domain associated endonuclease activity of Rrp44 (see below) was not synthetic-lethal with xrn1Δ (Figure 4B).
In addition to Rrp44, yeast contains two other proteins with homology to the RNase II family. Of these, Dss1 is mitochondrial and seemed unlikely to function redundantly with Rrp44. In contrast, Ssd1 is localized to the cytoplasm (34,35) and shows genetic interactions consistent with functions in RNA turnover and/or surveillance (36,37). Laboratory strains of yeast are polymorphic for Ssd1 synthesis (38); strains derived from S288c that were used for the systematic sequencing project and construction of the gene deletion collection, including BY4741 that we used for our initial functional analyses of Rrp44, harbor the full-length protein (Ssd1-v), whereas the widely used W303 strain expresses a truncated version of the protein (Ssd1-d). In comparison to GAL::rrp44 strains derived from BY4741, expression of Rrp44-exo in the W303 background consistently supported slightly less efficient growth when intact Rrp44 was depleted by incubation on glucose medium (data not shown). To determine whether this was due to the lack of intact Ssd1, the full-length form of Ssd1 was expressed from a plasmid. However, this failed to clearly improve growth of W303 strains expressing only Rrp44-exo.
Strain differences between BY4741 and W303 have a modest but reproducible impact on sensitivity to loss of Rrp44 exonuclease activity. Similar genetic background effects may be responsible for the fact that we found strains lacking the exonuclease activity of both Rrp44 and Rrp6 to be viable in BY4741, whereas they were previously reported to be synthetic lethal in strain BMA 64, which is related to W303 (4).
Comparison of the RNA processing phenotype of Rrp44-depletion to that shown by strains expressing Rrp44-exo (Figure 1D) suggested that some residual nuclease activity remained. Recent analyses of the PIN domain proteins, human Smg6 and yeast Swt1, had revealed that robust in vitro endonuclease activity required the presence of manganese ions (8,9). Four conserved acidic residues (Aspartate/D or Glutamate/E) coordinate the metal ion in the active site. All four residues are present in the N-terminal PIN domain of Rrp44 (Figure 1A and see below) and we therefore assessed whether Rrp44 also exhibited endonuclease activity in the presence of Mn2+ (Figure 5).
In vitro nuclease assays were first performed in the absence or presence of 5 mM Mn2+ using recombinant, GST-tagged proteins and a 5′-end-labeled A30 RNA substrate (Figure 5A). Protein preparations used are shown in Figure S2. Consistent with previous results (6), WT Rrp44 (lane 2) exhibited strong 3′-exonucleolytic activity in the absence of Mn2+, completely degrading the substrate to a 4 nt product. In contrast, the Rrp44-exo mutant showed little degradation activity (lane 3). However, in the presence of Mn2+, both WT and mutant proteins cleaved the RNA, generating very similar degradation products (lanes 5 and 6). From these results, we concluded that Rrp44 harbors two distinct ribonuclease activities, which respond differently to Mn2+ ions under in vitro conditions. The presence of Mn2+ inhibited 3′-exonuclease activity, whereas a second activity was stimulated.
To further characterize the novel activity, we performed in vitro nuclease assays using the exonucleolytically inactive Rrp44-exo mutant and 5′- and 3′-labeled A30 RNA substrates (Figure 5B). Rrp44-exo generated a ladder of degradation intermediates from both substrates in the absence of added Mn2+ ions (lanes 2 and 6), likely due to co-purification of a low level of Mn2+ bound to the recombinant protein, but the degradation activity was strongly stimulated in the presence of added Mn2+ (lanes 4 and 8). The ability of the Rrp44-exo mutant to generate degradation intermediates from both 3′-labeled and 5′-labeled substrates shows that it either carries both 5′-exonuclease and 3′-exonuclease activities or, more likely, possesses an endonuclease activity.
To assess if this potential endonuclease activity was associated with the PIN domain of Rrp44, point mutations were introduced at each of the four conserved active-site amino acids (D91N, E120Q, D171N, D198N), to create the Rrp44-endo mutant. In addition, the Rrp44-exo mutation was combined with the four PIN-domain mutations (Rrp44-endo–exo) and with a point mutation in the S1 RNA-binding domain (Rrp44-exo-S1) (6).
In vitro nuclease assays were performed using recombinant, GST-tagged proteins and a 5′-labeled substrate RNA derived from the 3′ region of the mouse 5.8S rRNA, which has a well defined, stable terminal stem structure (Figure 5C and D). WT Rrp44 (Figure 5C, lane 2) partially degraded the substrate to a 10 nt product. In contrast, Rrp44-exo generated a set of fragments with end-points in the loop-region of the substrate RNA (Figure 5C, lane 3). These fragments were not generated by either the Rrp44-endo (Figure 5C, lane 4) or Rrp44-endo–exo proteins (lane 5), indicating that their formation required a functional PIN domain. Rrp44-exo-S1 did generate the loop fragments, but lacked the major 10 nt product, suggesting that S1 RNA-binding activity might be more important for the exonuclease activity of Rrp44 than for endonuclease activity. A prominent product that is truncated by ~10 nt was detected in some samples. This appears to be due to a contaminating nuclease that is associated with purification of GST-fusion constructs from E. coli, as the same band was seen in analyses of other fusion proteins (data not shown). The truncated fragment appears to be very susceptible to degradation by the exonuclease activity of Rrp44, giving rise to the observed variations in signal. The PIN domain alone did not show in vitro activity. However, when purified from E. coli the PIN domain co-purified, apparently stoichiometrically, with the bacterial chaperone GroEL (indicated with an asterisk in Figure 7B), strongly indicating that it is largely misfolded. We conclude that recombinant Rrp44 shows an endonuclease activity that requires Mn2+ ions and the metal-binding amino acids of the PIN domain.
The effects of the rrp44-endo mutation were also tested on viability. In the GAL::rrp44 strain, expression of Rrp44-endo clearly supported growth when WT Rrp44 was depleted by growth on glucose medium. In contrast, expression of Rrp44-endo–exo failed to support growth (data not shown), indicating that the endonuclease and exonuclease activities of Rrp44 share some redundant essential function. In plasmid shuffle experiments, expression of Rrp44-endo, but not Rrp44-endo–exo, supported viability in an rrp44Δ rrp6Δ double mutant strain (data not shown, but see Figure 6B, lane 6). A low number of rrp44Δ strains complemented by the plasmid expressing Rrp44-endo–exo could be isolated by plasmid shuffle (data not shown). Rare cells are therefore able to adapt to loss of both the endonuclease and exonuclease activities of Rrp44 by epigenetic or physiological mechanisms.
Northern analyses were performed on the GAL::rrp44 strain expressing Rrp44-endo and Rrp44-endo–exo during Rrp44 depletion (Figure 6A). These showed that the 5′-extended forms of the 5.8S rRNA and truncated forms of the 5′-ETS observed in strains expressing Rrp44-exo were suppressed in the rrp44-endo strain and altered in the rrp44-endo–exo strain. In particular, 5′-ETS fragments accumulating in the rrp44-exo strain (Figure 6A, lane 4) were clearly reduced in strains expressing Rrp44-endo–exo (Figure 6A, lane 8). This suggested that the endonuclease activity of Rrp44 is involved in formation of intermediate species that are stabilized by loss of Rrp44 3′-exonuclease activity. The longer form of the 5.8S + 30 RNA seen in the rrp6Δ strain expressing Rrp44-exo (Figure 6B, lane 5) was not observed in the rrp6Δ strain expressing Rrp44-endo (Figure 6B, lane 6). Accumulation of the truncated form of the 5S rRNA, seen in rrp44-exo strains, was not suppressed in the rrp44-endo–exo strain. However, the truncated 5S was accumulated in rrp6Δ strains, even in the presence of the rrp44-endo mutation.
Expression of only Rrp44 lacking both the endonuclease and exonuclease activity resulted in a phenotype that closely resembled that seen on depletion of Rrp44 (vector control in Figure 6A, lane 10). Together the data indicate that the PIN domain of Rrp44 exhibits endonuclease activity in vitro and in vivo.
The archaeal exosome lacks a homologue of Rrp44, and yeast Rrp44 can be removed from the remaining nine components of the exosome by washing with high salt. This indicates that Rrp44 associates with a highly stable, nine component exosome core structure. A two-hybrid interaction was detected between the PIN domain of human Rrp44 and the RNase PH-homologue OIP2 (the human homologue of yeast Rrp43) (39) suggesting that the PIN domain might be involved in protein–protein interactions that tether Rrp44 to the core structure. This model would be consistent with the absence of PIN domains from E. coli RNase R and II (Figure 1A), which function as monomers.
The association of Rrp44 with the exosome core was initially tested by glycerol gradient centrifugation of yeast cell lysates (Figure 7A). WT, protein A-tagged Rrp44 largely co-sedimented with the core exosome component Rrp43 in ~14S complexes (fractions 14–17), with a small peak corresponding to the position of free Rrp44 (fractions 7–8). In contrast, protein A-tagged Rrp44 lacking the PIN-domain (Δ aa 86–203), was expressed at levels substantially below that of WT Rrp44, but predominantly sedimented as free protein. In Rrp44-depleted cells, Rrp43 becomes enriched in ~60S gradient fractions, reflecting stabilized exosome association with pre-ribosomes (L. Milligan and D.T., unpublished results), giving rise to the strong signal in the pellet fractions on the gradient shown.
Binding of Rrp44 to the core exosome was also assessed by in vitro binding assays using recombinant GST-tagged Rrp44 constructs expressed in E. coli (Figure 7B) and core exosome purified from yeast, with or without removal of Rrp44 by washing with 800 mM MgCl2 (Figure 7C). The GST-tagged constructs were bound to glutathione sepharose and incubated with the exosome preparations. The association of the exosome with the GST-fusion proteins was assessed by western blotting using antibodies specific for Rrp43 (Figure 7D) and Rrp4 (data not shown). Exosome binding to the full-length Rrp44 (FL) (Figure 7D, lane 2) was substantially stronger than to Rrp44 lacking the N-terminal PIN domain (ΔPIN) (Figure 7D, lane 4), which was not above the GST background (Figure 7D, lane 6). Notably, exosome binding was not affected by four individual mutations in the PIN domain (Rrp44-endo, see Figure 5) (Figure 7D, lane 3). The PIN domain alone (PIN) (Figure 7D, lane 5) gave a lower signal, but this clearly was above the GST background. As noted above, the low signal may reflect poor folding of the isolated PIN domain, as suggested by its copurification with bacterial GroEL. The signal from the Rrp44-depleted exosome (exosome –44) was stronger than for exosome purified at lower salt concentrations (exosome +44). However, some binding to Rrp44 was seen even without the high-salt wash, suggesting that Rrp44 is substoichiometric in the purified exosome.
We conclude that the PIN domain of Rrp44 plays a dual role in that it harbors endonuclease activity and also functions in tethering Rrp44 to the remaining nine subunit core of the exosome.
Genetic depletion of RRP44 is lethal (21), whereas strains expressing the Rrp44-exo mutant are viable with only a modest growth defect, at least in some strain backgrounds, showing that its exonuclease activity is dispensable for growth. It is, of course, difficult to be certain that any mutation fully inhibits enzymatic activity in vivo. However, this seems probable, as the D551N mutation (rrp44-exo) abolished detectable in vitro activity on more than five different substrates (4–6) while the equivalent mutation (D209N) abolished the activity of E. coli RNase II (40).
The exonuclease activity of Rrp44 does play an important role in pre-rRNA processing, participating in both the maturation of the 7S pre-rRNA to 5.8S rRNA and the degradation of the excised 5′-ETS region. However, on both substrates the phenotype of strains expressing only Rrp44-exo was substantially weaker than that seen in strains lacking Rrp44. The only substrate tested that showed apparently identical accumulation in Rrp44-depleted and rrp44-exo strains was a 3′-truncated form of the 5S rRNA, which was previously identified as a TRAMP/exosome substrate (24).
Strain heterogeneity was observed in sensitivity to the rrp44-exo mutation, which conferred a stronger growth phenotype in strain W303 than in a BY4741 strain background, even though both are widely used 'wild-type' strains. We tested W303 because it expresses only a truncated form of the RNase II-related protein Ssd1, and we speculated that this might show genetic interactions with loss of the exonuclease activity of Rrp44. In the event, expression of full-length Ssd1 failed to suppress the sensitivity of W303 to the loss of Rrp44 exonuclease activity.
The essential function of Rrp44 might be the maintenance of the structure of the exosome complex or binding to either substrates or cofactors. Recombinant GST-Rrp44-exo protein was able to bind to RNA (6), and the yeast exosome containing Rrp44-exo might thus still be able to recruit RNAs that are normally substrates for Rrp44. Substrates recognized and bound by Rrp44-exo might then be degraded by other nucleases. Loss of Rrp44 exonucleolytic activity is synthetic lethal (sl) with deletion of the nonessential cytoplasmic 5′–3′ exonuclease Xrn1, due to the synergistic inhibition of cytoplasmic mRNA degradation (33). This indicates that the rrp44-exo mutation blocked the 3′ degradation of at least some mRNA species in vivo. In contrast, BY4741 strains expressing only the Rrp44-exo mutant were viable in the absence of the other exosome-associated 3′-exonuclease Rrp6. This finding differs from previous observations made in a W303-related strain (BMA 36) (4), presumably reflecting the differences in strain background. Recent analyses (41) indicate that functional interactions between Rrp6 and Rrp44 are important for only a subset of Rrp6 substrates.
The exonuclease activities of the exosome in the nucleus (Rrp44 and Rrp6) likely form part of a redundant network of 3′-exonucleases, including the proteins of the Rex family (42), which could, for instance, be needed to fine-tune the balance between pre-ribosomal RNA processing and decay. In the cytoplasm, such a fine balance might not be necessary, since RNA decay is the predominant pathway.
The differences in RNA processing phenotypes between strains depleted of Rrp44 and those expressing Rrp44-exo, suggested that Rrp44 might harbor additional activities. Recent reports of manganese-dependent endonuclease activities associated with other PIN-domain proteins prompted us to determine whether this might also be the case for Rrp44. The endonuclease activity of Rrp44 failed to be observed in previous analyses, probably because in the absence of manganese the exonuclease activity is much more prominent. The concentrations of 0.5 or 5 mM Mn2+ that were used to inhibit exonuclease activity and stimulate endonuclease activity of Rrp44 are much greater than the physiological concentration (43). It seems likely that the in vivo endonuclease activity of Rrp44 is stimulated by one or more of the many exosome cofactors, allowing it to act at lower Mn2+ concentrations. However, we cannot fully exclude the possibility that the PIN domain primarily functions in RNA substrate binding in vivo, with a non-physiological cleavage activity that is induced by high Mn2+ levels in vitro. The range of in vivo substrates for the activity of the Rrp44 PIN domain remains unclear but comparison of the RNA processing phenotypes of strain expressing Rrp44-exo and Rrp44-endo–exo strongly indicates that these are likely to include the 5′-ETS region of the pre-rRNA. However, the putative target sites do not appear to correspond to the known A0 cleavage at position +470. This suggests that the activity of the Rrp44 PIN domain does not act during pre-rRNA maturation, but rather participates in the degradation of the excised spacer region following A0 cleavage. Several early acting ribosome synthesis factors are released from the pre-ribosomes in association with the excised 5′-ETS (44). Very efficient degradation of the 5′-ETS is therefore likely to be important for the release and recycling of ribosome synthesis factors.
Analyses in vivo and in vitro showed that the PIN domain of Rrp44 was both necessary and sufficient for association with the 9 subunit exosome core structure. Previous EM analyses indicated that both the Rrp44 N-terminus (where the PIN domain is located) and the C-terminal region form interactions with the exosome core (45). The data presented here indicate that the N-terminal interactions are more crucial for stable binding. Alterations in the relative positions of the N- and C-terminal regions of Rrp44 seen on association with the core exosome are proposed to be crucial for regulation of its exonuclease activity (45), and interactions between the C-terminal region of Rrp44 and the exosome may play an important role in this regulation.
Within Rrp44, the PIN domain appears to have a dual role, acting to link Rrp44 to the remainder of the exosome in addition to its apparent cleavage activity. This evolution of the region therefore appears to play a crucial part in the functional differences between bacterial RNase R and eukaryotic Rrp44. The structure of the exosome core is related to that of bacterial PNPase (5,46,47). In E. coli, PNPase can associate with RNase E and other proteins in the degradosome complex, that shows both 3′-exonuclease and endonuclease activity (48). The finding that the eukaryotic exosome also possesses both 3′-exonuclease and endonuclease activity underlines the conservation of the RNA processing machinery.
Supplementary Data are available at NAR Online.
This work was supported by the Wellcome Trust (to D.T.) and BBSRC (to J.D.B.), a long-term HFSP fellowship (to C.S.) and a PhD studentship from the BBSRC (to E.L.). Funding for open access charge: Wellcome Trust.
Conflict of interest statement. None declared.
We thank J. Houseley for yeast strains and reagents and critical reading of the manuscript.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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It is with great excitement that we announce this summer's Vacation Bible School, "Hero Central." Please mark your calendars and register for VBS on June 5 through June 8, 2017.
FPC Vacation Bible School offers a highly tailored program of Bible exploration. All children investigate the same daily Bible stories in various age-appropriate ways. For preschoolers, the program is offered in classroom space and at a pace appropriate to children ages 4 and 5. Elementary children find themselves traveling the campus and meeting with various leaders in workshop fashion. Rising 5th and 6th graders (VBS Plus) experience the Bible stories through the lens of mission and service. They often leave the campus to work and serve throughout the local community. The morning includes storytelling or Bible study, puppets, music, crafts and recreation for everyone.
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Already a volunteer and need nursery care? Please download the nursery registration form below and return it to Katie Resumovic in the church office. Nursery care is only available for VBS volunteers.


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Description: New York native Karmen Bella is a hot brunette babe that owes her fantastic looks to a mix of Indian and Romanian blood, but her lusty nature we have yet to find a cause for. Before we could get to know her, she got horny and got a doggy style dicking after begging for it.
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Here is a classic High ROI entry into a Parabolic Daily Move
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Profitable Trading Pattern:Eur/Cad
Perfect 10XROI Trade on Aud/NZD
10XROI GBP/USD Trade: Price Action
50-20,000 in One Week MM Case Study
The Holidays are Over and it's Back to Trading
Here is my first trading video after the summer break. High ROI trades on US Oil.
Post Brexit GBP/USD Final Update
Here is the last of the three post-Brexit videos featuring the fall and rise of the GBP/USD and I am doing this post after the surprise announcement from the BOE to keep interest rates unchanged confounding the markets who widely expected a rate drop. The question is could you predict the results and trade them using my systems and in this video I show that in my opinion price moves in very obvious ways that can be easily predicted regardless of the fundamentals.
Click here to watch this Post-Brexit trading video on Youtube.
Brexit Trade Using 30 Minute Push-Pull Day Trade Entry
Here is an update to the previous Brexit video using GBP/USD
Post Brexit GBP/USD What next?
Here is my first Brexit Video, where I think the GBP/USD will go next.
Click here to watch this Post- Brexit trading video on Youtube
10XROI Trade on Eur/Cad Rinse and Repeat
Sometimes I get the feeling that I am just repeating myself showing 10xroi videos because the setups are doing the same thing again and again, but that is what trading is like, doing the same things again and again and doing them well. Enjoy…
10XROI Trade on Aud/USD
Here is an interesting 10XROI Trade, it is all about the decision process behind taking a trade that may not look ideal, enjoy!
New Trading Psychology Book Out Only $0.99
My new trading psychology books is out new and comes with a FREE six-part video series on trader-psychology I recorded a while back
you can grab it Here now for only $0.99.
High ROI Trade on Weekly Chart
Here is this month's trade analysis using the weekly chart as a basis for a push-pull swing trade. Enjoy!
Click here to watch this video on Youtube
Lessons in Bravery
I have just been listening to a Tim Ferris interview with Caroline Paul (author of The Gutsy Girl -Escapades for Your Life of Epic Adventure) who is one of the few female firefighters in the San Francisco Fire Department as well as a pilot, white water rafter and all-around adventuress.
What really caught my attention though was how she described dealing with fear, she observed fear as though she was looking at at wall of bricks and fear was one of the bricks, she then asked herself what weight she wanted to give the fear in what she needed to do, she then remembered the importance she attached to feeling brave, she then pushed the fear somewhere else at the back of her mind as she decided not to allow it to be of importance.
To have bravery as one of her core values and then choose to be brave and place low emphasis on fear during high risk situations is a strategy that a trader can learn from. Can you create an image of your emotions which you can manipulate such as a pile of bricks and assign emotions to them and move them about according to importance?
Maybe use another mental construct if bricks don't do it for you, something that you can move around and control. This all depends of course on being able to distance yourself from your emotions rather than being hijacked by them; a key skill for traders.
Multiple High ROI Trades in Oil
Here is a quick video showing how to find multiple ways to enter into the latest oil slump.
Create Your Trading Success in 2016
This month saw the launch of a new Trading Psychology self-help course "Create Your Trading Success in 2016". You can check it out HERE.
3 Easy Trades on NZD/JPY
Here are some easy entries into a move using the 4-hour chart for a base where on the daily chart it would have been difficult to spot.
Here is a quick video answer to a recent student question.
"A quick update since starting your course.I'm going through the initial 10 x ROi again as I didn't manage to nail it down the first time. I have been struggling to identify which candle would be number one as a push pull and why."
Three High ROI Trades on Gold
In this video you can see multiple opportunities for 1-10 and 1-5 ROI trades.
Easy 1-10 ROI on US Oil
Here is a quick video showing an easy 10XROI Trade on US Oil, enjoy!
How Can I Trade When I'm Asleep?
Here is a question from one of my students and you will see my answer below
"I have read the 10XROI book, and it has inspired me to work on my own setups. The one difficulty I have, which would be the same difficulty as with your system, is that going down in time frame from the daily charts to the hourly charts is a bit problematic for me. I live in New Mexico and I'm asleep during the early trading hours in London so I miss the reversals that occur then.
For example I went long the EUR/GBP at 10:00AM NY time on 11/23, on the hourly pullback after price broke the horizontal range. It was moving in my favor nicely until the early trading hours in London today, when the Italian retail sales numbers fell short. I was shooting for the 10XROI target, made it to about 7XROI. I was taken out at breakeven, had I been watching I could have made 30 or 40 pips.
I had the fleeting thought before I went to bed that I should move my stop, because of the coming holiday, and low liquidity, but I didn't. I realize this is something you warned about in your book, and I get to experience it on my first trade! (Thanks for that trading Gods)
However, my interest has been peaked and I'm really excited about moving forward with my version of the 10XROI system. One modification I'm thinking of making is trailing on pivots on the 4 hour, or 60 minute charts, what are your thoughts about that type of trailing stop?
Best Regards,
Michael Morse"
My answer to Michael is this
Michael, When you are setting a take profit you need to take into account certain things and adjust your take profit accordingly but it should never fall below a 1-7 ROI, I don't trade the EUR/GBP because in my opinion it is not a good value pair with a low daily range and a comparatively high spread, if you had been trading another pair you would probably got your 1-8 or 1-10 ROI.
You should not have to stay awake to take your profit, your stop loss is in place and unless an unusual event occurs such as a large parabolic move followed by a pin candle (by which time you should have hit your profit target) there is no reason to exit the trade. These trades are designed to be low stress and with low screen time.
The problem with using a trailing stop is that you can be taken out at a 1-3 roi, 1-5 roi etc with pull backs which completely skews the numbers of the system.
However here is the caveat, you are adapting the 10XROI system to suit your way of trading and if it is working for you and making you profit after you have adapted it then that is fine too.
I'm 260 Pips Up, What do you think?
Here is a message I just recieved from a student and im going to give some advice which you may find helpful.
(see message below)
Hi LR
Just thought I'd update you on a 10xROI trade I'm currently in. Up about 260 pips so far (1:5R). It's progressing quite well. Let me know what you think.
10XROI Trade – 19/11/15
Daily Setup
Push-Pull setup after a momentum move with candles hugging 3MA in a down trend
Hourly Setup
Break of mini support
50 pip SL, 500 pip TP
currently +261 pips -moving to be
My first comment is well done to Kevin as he is pretty new to this system and he's doing great so far!
1) Here is how I would handle this as it is over a weekend, I would close the trade and re-enter at a better price if possible, unless Kevin is based in the UK and can use a spread betting broker with a guaranteed stop.
The reason is that there can be a gap over the weekend and it may not be in his favour, also the trade has already moved a long way and is due for a pull back as you can see by the last candle which is a long candle.
Generally price opens at around the same point as it closed on Friday but this protects your account in case of weekend gapping.
2) I would look at the context of the trade which is at the end of a run and adjust my expectations accordingly, as you can see price had already moved a long way down and had bumped up against some previous support so I would be minded to start moving my stop loss and be willing to exit at a 1-7 or 1-8 ROI.
But in any case well done to you Kevin!
Multiple Time Frame Analysis: 1-15 ROI on EurJpy
Here is an easy Eur/Jpy trade that you could get a 1-8 ROI or a 1-15 ROI by using my pyramiding techniques, it shows how to use a multi-candle push-pull.
Click here to watch this video on Youtube.
Easy 1-20 ROI Pure Price Action
Easy way to get a 1-20 return on a trade taking very little time and using a day trading entry. watch the video to learn how.
PRICE ACTION TRADING 1-30 RISK to REWARD
Here is a great and simple trade which would have given a minimum of 1-8 ROI with a potential 1-30 ROI or even more if held till the daily support .
This Makes my Job Worthwhile
When I get a letter like this (see below) I can really see what the impact of my teaching is, it just makes the effort feel worthwhle. ——————————————————————————— LR Thomas Hello Ms. Thomas, I'm sending you a quick email, although…
Example of a Day Trade using a Daily Push-Pull
See an example of one trade using the Daily Push-Pull as a foundation, there are 6 different types of setup available in this course so you should be able to find High ROI Day Trades nearly every day.
Here we can see the context of the trade an upward move followed by an upward daily channel with a third bounce off the lower daily trendline.
Here we see the pushpull candle 1 is a small candle followed by candle 2 the much larger push candle, candle 3 pulls back to the close of candle one. the pushpull zone is between the open and close of candle one which is where we look for an entry which we find on candle 4 the following day.
We go down to the 30 minute chart to look for an entry and see an overnight megaphone channel on the 30 minute chart
the orange dashed line is the 30 min resistance which is broken. the dashed green line is the push-pull level.
On the 5 minute chart there are 3 entries
The take profit could be a minimum of 1 -3 ROI
High ROI Day Trade on USD/JPY
Eur/Jpy High Probability Trades:Price Action
10XROI Trade on USD/JPY
Trading Opportunities April Recap
Student is 1002 Pips Up in the Last 2 Weeks
Mindset Video Post: A Good Trade
Easy 10XROI on the Eur/Nzd
Adding Weekly Push-Pulls to the 10XROI System
One of the biggest complaints I get about the 10XROI system is that there aren't as many trades as my students would like and sometimes you may have to wait weeks for a trade. In this video i go through how to use weekly push-pulls and show how you can really increase the volume of trades by adding in these weekly setups. ps There is also nothing to stop you adding in monthly push-pulls as well!
How to Stop Over-Trading Now in Audio
My trading psychology book How to Stop Over-Trading is now available in audio book format at the link below.
Price Action Forex Trading..Lovely 10XROI Trade
Losing Trade Analysis on Eur/Aud
Overcome Your Fear in Trading Now in Audio!
My trading psychology book Overcome Your Fear in Trading is now available as an audio book on Audible, Amazon and Itunes
Here is a UK link http://www.audible.co.uk/pd/Business/Overcome-Your-Fear-in-Trading-Audiobook/B00SYVSSMY/
Here is a US Link http://www.audible.com/pd/Business/Overcome-Your-Fear-in-Trading-Audiobook/B00SYVXHA2/
Why Focus on High ROI Trades?
One hidden benefit of a high ROI trading system over a high Probability trading system is that you don't need to be the best trader in the world to make money. Some time ago I bought a day trading course…
Aud/Usd Trade using Technicals AND Fundamentals
Flow Like Water in Trading
How to Trade During the CHF Announcement
I was looking at a trade yesterday to see what was happening to the markets around the time of the CHF announcment which removed the 1.2 level which the Swiss had been defending for the past three years. Here is a short video below showing what i found.
Trading Success 2015-Webinar Replay
Control Your Inner Trader Now Available in Audio
For traders who like to listen to books rather than read them, Control Your Inner Trader is now in Audio Book Format on Amazon
Click Here For More Info..
How to Avoid Price Spikes
Kid makes $72 Million Trading the Stock Market
I noticed this article about this Kid who was supposed to have made $72 Million trading the stock market. There is a lot of debate whether the boy Mohammed Islam actaully made that money although the latest news seems to be that he has made 8 figures, who knows?
Anyway the thing that I found really impressive was the http://leadersinvestmentclub.com/the-team/, (the members have banned Mohammed for misleading the public, the plot thickens),none of these guys are over 25, amazing!
What is it about young people that enables them to suceed, maybe they know no fear? maybe they don't have all the baggage of years holding them back. In any case check out the site it's interesting!
Go For Low Hanging Fruit
New Market Rhythms Book Release
I have just released the book Market Rhythms :Learn How to Read and Profit From the Price Rhythms of the Market
Only $2.99 on Amazon. This book will teach you how to understand market rhythms and how to integrate them into your trading. Market rhythm is applicable whatever time frame, trading instrument or trading system you use and will help you both increase profits and just as important, reduce losses!
This book was created because there is very little usable information available on market rhythms and how they affect your trading and yet it is a vital part of any trading decision that you make. Market rhythms apply to the movement of the market, candle patterns and chart patterns.
Price action does not form in a vacuum; there are transitions from one type of price action to another. The market tends to form rhythmic price action which then transitions through to non-rhythmic price action. By understanding what the market is telling you, you can make better trading decisions.
The book is presented in a nice and simple to understand fashion. Click Here to Check it Out
10XROI Trade Using a Scalping Entry
Watch this video to learn how to turn a scalping trade into a 10XROI trade or even more if you use pyramiding techniques to hold the trade.
Pure price action, technical analysis using patterns and breakouts.
For Forex traders who want to learn how to become more profitable you need to go after low risk and high reward trades.
Watch the Video and Enjoy!
How to Catch the Big Moves in Forex
I have just released my new eBook which is a short and to the point guide showing how to catch the big Moves in the Forex Market
Click here to check it out.
Using Stochastics in Your Price action Trading
Price Rhythms Helping With Four Hour Price Action Trade
Here is a trade I took today which could have been a swing trade on the Eur/Usd but instead I took it as a five minute scalp trade. It is interesting to see how precise patterns can be on the smaller time frames, which can help you tap into the momentum of the higher time frames which helps get to take profit more quickly and with higher certainty.
Pure Price Action:How to Avoid False Breakouts
What I Love About Trading
I know that there are a lot of difficulties in becoming a profitable trader, but there are a lot of difficulties in running any business profitably and in my other business ventures I am experiencing some of these difficulties at the moment. When these issues come up it hits me what great advantages there are in trading as a business compared to other businesses and in case anyone apart from myself needed reminding, I thought I'd just do a quick list here.
1. Easy to scale.. you can start with a tiny account and test out your system..if you lose money you have only lost a little but if you find a winning strategy you can scale more or less as big as you like. In other businesses this is much harder and if it is possible can require a large investment in marketing.
2. No customers,no employees,no employer,no office politics..in other words not having to deal with people who do your head in.
3. Time freedom, even if you dont make a huge living trading you can work hours that suit you, if you only want to work three days a week you can, if you only want to check charts at the end of the day you can, bringing your work time down to two hours or less per week!
4. Your risk is quantified, when you start up a business where you may have to invest in premises,stock,staff etc, you have no real idea what your risk is going to be, in trading you know.
5. No competition,in other types of business you always have the competition coming up behind you to try and grab your business, in trading you always have a market however many traders there are. It makes no difference how many traders flood the market as regards how profitable you can be.
6.No expenses except your trading account and maybe a trading platform, most businesses require outlay.
I know this is a very short list and there are probably a lot more advantages to trading but for me these are the main ones.
Bye for Now and Good Trading…
One Chart Only Technical Analysis on Aud/Jpy 10XROI Trade
High ROI Trade on the EUR/JPY
Here is a video of a High ROI Trade on the Eur/Jpy that could have been taken using the 10XROI System in conjunction with The High ROI Scalping System
Second Cad/Jpy Trade
Cad/Jpy 10XROI Day Trade
Getting Out of Your Own Way
Aud/Chf Update Re-entry on Break
In my last video post I talked about my 1-3 ROI trade on the Aud/Chf and talked about why I exited the trade. However I kept an eye on the pair and after much sideways movement at that four houly trend line area it eventually broke. I then entered on the 15 minute time frame and have just exited at the 0.8600 level.
Aud/Chf Trade 8th September 2014
Eur/Nzd News Trade Taken Today
GBP/JPY High ROI Scalping Trade Taken Today Video Post
I found this GBP/JPY trade today which gave me an easy1-3 ROI, I forgot to put in the video that I took the trade at 1.30 about an hour and a half before the Cad Interest Rate announcement, however I did some research and it looked like there were no suprises dueplus I moved my SL to break even just before it hit.
Recent 7-30 ROI Trade Spotted on Eur/Jpy Video Post
Eur/Aud Summer Trade Using Two Combined Systems
Student Goes From Bacon to Filet Mignon With USD/CAD Trade
Student Uses Weekly Push-Pull to Bring in Bacon
In my last post and in my monthly newsletter I talked about the Eur/Cad as being a pair to watch in July, I also covered the two weekly push-pull setups in my last video. Well a new student of mine set me this reply together with his chart, which I am reprinting with his permission.
"Thank you for the interest in how I was doing since I started the High ROI Forex Trading Course. Well I was successful with 2 trades! The EUR/CAD and the AUD/CAD. especially the first one brought us bacon for a whole month. Just wondering how long it will take for such a set up it might take weeks. Do you see and possible trades that could develop in the near future?"
Here is my answer to him
"To answer your question , its vital that you don't force trades whatever time frame you are trading. The more systems you trade in tandem the more trades there will be, however what is important is to master one system at a time.
Just be patient and build your account, compounding and high ROI trades will do the work. You just focus on taking the best trades you can find and continual improvement and using good money management. I do a newsletter which goes out monthly where I analyse the upcoming levels for the month as well as analyse the trades for the past month. Here is the link if you are interested,
Here is the chart showing the four hour entry on the Eur/Cad weekly push-pull.
Multiple Trades on the Eur/Cad Video
Process Versus Results Thinking Chris Mathis From ForexMentor Agrees!!
How Many Trading Filters Should You Use?
One of the biggest problems I see with traders and trading gurus is that they overcomplicate trading by using too many filters. The problem with adding more and more filters to your trading is firstly that filters will start to contradict each other, secondly you will become overwhelmed with the amount of information needed to be taken into account before taking a trade and thirdly there is no proof that having overly complicated systems actually improves your results. What type of filters am i talking about? Here is a list…
Lower and higher time frame support and resistance
Fibbonacci levels
Pivot Points
Murray Maths
Moving Averages
Elliot wave
Candle Patterns
Chart Patterns
Multiple Indicators
Round Numbers
Average daily Range
Trend lines
Harmonic patterns
Gartley patterns
Higher time frame momentum
Correlation of other related pairs
Correlation of the Indices
Correlation of commodities
The more filters that are added in the harder it is to make a decision, so here is a bit of advice. Go for the least amount of filters rather than the most.
You can trade successfully using support and resistance and candle patterns only.
You can trade successfully using chart patterns combined with a few candle patterns.
Go for simplicity rather than complexity, rememmber the only decision you are really making is will price go up or down and have you found the optimum place to enter that is low risk and high reward. Those questions can be answered using very simple systems with very few filters. I bought a day trading system some time ago which was created by a fantastic short term day trader, but she has multiple filters which she uses to make a decision which made the system too subjective and complicated for me. I like simple, simple can still give you a profitable system and much less of a headache.
Two Day Trading Systems Combined for 1-5ROI
Here is a channel trade using my new upcoming High ROI Day-Trading System which works alongside my High ROI Scalping System. You can see how exact the entries and exits can be when you combine channel trendlines, chart patterns, candle patterns and the ADR.
Businesses You Can Run While Waiting for a Trade
I have received a good amount of requests for some information on running multiple businesses while you trade. I have created a video on this topic and because it is rather lengthy I have only covered three businesses that are…
Trading and Black Jack
I have just been reading 'Beat the Dealer' by Edward Thorp where he describes his system for beating the casinos at Black Jack. The book was written in 1962 and in order to find their mathematical edges they had to…
Trade Management of GBP/AUD Trade (Video)
Here is a video showing how and why I closed my recent GBP/AUD trade.
Lovely 10XROI Trade on GBP/AUD (Video)
Here is my analysis of a trade I took this morning, using multiple time frame analysis from the monthly down to the five minute charts.
Autoresponder is Down
>>Use the New Subscribe Box on the Right<< Many apologies to people who have tried to sign up or click links. The GetResponse website is down and none of their services are working. Hopefully they will get it fixed soon.…
Building Other Businesses While You Trade
I advise my students not to depend on solely trading income if they want to leave their jobs. The time spent waiting for trade set-up can be usefully spent creating other income streams that allow time flexibility and stability of monthly…
Why Trading Mindset is Critical to Success
My trading mind-set books are the lowest sellers of my Amazon eBooks. The reason for that is simple, traders generally want to learn how to make money and are attracted by the idea of a trading system with a high…
How to Stop Over-Trading
Just to let you know that my new eBook 'How to Stop Over-Trading' is now live on Amazon. If you have ever taken a trade that is outside your strategy,then this book was written to stop that behaviour dead in…
Recommended Blog
Today I have one recommendation to make re. a blog that I discovered through one of my students FXOutlier. It stands for everything that I have come to learn (the hard way) about trading. This blog is a great addition…
Combo 10XROI trade on AUD/JPY
I have been talking a lot about the AUD/JPY of late, here is the link to a previous post. Click Here Here is a video of a trade I closed yesterday on the AUD/JPY and my reasoning. (corrections) 4.06 exit not enter,4.24 my stop loss not roi
Announcement re Free Coaching session
Thanks to everyone who replied to the request for feedback about their over-trading issues. I also got a lot of responses from people explaining why they thought other people over traded. Every reply was extremely useful and everyone who responded…
Potential Short Term and Medium Term AUD/JPY Trades
The AUD/JPY is hitting the upper trend line in an upward moving daily channel, in the short term it may provide a nice reversal counter move which could be entered on the lower time frames the four hour or the…
New Book Coming Out!
I am in the process of writing my next book, How to Stop Overtrading! I will be giving free copies of the book to those people who can add to the book by sending me their specific over trading scenarios,…
Using the 61.8 Fibonacci Level on a High ROI Scalp Trade
As a continuation to my previous comments about Fibonacci levels I show how I would use them if I were trading a High ROI scalping setup.


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ByWater Solutions announced this week that they will offer support, installation and hosting for Libki. To learn more about Libki please visit booth #117 at the ALA Annual Conference in Chicago, IL to meet the ByWater team, see a demo or ask questions.
Organized Libki demos will be offered at these times:
- Friday, June 28: 5:15pm
- Saturday, June 29: 11:30am
- Saturday, June 29: 2:00pm
- Sunday, June 30: 11:00am
- Sunday, June 30: 4:00pm
- Monday, July 1: 9:30am
We'll of course show you anything specific should you stop by at any time!


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Music Sensation GREYSON CHANCE Signed Autographed 11x14 Photograph
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***Item Description***GREYSON CHANCE hand signed 11x14 Photograph Beautifully Hand Signed by music sensation GREYSON CHANCE Type of Pen: BLACK SHARPIE Date and location of signing will be included with Certificate of Authenticity Proof photos that are shown in the auction are posted for authentication purposes only and are not included with the item. ***Authenticity*** Each item we sell comes with a Certificate of Authenticity issued by SIGNED Sports and Entertainment Memorabilia documenting the date and location each item was signed. In addition, each item comes with a SIGNED AUTHENTICS hologram that has a unique serial number that is registered in our database. The certificate of authenticity will include a matching hologram to the one affixed to the item. All of our items are guaranteed to pass any eBay approved 3 rd party authentication process including PSA/DNA, James Spence Authentication JSA, or Global Authentication GAI. ***Payment*** Paypal, the safe, secure way to pay online, is our preferred method of payment. All Paypal payments should be sent through eBay to the account linked with this auction. If you do not wish to use Paypal , please contact us to make other arrangements. Payments not received within 7 days of auction end will ... read more


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Hamatora The Animation Episode 1 English Subbed
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"File 01: Egg of Columbus"
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Episode Summary: Nice and Murasaki take two job requests from Koneko to investigate the recent kidnapping of college girls and another by Shinji Toyosaki to protect a safe from thieves. At the same time, PIs Birthday and Ratio head off on a bodyguard request to protect a client. With help from Art, Nice deduces that the kidnappers were targeting the girls for information and theorizes that their cases may be linked. Nice then calls Birthday and Ratio for confirmation just as they are attacked by a Gravity Minimum Holder who forces their client, Azuza to reveal a code. Afterwards Azuza reveals that she and her college friends had been seeing the somniloquous Toyosaki patriarch from whom she accidentally learnt the code. Realizing that the kidnappers and thieves are the same group after the safe Nice, Ratio and Birthday head over to the Toyosaki mansion just as the thieves make their move. While Ratio and Birthday take out the thieves, Nice shows up during Murasaki's stare-down with the hired Gravity Holder and defeats the latter with his Holder ability. Afterwards, as the police and Art apprehend the thieves, the latter contemplates on telling Hamatora of a serial murderer targeting Minimum Holders.


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