high - throughput transcriptome analyses in eukaryotes have revealed pervasive transcription at most , if not all , genomic loci . this generates many long noncoding rnas ( lncrnas ) , which lack protein - coding capacity and are distinct from well - characterized structural rnas ( rrnas , trnas , snrnas , and snornas ) or small regulatory rnas . lncrnas arise from intergenic , antisense , or promoter - proximal regions and range in size from 200 nt to > 20 kb . many features are shared between lncrnas and mrnas ; both classes of rna possess 5-methylguanosine caps ( neil et  al . , 2009 ) and poly(a ) tails ( david et  al . , 2006 . moreover , both are transcribed by rna polymerase ii ( pol ii ) from similar preinitiation complex assemblies ( rhee and pugh , 2012 ) and can be regulated by common transcription factors . despite these similarities , the fates and functions of lncrnas and mrnas are substantially different . most mrnas are rapidly exported to the cytoplasm , where they engage with the protein synthesis machinery . in contrast , diverse nuclear functions have been attributed to lncrnas , including the assembly of nuclear domains , directing chromatin - modification , resetting of epigenetic marks , and the regulation of mrna transcription . for example , stable unannotated transcripts ( suts ) are detectable in wild - type yeast , whereas cryptic unstable transcripts ( cuts ) are apparent only in the absence of the nuclear surveillance factor rrp6 , and xrn1-sensitive unstable transcripts ( xuts ) are apparent only in the absence of the cytoplasmic exoribonuclease xrn1 ( van dijk et  al . , 2011 ; xu et  al . , 2009 ) . this indicated that distinct classes of lncrnas can be distinguished from each other , as well as from mrnas , but the features that might differentiate these species were unclear . all mrnas interact with a defined series of protein factors during their transcription , packaging , processing , export , and turnover ( see figure  1a ) , forming ribonucleoprotein particles ( rnps ) . we hypothesized that lncrnas and mrnas must diverge at some point along this maturation pathway . we therefore systematically analyzed the in  vivo , transcriptome - wide targets of key factors in this pathway in budding yeast ( figure  1a ) . we anticipated that this atlas of rnp compositions would provide a comprehensive picture of the dynamic events during canonical messenger rnp ( mrnp ) assembly , and give insights into the definition and behavior of different classes of mrnas and lncrnas . overall , our data reveal how distinct transcript classes are defined and how rnp composition relates to function , and have enabled us to begin to tackle the overwhelming complexity of the transcriptome . to establish how and when different classes of pol ii transcripts are distinguished in the cell , we determined the transcriptome - wide targets for 13 key mrna biogenesis and turnover factors ( listed in figure  1a and table 1 , with references therein ) . we included nuclear surveillance factors ( mtr4 and trf4 ) , a component of the nuclear cap - binding complex ( cbc1 ) , components of the trex rna packaging complex ( gbp2 and tho2 ) , pre - mrna 3 cleavage and polyadenylation factors ( hrp1 , nab2 , and pab1 ) , a nuclear - cytoplasmic export factor ( mex67 ) , an mrna localization factor ( hek2 ) , a cytoplasmic translation factor ( tif1 ) , and cytoplasmic mrna turnover and surveillance factors ( xrn1 and ski2 ) . hrp1 and nab2 function in mrna cleavage and polyadenylation but additional roles are reported for nab2 in mrna packaging ( batisse et  al . , 2009 ) , export ( iglesias et  al . , 2010 ) , and nuclear surveillance ( schmid et  al . , 2012 ) , and for hrp1 in nuclear and cytoplasmic surveillance ( gonzlez et  al . , 2000 ; kuehner and brow , 2008 ) . for target site identification , we used the crosslinking and analysis of cdna ( crac ) technique ( granneman et  al . , 2011 ) . actively growing cells expressing htp - tagged ( his6-tev - protein a ) proteins under the control of the endogenous promoter were uv irradiated to fix direct protein : rna contacts . after stringent , multi - step affinity purification , mild rnase digestion , and radiolabelling , rnps were isolated by sds - page ( figure  s1a available online ) . bound rna fragments were amplified by rt - pcr and analyzed by high - throughput sequencing . identical conditions were used for all proteins tested , and in most cases replicate data sets acquired ( table s1 ) . we also repeated the analysis for the poly(a)-binding protein nab2 in an rrp6 background , which is reported to stabilize its transient binding ( schmid et  al . , 2012 ) . comparison of the number of reads mapping to each annotated transcript in replicate data sets revealed good reproducibility , with most spearman rank correlation coefficients   0.75 ( figure  s1b ) . furthermore , the most highly enriched 10% of mrnas in hrp1 , nab2 , and hek2 data sets showed significant overlap with published immunoprecipitation analyses (   0.001 ) ( batisse et  al . , 2009 ; hasegawa et  al . , 2008 ; kim guisbert et  al . , 2005 ) . a breakdown of hits by transcript class revealed a broad range of substrate specificities for the tested proteins ( figure  s1c ) . the poly(a)-binding protein pab1 predominantly bound to mrnas , whereas trnas were prevalent targets of the nuclear surveillance factor mtr4 , consistent with reports of extensive nuclear pre - trna degradation ( gudipati et  al . , 2012b ) . in hrp1 data sets , snornas were abundant , supporting a role in snorna biogenesis originally indicated by a chip study ( kim et  al . , 2006 ) . many proteins crosslinked to rrnas at regions distinct from those typically detected as background ( figure  s1d ) . mex67 is reported to bind 60s and 40s ( faza et  al . , 2012 ) preribosomal particles , in good agreement with our data , and the peak of xrn1 binding in its1 supports its role in cytoplasmic degradation of this excised spacer region . the nuclear surveillance factors mtr4 and trf4 assist in the degradation of the 5 ets , and we identified binding within this region . the specific rrna association of the cytoplasmic helicase ski2 is likely to reflect interactions with translating ribosomes during mrna degradation by the exosome . other proteins showed distributed binding on the pre - rrna ( figure  s1d ) , but the significance is currently unclear . analyses of hits in mrnas provided a high - resolution picture of the dynamic assembly of mrnps . for each protein , we calculated the ratio of reads mapping across exon - exon ( ee ) versus intron - exon ( ie ) junctions ( figure  1b ) ( schneider et  al . , 2012 ) . ie junctions are exclusively present in unspliced pre - mrna , and ee junctions in spliced mature mrnas , so the ee / ie score indicates when , relative to splicing , each protein associates with the mrnp . low scores were found for the nuclear cap - binding protein cbc1 , trex components tho2 and gbp2 , and nuclear surveillance factors mtr4 and trf4 , consistent with these factors acting during or shortly after transcription . in contrast , high scores for the translation initiation helicase tif1 and cytoplasmic surveillance factors xrn1 and ski2 indicate that they function late in the mrnp lifecycle . the slightly lower score for xrn1 is consistent with its role in surveillance of unspliced pre - mrnas and lariat intermediates ( hilleren and parker , 2003 ) . for example , mex67 and nab2 load onto mrna in the nucleus and contribute to export ( iglesias et  al . , 2010 ) and are removed at the cytosolic face of the nuclear pore ( lund and guthrie , 2005 ; tran et  al . , 2007 ) . the lower ee / ie scores for nab2 suggest that nab2 enters the mrnp before mex67 , which is perhaps only recruited when cleavage and polyadenylation is complete . nab2 and pab1 are both implicated in poly(a ) tail length control , but it has been unclear which acts first . the ee / ie score for pab1 is lower than that of mex67 and similar to that of nab2 , supporting early roles for both nab2 and pab1 in the nucleus . the cleavage factor hrp1 also shuttles and can contribute to cytoplasmic surveillance ( gonzlez et  al . , 2000 ) . the low ee / ie score , however , suggests that hrp1 primarily functions early in mrnp biogenesis , consistent with prolonged cytoplasmic binding of hrp1 denoting an aberrant mrnp . conversely , the high score for hek2 , which contributes to translational regulation and mrna localization ( irie et  al . , 2002 ; paquin and chartrand , 2008 ) , suggests that it binds late in the nucleus and is predominantly associated with cytoplasmic mrnps . although the architecture of several ribonucleoprotein complexes , such as the ribosome and spliceosome , has been studied in detail , little is known about the topology of mrnps . we therefore examined their organization by plotting the average binding distribution ( hit density ) of each protein across its top 1,000 mrna targets ( figure  1c ) and across individual mrnas ( figures 1j1l ) . cbc1 and tif1 reside in complexes that interact with the 5 cap and predominantly crosslinked to mrna 5 ends , whereas the poly(a)-binding protein pab1 bound at mrna 3 ends . for the trex components gbp2 and tho2 and export receptor mex67 hits mapped across mrna bodies , consistent with their proposed ability to bind at multiple sites . the even hit distribution for mex67 suggests that it binds full - length mrnas , whereas the 5 enrichment for gbp2 and tho2 presumably reflects binding to nascent transcripts at various stages of elongation . the mrna localization factor hek2 is homologous to the human poly(c)-binding proteins and preferentially binds ( cnn)n motifs ( hasegawa et  al . , 2008 ; wolf et  al . , 2010 ) . consistently , hek2 showed no overall positional bias but a strong specificity for cnn repeats within individual mrnas ( figure  1l ) . nab2 and hrp1 contribute to mrna cleavage and polyadenylation and , consistent with this , bound the 3 ends of mrnas . unexpectedly , they also recovered many 5-proximal rna fragments , as did the surveillance factors trf4 and mtr4 ( figure  1c ) and the exosome - associated nucleases rrp44 and rrp6 ( schneider et  al . , 2012 ) , which participate in 3-5 nuclear decay . we propose that this reflects binding to unstable promoter - proximal rna fragments rather than to the 5 ends of full - length mrnas ( see below ) . nab2 binding was observed throughout the body of mrnas , consistent with previous chip analyses ( gonzlez - aguilera et  al . , 2011 ) . in addition to binding poly(a ) , nab2 shows nonspecific rna - binding activity ( viphakone et  al . , 2008 ) and may be an architectural component of mrnps ( batisse et  al . , 2009 ) . in the cytoplasm , the 5 to 3 and 3 to 5 mrna decay pathways are preceded by deadenylation of the poly(a ) tail to 1012 nt . pab1 is then displaced and the 3 end becomes accessible to the ski2/3/8 complex and exosome for 3 degradation , or the lsm1 - 7/pat1 complex that activates decapping and 5 degradation by xrn1 . we observed prominent peaks at the 3 end of mrnas for ski2 and , less expectedly , for the 5 to 3 exonuclease xrn1 . this indicates that the oligo(a ) tail is the site of a rate - limiting step in mrna turnover , perhaps reflecting assembly of the surveillance machinery or regulated initiation of decay . indeed , xrn1 interacts with the lsm1 - 7/pat1 complex that crosslinks primarily to mrna 3 ends ( mitchell et  al . the 3 peak of xrn1 hits might also reflect slowed degradation of the 3utr due to the presence of rna - binding proteins not displaced by translating ribosomes . the absence of clear peaks of xrn1 crosslinking elsewhere along the mrna body is consistent with its high processivity , perhaps following the last translating ribosome ( hu et  al . , 2009 ) . supporting this model , we observed a moderate accumulation of xrn1 upstream of mrna stop codons ( figure  s2a ) . conversely , ski2 binding was distributed across the body of mrnas , suggesting that 3-5 decay is slower or is more prone to pausing , perhaps due to collisions with translating ribosomes . having obtained a picture of an  average  mrnp , we next investigated how rnp composition varies between mrnas and between classes of pol ii transcripts . for each protein tested , we extracted all hits in pol ii transcribed rnas ( table s1 ) and plotted the proportion mapping to snornas , cuts , suts , xuts , and two mrnas . combining the data for all 13 proteins produced cbc1 , gbp2 , and tho2 were moderately abundant in all six rnp profiles and bind with similar distributions along mrnas , cuts , and suts ( figures 1 m and s2b ) , suggesting they are universal rnp components and that early rnp assembly is similar for mrnas and lncrnas . these data corroborate reports that the cbc is present in snornp assembly intermediates ( schwer et  al . , 2011 ) , and that the tho complex regulates snorna expression by binding at the 3 end ( figure  s2c ) ( larochelle et  al . , 2012 ) . hrp1 and nab2 have multiple functions in rna metabolism and were present in snornps , lncrnps , and the htb2 mrnp , indicating that they too are ubiquitous constituents of rnps . despite these similarities in early rnp assembly , there were striking differences in binding of different transcript classes to cytoplasmic and nuclear surveillance factors . rpl3 was strongly bound by xrn1 and ski2 , but not mtr4 or trf4 , indicating that it is predominantly degraded in the cytoplasm , whereas the reverse was seen for snornas , suts , and particularly cuts . furthermore , rpl3 and htb2 were bound more extensively by the export receptor mex67 than were snornas , cuts , and suts . collectively , these data suggest that cuts and suts , like snornas , are predominantly confined to the nucleus and that the distinction between lncrnps and mrnps occurs after early rnp packaging but prior to mex67 recruitment . notably , cuts and suts were significantly bound by pab1 , hrp1 , and nab2 suggesting that 3 end formation on lncrnas initially resembles that of mrnas but culminates in nuclear retention rather than export . in addition to differences in rnp composition between transcript classes , we identified heterogeneity within each class . for example , trf4 bound to htb2 more strongly than rpl3 , consistent with reports that trf4 regulates htb2 expression ( reis and campbell , 2007 ) . this heterogeneity raised the question of whether the scarce lncrna hits in xrn1 , ski2 , and mex67 data sets arise from a general low level of binding to lncrnas or robust interactions with a few atypical lncrnas . to assess heterogeneity among mrnps and lncrnps , we performed a k - medians clustering analysis of mrnas , cuts , and suts based upon their individual rnp profiles ( figure  2a ) . these profiles were derived from the number of hits for each transcript in cbc1 , mtr4 , nab2 , mex67 , xrn1 , and ski2 data sets ( figure  2a , columns 38 ) , with hits normalized for each row ( transcript ) . within the pab1 data set , some transcripts with relatively low numbers of total hits displayed a sharp peak of binding at the 3 end . to distinguish site - specific binding at these putative polyadenylation sites , from broadly distributed , potentially nonspecific , interactions across transcript bodies , we identified the highest peak in each transcript and divided the value of this by the maximum obtained when reads in the surrounding 400 nt region were randomly placed . the cluster analysis recapitulated the major findings from figure  1 , with cut and sut classes of lncrnas predominantly falling into clusters ( i  iii ) distinct from mrnas ( iv  x ) . furthermore , whereas cluster i was enriched for cuts , clusters ii and iii contained more suts ( table s2 ) , revealing differences not readily apparent from the class - wide analysis in figure  1 . in comparison to the  cut cluster ( i ) , the  sut clusters  ( ii and iii ) had more specific pab1 binding . this suggests that although all lncrnas are predominantly retained and degraded in the nucleus , cuts are more rapidly degraded so might represent byproducts of functional transcription , whereas suts are more stable so perhaps function as transcripts . furthermore , 34.4% of suts fell into mrna clusters ( iv  x ) , compared to just 6% of cuts , indicating that cuts are distinct from mrnas but suts and mrnas often overlap . the pho84-as lncrna , which falls into an mrna cluster , functions in trans ( camblong et  al . , 2009 ) and accumulates in the cytoplasm ( castelnuovo et  al . mrna - like  suts ( clusters iv  x ) bound more strongly to mex67 than suts in the lncrna clusters ( i  iii ) ( figure  s2d ) . the  mrna - like  lncrnas therefore appear to behave and function differently from the  standard  lncrnas in clusters i  iii , perhaps representing functional transcripts exported to the cytoplasm . x suts accumulate less than cluster i  iii suts in nuclear surveillance mutants ( figure  s2e ) ( gudipati et  al . , 2012b ) , suggesting they are degraded in the cytoplasm by xrn1 , with which they interact ( figure  2a , column 7 ) . hek2 has a role in mrna stabilization ( mauchi et  al . , 2010 ; vogel et  al . , 2011 ) and localization , and suts in clusters iv  x bound more abundantly to hek2 than those in clusters i  iii ( figure  s2d ) . hek2 might therefore be one factor that helps discriminate mrnas and  mrna - like  lncrnas from  standard  lncrnas , based on sequence - specific binding . indeed , suts in clusters vi  x have particularly long cnn repeats ( figure  s2f ) to which hek2 binds ( figure  s2 g ) . clusters i  iii contained 411 mrnas ( table s2 ) , and these are likely to behave like lncrnas , with retention and/or degradation in the nucleus . rrp6 is active in cut surveillance ( neil et  al . , 2009 ; xu et  al . , 2009 ) , and cluster i  iii mrnas are highly stabilized in strains lacking this exonuclease ( figure  s2d ) . furthermore , nrd1 ( cluster i ) transcripts undergo attenuation dependent on the nrd1nab3 complex ( kuehner and brow , 2008 ) , which functions in the termination and nuclear surveillance of many cuts . additional cluster i  iii mrnas , such as cth2 ( ciais et  al . , 2008 ) and rpl9b ( gudipati et  al . other cluster i  iii mrnas including ura8 and imd2 are regulated by promoter - proximal cuts ( kuehner and brow , 2008 ; thiebaut et  al . , 2008 ) . we predict that the remaining cluster i  iii mrnas either behave like lncrnas , with retention and degradation in the nucleus , or are regulated by overlapping lncrnas . indeed , comparison to ribosome profiling data ( brar et  al . , 2012 ) ( figure  s2h ) reveals that cluster i  iii mrnas are 5-fold less abundant on ribosomes than cluster iv x mrnas , relative to their transcription rate ( gauged by gbp2 binding ; figure  s2h ) . cluster iv was most similar to the lncrna clusters ( i  iii ) , with binding to mtr4 as well as cytoplasmic surveillance factors . several cluster iv mrnas overlap cuts ( ura2 , ser3 , ade12 , imd3 , and leu4 ) ( davis and ares , 2006 ; thiebaut et  al . , 2008 ) , and we suggest that the  mixed  rnp profile of cluster iv reflects genes where mrnas and lncrnas are transcribed concurrently ( perhaps in distinct subpopulations of cells ) . go term analyses ( table s3 ) revealed that some mrna clusters were enriched for transcripts related to particular cellular processes , suggesting that mrnp composition is linked to the function of the encoded protein . for example , transcripts encoding ribosomal proteins were prevalent in cluster v , which showed high binding by nab2 ( figure  2a ) and strong stabilization in rrp44 mutants ( figure  s2d ) ( gudipati et  al . , 2012b ) . this is consistent with reports that nab2 and rrp44 act in the nuclear surveillance of ribosomal protein gene pre - mrnas ( bousquet - antonelli et  al . , 2000 ; gudipati et  al . transcripts encoding proteins with functions critical to the nucleus , such as nucleic acid metabolism , were enriched in clusters vi and vii , with high cbc1 and nab2 binding , whereas those encoding proteins participating in predominantly cytoplasmic processes such as glucose metabolism were enriched in cluster x , with high levels of ski2 , xrn1 , and mex67 binding . this suggests that mrnas are preferentially regulated in the cellular compartment most appropriate to the function of their encoded protein , perhaps facilitating rapid feedback regulation . the largely distinct behavior and rnp composition of mrnas , cuts , and suts lead us to question how they are distinguished in the cell . both cuts and suts bound early , cotranscriptionally recruited , mrnp packaging components ( cbc1 , trex , hrp1 , and nab2 ) but were underrepresented ( particularly cuts ) among mex67 targets . these results indicated that the distinction between cuts , suts , and mrnas is made following transcription elongation but prior to the acquisition of export competence . this suggested that there might be crucial differences in 3 end formation , which generally proceeds via one of two possible mechanisms : stable mrna 3 ends are generated via cotranscriptional cleavage and polyadenylation , whereas the 3 ends of some cuts arise directly from nrd1-dependent transcription termination coupled to oligoadenylation and turnover . plotting the average distribution of pab1 hits across mrnas , cuts , and suts ( figure  3a , red ) revealed 3 peaks for mrnas and suts but distributed binding across cuts , consistent with the lower pab1 peak scores in the cut cluster ( figure  2 , cluster  i ) . thus 3 end processing only of mrnas and suts results in the acquisition of a stable , pab1-bound poly(a ) tail . together with the clustering analysis in which suts and mrnas overlapped and were distinct from cuts , this leads to a working model in which suts undergo cleavage and polyadenylation like mrnas . in contrast , cuts are terminated by a distinct mechanism , most likely dependent on nrd1-nab3 , coupled to rapid turnover . cleavage and polyadenylation of pre - mrnas is dependent on the recognition of a precise configuration of sequence motifs by a large multicomponent complex . to investigate whether sut 3 ends are defined by similar motifs , we needed to precisely localize sites of 3 end formation on suts and mrnas . we therefore searched the pab1 data set for chimeric reads in which the mapped sequence is followed by adenosine residues that are not genome encoded ( nonencoded a - tails ) . these represent junctions between mrna 3 ends and poly(a ) tails , and extracting the genomic coordinate of the last encoded nucleotide provided us with a transcriptome - wide set of precise poly(a ) ( pa ) sites . plotting these sites across mrnas and suts gave sharp 3 peaks ( figure  3a , blue ) , indicating that 3 ends were detected with high precision . for many transcripts , we found multiple pa sites , consistent with a recent transcript isoform sequencing study ( pelechano et  al . , 2013 ) and indicating that alternative pa sites are prevalent . to compare the sequence features defining mrna and sut 3 ends , we generated logos for the genomic sequence flanking the most frequently identified pa site for each mrna and sut ( figure  3b ) . these were similar for mrnas and suts , with an au - rich region extending 80 nt upstream of the pa site , and a bias toward adenosine as the last encoded residue ( or immediately 3 to the last encoded residue , because these can not be distinguished ) . a motif search identified uauaua as highly enriched in the 3 regions of sut and mrna genes , most frequently located 3070 nt upstream of the pa site ( figures 3c and 3d ) . this motif corresponds to the efficiency element , originally identified 50 nt upstream of mrna cleavage sites . 3 ends are also defined by the positioning element , aauaaa , 1030 nt upstream of the pa site , and u - rich regions flanking the pa site . we detected both of these features in genes encoding suts and mrnas when we plotted the frequency of aauaaa motifs ( figure  3e ) or nucleotide base composition ( figure  3f ) around pa sites . we conclude that mrnas and suts possess stable pab1-bound poly(a ) tails , and their 3 ends are defined by common sequence elements . the mode of 3 end formation of mrnas and suts is apparently distinct from cuts , so it was surprising that cuts bound to the mrna cleavage and polyadenylation factors hrp1 and nab2 . hrp1 specifically binds the uauaua efficiency element via its tandem rrm domains ( prez - caadillas , 2006 ) . this motif was enriched at hrp1-binding sites in mrnas but also within suts and cuts ( figure  4a ) . single - nucleotide deletions in cdna reads indicate the precise nucleotide crosslinked to the bait protein , and for hrp1 hit density and sequence deletions were elevated over uauaua motifs in all three classes of transcript indicating that this is a direct binding site ( figure  4b ) . however , whereas mrnas and suts displayed a 3 peak of hrp1 binding , cuts did not ( figure  4c ) . furthermore , 40% of hrp1-binding sites at the 3 end of mrnas and suts possessed a uauaua element , but there was no such enrichment among the low number of hrp1-binding sites at the 3 end of cuts ( figure  4d ) . this indicates that hrp1 binds directly to the efficiency element to promote 3 processing of mrnas and suts , but binds in a more distributed manner throughout cuts . notably , even within suts and mrnas , the majority of hrp1 is bound to promoter - proximal regions ( figure  4c ) , and 90% of binding sites lack a uauaua motif ( figure  4a ) . this suggested that hrp1 has additional functions unrelated to cleavage and polyadenylation and independent of binding to uauaua . the poly(a)-binding protein nab2 also participates in mrna 3 end formation and showed increased binding at the 3 ends of mrnas and suts . however , like hrp1 , nab2 binding was more distributed over cuts , with additional binding toward the 5 ends of mrnas and suts ( figure  4c ) . the lack of binding to the 3 end of cuts was not simply due to their rapid turnover , as a 3 peak of nab2 binding was still absent from full - length cuts stabilized in an rrp6 strain ( figure  4c ) . we conclude that both hrp1 and nab2 function in mrna and sut 3 end formation but act in a different manner on cuts and promoter - proximal regions . to compare the functions of nab2 and hrp1 when bound to mrnas and cuts , we constructed strains with endogenous nab2 and hrp1 genes under the control of glucose - repressible pgal promoters . the effects of hrp1 and nab2 depletion were assessed for transcripts identified as targets in the crac analyses . northern analysis of ccw12 , which falls into a typical mrna cluster ( x ) , revealed a slight increase following nab2 depletion and dramatic decrease upon hrp1 depletion ( figure  4e ) . this is consistent with the essential role of hrp1 in mrna synthesis and with the mild general increase in mrna expression observed in a previous analysis of nab2 depletion ( schmid et  al . , 2012 ) . in contrast , the abundance of two tested cuts , cut479 and cut200 , was increased up to 15-fold following depletion of nab2 or hrp1 ( figure  4e ) . many of the rna fragments isolated with hrp1 or nab2 , including those mapping to cuts , possessed short nonencoded oligo(a ) tails ( figures 5a5c ) . these are hallmarks of nuclear decay intermediates , suggesting that cuts bound by hrp1 and nab2 were undergoing active degradation . previous analyses showed that nab2 binds the surveillance factors rrp6 and trf4 ( schmid et  al . , 2012 ) and participates in degradation of intron - containing pre - mrnas , whereas hrp1 was implicated in nrd1-dependent termination coupled to pre - mrna turnover at the nrd1 and hrp1 loci ( kuehner and brow , 2008 ) . we conclude that hrp1 and nab2 participate in the nuclear turnover of cuts , in addition to their roles in the generation of stable mrnas and perhaps suts . these dual roles support a model in which 3 end processing is a key step in determining transcript fate . finally , we sought to determine the origin of the 5 proximal binding of surveillance factors to mrnas . rnas carrying nonencoded a - tails were identified for many proteins ( figure  5a ) and indicate that transcripts have been released from the polymerase . generally , stable mrnas possess long pab1-bound poly(a ) tails that promote export and translation but are deadenylated to 1012 adenosines prior to cytoplasmic turnover by xrn1 or the ski complex and exosome . in contrast , short ( 45 nt ) oligo(a ) tails mapping throughout a gene arise from the adenylation activity of the tramp complex and characterize nuclear surveillance intermediates . in agreement with these roles , ( 1 ) a - tails in xrn1 and ski2 data sets were 112 nt long ( figures 5b and 5c ) , absent from transcript classes such as trnas ( figure  5d ) , and almost exclusively present at the 3 end of mrnas ( figure  5e ) ; ( 2 ) pab1 bound to long poly(a ) tails ( present on 74.2% of recovered fragments ) but not oligo(a ) < 10 ( figures 5b and 5c ) ; and ( 3 ) mtr4 substrates universally possessed short ( 45 nt ) oligo(a ) tails ( figures 5b5d ) , some of which mapped across mrnas ( figure  5e ) . therefore , oligo(a ) tails are a universal feature of tramp activity and are exclusively associated with nuclear surveillance , whereas the longer a - tails in xrn1 and ski2 data sets reflect 3 poly(a ) tails on mrnas and some other pol ii transcripts . the abundant short oligo(a)-tails in nab2 data sets ( figure  5b ) support a noncanonical role in surveillance . most oligo(a)-tailed mrna reads in mtr4 data sets mapped to promoter - proximal regions ( figure  5e ) and even for long genes did not extend beyond the first 400 nt ( figure  5f ) . promoter - proximal mtr4-bound fragments are therefore unlikely to represent decay intermediates of full - length mrnas but might instead arise from high levels of early transcription termination . in nab2 data sets , a - tails were shorter for promoter - proximal reads than for reads mapping to mrna 3 ends and largely absent from reads mapping to central regions of mrnas ( figure  5 g ) . nab2 therefore binds to poly(a ) tails on full - length mrnas , and to oligo(a)-tailed unstable transcripts from promoter - proximal regions , whereas interactions with mrna central regions apparently reflect an adenosine - independent - binding activity . promoter - proximal transcripts of a similar length were previously identified in yeast depleted of rrp6 and trf4 , which stabilizes full - length cuts ( figure  5h ) ( neil et  al . , 2009 ) . this supports the notion that promoter - proximal transcripts do not originate from longer precursors . to test whether promoter - proximal fragments arise from early termination , we compared the distribution of pol ii ( churchman and weissman , 2011 ) , the termination factors nrd1 and nab3 ( figure  5h ) ( wlotzka et  al . , 2011 ) , and the 5 exonuclease rat1 ( granneman et  al . , 2011 ) across mrnas . the promoter - proximal enrichment of pol ii has been interpreted as stalled elongation complexes that are competent to resume transcription . however , nrd1 , nab3 , and rat1 are all enriched in this region , which coincides with the promoter - proximal oligoadenylated fragments . this suggests that stalled pol ii is susceptible to early termination , triggered either by nrd1-nab3 or by cleavage / decapping followed by a rat1-dependent  torpedo  mechanism . the resultant 5 fragments account for most mtr4 , trf4 , nab2 , and hrp1 hits mapping to protein - coding genes , thus removal of early termination products is a major function of the nuclear surveillance machinery . furthermore , these transcripts bind the same factors as cuts ( mtr4 , trf4 , hrp1 , nab2 ) , indicating that protein - coding loci give rise to both classical mrnps and an abundant class of promoter - proximal transcripts that assemble and behave like unstable lncrnas . our data reveal that distinct transcript classes are defined during 3 end formation , with rnp compositions tailored to the functions and fates of the transcripts . these classes loosely align with existing annotations , but we identified hundreds of exceptions . to address the extensive overlap between , and heterogeneity within , annotated transcript classes , we suggest an improved rnp - based classification , which reflects how transcripts are regulated and how they might function . we find that cuts and suts are predominantly retained and degraded in the nucleus , suggesting that their functions primarily arise from the act of transcription rather than the transcript itself . this refutes the notion that suts are generally  stable ,  but agrees with recent analyses identifying suts among exosome substrates ( gudipati et  al . however , suts were mildly less prone to nuclear turnover than were cuts , and their rnp composition overlapped with that of mrnas , suggesting that some suts might function as stable transcripts . xrn1 and the cytoplasmic exosome do not appear to function widely in bulk lncrna turnover , but we suggest they degrade mrna - like suts and provide a fail - safe for leaky nuclear surveillance . a substantial lncrna class , termed xuts , was reported to be exported and degraded in the cytoplasm by xrn1 ( van dijk et  al . , 2011 ) , but we see little evidence for this ( figure  1i ) . we also uncovered extensive heterogeneity in mrnp composition , with one mrna class regulated by lncrnas or subject to lncrna - like turnover in the nucleus , and another regulated by nab2 and rrp44 ( gudipati et  al . we suggest that tailored rnp compositions enable transcripts to be regulated and localized in a way appropriate to their function ( or that of the encoded protein ) . conventional transcriptome profiling experiments ( e.g. , rna - seq ) struggle to distinguish overlapping transcripts , particularly where one is less abundant . by combining high - resolution binding data for many rnp proteins multicolored  view of the transcriptome and could readily distinguish overlapping transcripts with different rnp profiles . most strikingly , this revealed that the nuclear surveillance machinery targets a major class of promoter - proximal lncrnas apparently generated by early transcription termination and with an rnp composition resembling cuts . our analyses suggest that this early termination is prevalent for  lncrna - like  mrnas , but occurs to some extent for most mrnas , and we speculate that this reflects a checkpoint in pol ii transcription . mtr4 hits peaked within 150 nt of mrna tsss , coincident with locations of pol ii pausing ( churchman and weissman , 2011 ) . here , transcription initiation factors exchange for elongation factors ( mayer et  al . , 2010 ) prior to the polymerase traversing the  + 2 nucleosome dyad 90 nt further downstream , which can impede elongation . if remodeling of the transcription complex is unsuccessful or slow , we suggest that pol ii transcription is terminated . termination might involve nrd1 , nab3 , and/or rat1 , which crosslink to promoter - proximal regions ( creamer et  al . are implicated in the early termination of some mrnas and lncrnas ( geisler et  al . , the oligoadenylated 3 ends that we detect suggest that termination generates an entry site for the tramp and exosome complexes . this is consistent with either an nrd1-dependent mechanism or endonuclease cleavage followed by rat1-dependent termination , which was recently identified in humans ( wagschal et  al . , 2012 ) . the distribution of mtr4 hits suggests that this checkpoint is restricted to the first 500 nt , consistent with the exclusion of termination factors from the midregions of genes by y1p modification of the pol ii ctd ( mayer et  al . , 2012 ) . the extent to which these early terminating transcripts function as ncrnas remains to be determined . our analyses also revealed characteristics by which transcript classes are distinguished in the cell , with 3 end formation emerging as a key step ( figure  6 ) . for mrnas and suts we detected the hallmarks of cleavage and polyadenylation , including an appropriate configuration of sequence elements , a pab1-bound poly(a ) tail , and hrp1 bound to an efficiency element 50 nt upstream of the pa site . in contrast , these were absent from cuts , which therefore undergo a distinct termination pathway . this is most likely nrd1-dependent termination , which is associated with exosome recruitment potentially explaining the inherently low stability of cuts ( vasiljeva and buratowski , 2006 ) . the rnp composition of cuts was related to that of snornas ( figure  1 ) , for which nrd1-dependent termination is well established . analyses of two mrna cleavage and polyadenylation factors , hrp1 and nab2 , revealed additional roles in the surveillance of cuts and promoter - proximal rnas , supporting our conclusion that 3 end formation is a key step in determining transcript fate . sequence nonspecific binding may be a default activity that is associated with recruitment of the nuclear surveillance system . in contrast , hrp1 bound to the uauaua motif and nab2 bound to poly(a ) at the 3 end of mrnas , in the context of a cleavage / polyadenylation complex , assist in correct mrnp maturation . functions for nab2 in rna surveillance are consistent with  its interactions with rrp6 and trf4 , and its role in intron - containing pre - mrna turnover and rrp6-dependent autoregulation of the nab2 transcript ( roth et  al . , 2009 ; schmid et  al . , a function for hrp1 in surveillance is consistent with reports that hrp1  participates in nrd1-dependent termination ( kuehner and brow , 2008 ) and cytoplasmic nonsense - mediated decay ( nmd ) ( gonzlez et  al . , 2000 ) . the extensive contacts made by hrp1 and nab2 across the body of transcripts is consistent with chip data ( gonzlez - aguilera et  al . , 2011 ; kim et  al . , 2004 ) , whereas the human hrp1 homolog tdp-43 is a ubiquitous rnp component with lncrnas among its targets ( tollervey et  al . , 2011 ) . these data suggest that nab2 , hrp1 , and tdp-43 function widely as rna packaging factors . nab2 is reported to fold poly(a ) tails into a particular configuration ( viphakone et  al . , 2008 ) , and in nab2 mutants mrnas are both hyperadenylated and aberrantly compacted ( brockmann et  al . , 2012 ) . this suggests that rna packaging and processing are tightly coupled , and appropriate rna folding by nab2 and hrp1 might regulate access to the  mrna 3 end and/or recruitment of surveillance and cleavage / polyadenylation factors . rna fate may be determined not only by the protein factors bound but also by the folding of the rna . the ability of suts to undergo mrna - like cleavage and polyadenylation may explain why suts are more stable than cuts ( figure  6 ) . however , unlike mrnas , most suts are retained in the nucleus , with only a minority showing  mrna - like  rnp compositions . we propose that additional layers of regulation following cleavage and polyadenylation determine whether a transcript is retained or exported . one candidate is hek2 , which bound to mrnas and some  mrna - like  suts but not to nuclear - restricted lncrnas ( figure  s2c ) . hek2 has roles in mrna stabilization and localization , and a human homolog ( cp2 ) enhances the 3 end processing and stability of h-mrna ( ji et  al . , 2011 ) . we suggest that hek2 contributes to a decision point associated with 3 end formation and selectively marks mrnas , and some suts , for export . we predict that other sequence - specific binding proteins assist in the export of the  mrna - like  suts that are not bound by hek2 . in conclusion , our systematic analysis of rnp composition sheds light on how diverse classes of transcripts are distinguished in the cell and provides insights into the functions of these transcripts and of key rnp proteins . we used the crac method as previously described ( granneman et  al . ,  2011 ) . in  vivo protein : rna crosslinks were generated by irradiating yeast cultures with uv light ( 254  nm , 100 s ) . illumina sequencing reads were aligned to the yeast genome ( sgd v64 ) using novoalign . to quantify hits for particular genomic features and identify binding motifs , we used the pycrac package developed by sander granneman and custom python / awk scripts ( available upon request ) . analyses of hits in spliced versus unspliced transcripts and of non - genome - encoded oligo(a ) tails  were performed as previously described ( schneider et  al . , 2012 ; wlotzka et  al . , 2011 ) , and k - medians clustering used cluster 3.0 . cerevisiae strains with genomically encoded tagged proteins were generated by standard methods and grown at 30c to a600  0.5 . crac strains expressed c - terminally htp - tagged ( his6-tev - protein a ) proteins under the control of their endogenous promoter and were grown in synthetic dropout media with glucose . depletion strains were grown in rich media with galactose , then cultures supplemented with 2% glucose to repress expression of n - terminally ha3-tagged hrp1 or nab2 under the control of the gal1 promoter . detailed strain information is provided in table s5.plasmids and oligonucleotidesthe plasmids and oligonucleotides used in this study are described in table s5.crosslinking and analyses of cdnas ( crac)we used the crac technique largely as previously described ( granneman et  al . , 2009 ; granneman et  al . , 2011 ) , but include full details here : cell pellets were vortexed with 1 ml tn150 ( 50  mm tris - hcl [ ph 7.8 ] , 150  mm nacl , 0.1% ( v / v ) np-40 , 5  mm -mercaptoethanol , edta - free protease inhibitor cocktail [ roche ] ) and 2.5  ml zirconia beads ( thistle scientific ) for 5x 1  min pulses , cooling on ice in between . cell lysates were diluted with an additional 3  ml tn150 , and debris removed by centrifugation ( 20  min , 4,600   g ; then 20  min , 20,000   g ; 4c ) . cleared lysates were incubated with 125  l igg beads ( igg sepharose 6 fast flow , ge ) , rotating at 4c for 2  hr . beads were washed with tn150 ( 2 10  ml ) then tn1000 ( 2 10  ml ; as tn150 , but with 1  m nacl ) , then his - tagged rna : protein complexes eluted by tev cleavage in tn150 ( 1.5  l homemade gst - tev , 2  hr , 18c ) . the eluate was treated with rnace - it ( agilent ; 0.1 units , 5  min , 37c ) to fragment protein - bound rna , and added to 400  mg guanidine - hcl to quench rnase activity . the solution was adjusted for nickel affinity purification by the addition of 27  l nacl ( 5.0 m ) and 3 ul imidazole ( 2.5 m ) , and added to 50 ul nickel beads ( ni - nta agarose , qiagen ) . after an overnight incubation ( 4c ) , the nickel beads were transferred to a spin column ( snap cap , pierce ) and washed three times with wbi ( 50  mm tris - hcl [ ph 7.8 ] , 300  mm nacl , 0.1% np-40 , 10  mm imidazole , 5  mm -mercaptoethanol , 6.0  m guanidine - hcl ) then three times with 1xpnk ( 50  mm tris - hcl [ ph 7.8 ] , 10  mm mgcl2 , 0.5% np-40 , 5  mm -mercaptoethanol ) . several on - bead reactions ( total volume 80  l in each case ) were then performed , washing once with wbi and three times with 1xpnk after each reaction:1.tsap ( promega ) phosphatase treatment 30  min , 37c , in 1xpnk.2.preadenylated 3 mircat-33 linker ( idt ) ligation using t4 rna ligase ( neb )  6  hr , 25c , in 1xpnk.3.5 end labeling with [ p]-atp using t4 polynucleotide kinase ( sigma ) 1  hr , 37c , in 1xpnk , with addition of 100  nmol atp after 40  min.4.5 linker ligation using t4 rna ligase ( neb ) 16  hr , 16c , in 1xpnk.the beads were then washed three times with wbii ( 50  mm tris - hcl [ ph 7.8 ] , 50  mm nacl , 0.1% ( v / v ) np-40 , 10  mm imidazole , 5  mm -mercaptoethanol ) , and rna : protein complexes eluted into eb ( 50  mm tris - hcl [ ph 7.8 ] , 50  mm nacl , 0.1% np-40 , 150  mm imidazole , 5  mm -mercaptoethanol ) and precipitated with tca ( 20% ( v / v ) final concentration ) . after washing with acetone , pellets were resuspended in nupage 1x lds sample loading buffer ( invitrogen ) and protein : rna complexes resolved by electrophoresis ( 4%12% bis - tris nupage gel , invitrogen ; 150 v ) . after electrophoretic transfer to a hybond c nitrocellulose membrane ( ge ) in 1x nupage transfer buffer ( 1.5  hr , 100v ; invitrogen ) , labeled rna was detected by autoradiography . the appropriate regions were excised from the membrane , and treated with proteinase k ( roche ) in wbii containing 1% ( w / v ) sds and 5  mm edta to release rna ( 55c , 2  hr ) . rna was isolated by phenol : chloroform extraction followed by ethanol precipitation , resuspending in 11  l water.the rna was reverse transcribed with superscript iii ( invitrogen ; 1  hr , 50c ) , using the mircat-33 rt oligo ( idt ) . after heat inactivation ( 15  min , 65c ) , samples were treated with rnase h ( neb ; 30  min , 37c ) . the cdna was amplified by pcr using la taq ( takara ; 1924 cycles , 52c annealing temperature ) . pcr products were precipitated using ethanol , resuspended in 1x gel loading dye ( neb ) and resolved on a 3% metaphor agarose gel ( lonza ) . a region corresponding to 120300  bp was excised from each lane , and dna extracted using a qiagen gel purification kit , eluting in 20  l water.the libraries were checked by sanger sequencing . briefly , 2  l of the purified pcr product was cloned into a pcr4 topo vector and transformed into top10 cells ( invitrogen ) according to manufacturer s instructions . colonies were picked , inoculated into lb medium with ampicillin , grown overnight at 30c , and plasmid dna extracted using a plasmid mini kit ( qiagen ) . sequencing reactions were performed using the big dye kit ( applied biosystems ) and the m13  f primer supplied with the pcr4 topo vector ( invitrogen).for solexa sequencing , libraries were sent to genepool ( university of edinburgh ) or source bioscience.northern analysesrna was extracted by hot phenol extraction , northern hybridizations with riboprobes performed using ultrahyb ( ambion ) , and signals detected using a fuji fla-5100 phosphorimager or by autoradiography.bioinformatic analysesquality filtering and read mappingraw data were preprocessed using the fastx toolkit , specifically the fastx_clipper to remove 3 sequencing adapters , fastq_quality_trimmer to trim low - quality positions from the 3 end of reads , fastq_quality_filter to remove reads without a high - quality score throughout , and fastx_artifacts_filter to remove homopolymeric sequencing artifacts . most of the 5 linkers used to prepare crac libraries contain a random 3 nt sequence , which enabled pcr duplicates ( reads amplified from a single cdna ) to be removed by collapsing identical sequences . the 5 linkers also contain a barcode , enabling samples to be multiplexed for sequencing . following preprocessing , we separated reads by barcode then mapped them to the yeast genome ( sgd v64 ) using novoalign , and we refer to mapped reads as  hits .  to remove pcr duplicates that were not collapsed during preprocessing due to sequencing errors or differential trimming at the 3 end by fastx_quality_trimmer , we collapsed any reads with the same random 3 nt tag in their 5 linker and with 5 ends mapping to the same genomic coordinate.we downloaded gene annotations from ensembl ( ef4.68 ) , and supplemented them with the coordinates of utrs , cuts and suts ( xu et  al . , 2009 ) , additional antisense and intergenic lncrnas ( granovskaia et  al . , 2010 ; yassour et  al . , 2010 ) and xrn1-sensitive unstable transcripts ( van dijk et  al . , 2011 ) . to count hits for each genomic feature we used the pycrac package developed by sander granneman ( source code and documentation available from https://bitbucket.org/sgrann/pycrac ) briefly , mapped reads from the novoalign output file are corrected for the presence of insertions , deletions or substitutions , then the corrected reads overlapping each genomic feature counted . we included flanking regions around mrnas of up to 50 nt at the 5 end and 300 nt at the 3 end to catch hits falling outside of misannotated features . hit table  for each sample . to assess the similarity between samples we calculated the spearman rank correlation coefficient between pairs of hit tables , considering all mrnas , cuts , suts , trnas , snrnas , snornas and rrnas detected in at least two data sets.plots of hit distributions across genomic featuresto examine the distribution of hits across the length of individual genomic features we used the pycrac package to count the number of mapped reads overlapping each nucleotide along the feature of interest . to examine the distribution of hits for a particular protein across all members of a transcript class ( e.g. , cbc1 hits in mrnas ) , we used two related approaches , both performed on the most abundantly bound members of the transcript class.in the first approach , which provides an average binding profile across all transcripts , each transcript was divided into 100 bins of equal length , and 100 nt 5 and 3 flanking regions divided into 10 bins ( 120 bins in total ) . considering the first transcript , hits were counted for each nucleotide , then divided by the length of each bin to obtain hit densities . this was repeated for 1,000 transcripts , then each transcript normalized by linear scaling so that the densities for that transcript summed to 100 . we then averaged the 1,000 individual profiles , and the resulting plot reflects the typical hit distribution across all 1,000 transcripts , with the normalization step ensuring that each transcript contributes equally.we complemented this approach with an analysis in which 1,000 transcripts were sorted by length , hits counted at each position and scaled to the maximum value for each transcript , and the data plotted as a two dimensional heat map ( figure  5f ) . here , each row represents a transcript , and each column the absolute position from the aligned tsss . this enables the individual hit distributions of 1,000 transcripts to be displayed on one plot , without scaling by length . we used a similar approach to plot the distribution of crac hits or other transcriptome - wide data in 300 - 900 nt windows aligned to mrna transcription start and poly(a ) sites ( figure  5h ) , but in this case plotted the average rather than individual distributions.motif analysesto search for sequence motifs , we used the pycrac package to calculate statistical overrepresentation scores for each possible k - mer according to the previously described algorithm ( wlotzka et  al . , 2011 ) . high z scores indicate that a motif is significantly more abundant within hits than would be expected by chance , taking into account the sequence composition of the transcripts to which the hits map . to avoid detecting spurious motifs arising from sequencing artifacts or adapters that were not removed , we only used reads for which the 3 adaptor was detected . to restrict the analysis to encoded motifs , we used the genomic sequence corresponding to each mapped read . we also excluded low - complexity reads ( with fewer than 7 nonmodal nucleotides , e.g. ,  gtccgaaaaaaaaa  would be excluded ) to avoid the artifactual detection of oligo(a ) motifs for reads with short non - genome - encoded oligo(a ) tails that can map to a - rich regions of the genome . having identified a motif , we then plotted the distribution of hits and deletions around all occurrences of that motif in the transcriptome using the pycrac package . using a novoalign file as input , this counts the number of mapped reads overlapping each nucleotide ( e.g. , 100 nt to  + 100 nt ) around all occurrences of a given motif , then sums the scores for each nucleotide . we used a similar strategy to plot hits around stop codons.to examine the presence of different length ( cnn)n repeats in different transcript classes , we counted the number of transcripts in which the longest cnn repeat was ( cnn)1 , ( cnn)2 , ( cnn)3 ,  ( cnn)n . we then plotted the proportion of transcripts for each value of n.pre-mrna analysesto identify hits in spliced mrnas versus unspliced pre - mrnas we mapped reads to a library of spliced transcripts and another of unspliced transcripts , as previously described ( schneider et  al . we considered only reads mapping to intron - containing genes , and calculated the ratio of hits across exon - exon junctions to hits across intron - exon junctions.clustering analysisto classify transcripts by their rnp profiles ,  we extracted the number of hits for each mrna , cut and sut from cbc1 , mtr4 , nab2 , mex67 , ski2 and xrn1 hit tables . each data set was then normalized to hits per million hits in mrnas , cuts and suts , and transcripts rejected if they did not have at least 50 hits per million in two data sets . to remove transcripts for which data were not reproducible , we rejected transcripts with spearman rank correlation coefficients of  < 0.37 when comparing two replicate sets of observations for the 6 proteins tested . for each transcript , we then averaged replicate observations ( mean ) to reduce the influence of experimental variation upon the clustering analysis . to account for differences in transcript abundance this produced a set of 4,960 rnp profiles , which reflect the relative binding of a transcript to each of the six tested proteins.we also included pab1 in our analysis , but instead of total hits used a measure of peak sharpness to distinguish bona fide  pab1 interactions at the 3 end of transcripts ( sharp peak ) versus nonspecific interactions across transcript bodies ( broad  distribution ) . briefly , for each transcript the nucleotide with the greatest number of pab1 hits was identified , and the height of this binding peak divided by the maximum height when reads in a 400 nt window centered on this peak were placed  randomly . transcripts were then clustered by their rnp profiles ( including the pab1 score ) using cluster 3.0 ( k - medians , k  = 10 , euclidean distance ) . the data were displayed as a heat map , with all replicates ( rather than averages across two or three replicates ) shown . the third replicate data set for cbc1 , mtr4 , nab2 and mex67 was not used in the initial spearman rank filtering step , enabling us to verify that the data had not been over-fitted.for proteins not included in the clustering analysis ( e.g. , hek2 , tho2 and gbp2 ) , the amount of binding to transcripts in each cluster ( or a set of clusters ) was examined using box - and - whisker plots to summarize the distribution of transcript hit totals ( raw hit numbers ) . the stabilization of mrnas or suts in surveillance mutants ( gudipati et  al . , 2012 ) was examined for each cluster by plotting cumulative frequency distributions , as described in ( gudipati et  al . , 2012 ) . binding to ribosomes was examined using ribosome profiling data ( brar et  al . , 2012 ) briefly , the first 25 nt was extracted from each raw read , mapped to the yeast genome using novoalign , and the number of reads for each annotated mrna or sut counted using pycrac . the raw number of hits are presented.go term analyses were performed using the sgd go term finder ( http://www.yeastgenome.org).analyses of non - genome - encoded oligo(a ) tailsto identify reads with non - genome - encoded oligo(a ) tails , we employed a pipeline developed by grzegorz kudla ( wlotzka et  al . , 2011 ) . we first selected reads where the 3 adaptor was identified and clipped in the data preprocessing steps . we then used  blastall to identify the region of the read mapping to the yeast genome . where the mapped region did not extend to the 3 end of the clipped read we selected reads where the non - genome - encoded portion contained two or more as , and fewer than one in five non - a residues . we then analyzed these reads in the same way described for total reads , to identify which transcripts they mapped to , where within transcripts they mapped , and the proportion of a - tailed reads in different transcript classes . the rna fragmentation step in the crac protocol preserves the length of a - tails , as rnase a and t1 only rarely cut after a residues , enabling the length distribution of a - tails to be analyzed for each sample . however , one limitation of this approach is that the adaptor , barcode and mapped portion of the read occupies 31 nt , and so long a - tails ( > 15 nt ) are underrepresented . to address this limitation , we removed the requirement for reads to contain a mapped region , and instead simply counted the number of a residues at the 3 end of each read . to avoid biases from different read length distributions in different data sets , we restricted this latter analysis to reads between 30 and 35 nt long ( after removal of the 3 adaptor).definition of transcript 3 endsto precisely locate sites of 3 end formation on mrnas and suts , we selected all reads in the pab1 data set with non - genome - encoded oligo(a ) tails , and extracted the genomic coordinate of the last genome - encoded nucleotide . we defined these as poly(a ) ( pa ) sites , and for each gene selected the most frequently identified pa site (  major pa site  ) . to exclude adenosine - rich reads mapping with low confidence to genome - encoded oligo(a ) tracts , we removed low - complexity reads ( which we defined as reads with genome - encoded portions containing fewer than 8 nonmodal nucleotides ) . we also removed reads mapping within 200 nt of a tss , as these may correspond to upstream transcripts . we then plotted the frequency of motifs ( e.g. , uauaua ) or individual nucleotide bases ( i.e. , a , u , g or c ) across the genomic region flanking each major pa site . s.  cerevisiae strains with genomically encoded tagged proteins were generated by standard methods and grown at 30c to a600  0.5 . crac strains expressed c - terminally htp - tagged ( his6-tev - protein a ) proteins under the control of their endogenous promoter and were grown in synthetic dropout media with glucose . depletion strains were grown in rich media with galactose , then cultures supplemented with 2% glucose to repress expression of n - terminally ha3-tagged hrp1 or nab2 under the control of the gal1 promoter . 2011 ) , but include full details here : cell pellets were vortexed with 1  ml tn150 ( 50  mm tris - hcl [ ph 7.8 ] , 150  mm nacl , 0.1% ( v / v ) np-40 , 5  mm -mercaptoethanol , edta - free protease inhibitor cocktail [ roche ] ) and 2.5  ml zirconia beads ( thistle scientific ) for 5x 1  min pulses , cooling on ice in between . cell lysates were diluted with an additional 3  ml tn150 , and debris removed by centrifugation ( 20  min , 4,600   g ; then 20  min , 20,000   g ; 4c ) . cleared lysates were incubated with 125  l igg beads ( igg sepharose 6 fast flow , ge ) , rotating at 4c for 2  hr . beads were washed with tn150 ( 2 10  ml ) then tn1000 ( 2 10  ml ; as tn150 , but with 1  m nacl ) , then his - tagged rna : protein complexes eluted by tev cleavage in tn150 ( 1.5  l homemade gst - tev , 2  hr , 18c ) . the eluate was treated with rnace - it ( agilent ; 0.1 units , 5  min , 37c ) to fragment protein - bound rna , and added to 400  mg guanidine - hcl to quench rnase activity . the solution was adjusted for nickel affinity purification by the addition of 27  l nacl ( 5.0 m ) and 3 ul imidazole ( 2.5 m ) , and added to 50 ul nickel beads ( ni - nta agarose , qiagen ) . after an overnight incubation ( 4c ) , the nickel beads were transferred to a spin column ( snap cap , pierce ) and washed three times with wbi ( 50  mm tris - hcl [ ph 7.8 ] , 300  mm nacl , 0.1% np-40 , 10  mm imidazole , 5  mm -mercaptoethanol , 6.0  m guanidine - hcl ) then three times with 1xpnk ( 50  mm tris - hcl [ ph 7.8 ] , 10  mm mgcl2 , 0.5% np-40 , 5  mm -mercaptoethanol ) . several on - bead reactions ( total volume 80  l in each case ) were then performed , washing once with wbi and three times with 1xpnk after each reaction:1.tsap ( promega ) phosphatase treatment 30  min , 37c , in 1xpnk.2.preadenylated 3 mircat-33 linker ( idt ) ligation using t4 rna ligase ( neb ) 6  hr , 25c , in 1xpnk.3.5 end labeling with [ p]-atp using t4 polynucleotide kinase ( sigma ) 1  hr , 37c , in 1xpnk , with addition of 100  nmol atp after 40  min.4.5 linker ligation using t4 rna ligase ( neb ) preadenylated 3 mircat-33 linker ( idt ) ligation using t4 rna ligase ( neb ) 1  hr , 37c , in 1xpnk , with addition of 100  nmol atp after 40  min . the beads were then washed three times with wbii ( 50  mm tris - hcl [ ph 7.8 ] , 50  mm nacl , 0.1% ( v / v ) np-40 , 10  mm imidazole , 5  mm -mercaptoethanol ) , and rna : protein complexes eluted into eb ( 50  mm tris - hcl [ ph 7.8 ] , 50  mm nacl , 0.1% np-40 , 150  mm imidazole , 5  mm -mercaptoethanol ) and precipitated with tca ( 20% ( v / v ) final concentration ) . after washing with acetone , pellets were resuspended in nupage 1x lds sample loading buffer ( invitrogen ) and protein : rna complexes resolved by electrophoresis ( 4%12% bis - tris nupage gel , invitrogen ; 150 v ) . after electrophoretic transfer to a hybond c nitrocellulose membrane ( ge ) in 1x nupage transfer buffer ( 1.5  hr , 100v ; invitrogen ) , labeled rna was detected by autoradiography . the appropriate regions were excised from the membrane , and treated with proteinase k ( roche ) in wbii containing 1% ( w / v ) sds and 5  mm edta to release rna ( 55c , 2  hr ) . rna was isolated by phenol : chloroform extraction followed by ethanol precipitation , resuspending in 11  l water . the rna was reverse transcribed with superscript iii ( invitrogen ; 1  hr , 50c ) , using the mircat-33 rt oligo ( idt ) . after heat inactivation ( 15  min , 65c ) , samples were treated with rnase h ( neb ; 30  min , 37c ) . the cdna was amplified by pcr using la taq ( takara ; 1924 cycles , 52c annealing temperature ) . pcr products were precipitated using ethanol , resuspended in 1x gel loading dye ( neb ) and resolved on a 3% metaphor agarose gel ( lonza ) . a region corresponding to 120300  bp was excised from each lane , and dna extracted using a qiagen gel purification kit , eluting in 20  l water . briefly , 2  l of the purified pcr product was cloned into a pcr4 topo vector and transformed into top10 cells ( invitrogen ) according to manufacturer s instructions . colonies were picked , inoculated into lb medium with ampicillin , grown overnight at 30c , and plasmid dna extracted using a plasmid mini kit ( qiagen ) . sequencing reactions were performed using the big dye kit ( applied biosystems ) and the m13  f primer supplied with the pcr4 topo vector ( invitrogen ) . for solexa sequencing rna was extracted by hot phenol extraction , northern hybridizations with riboprobes performed using ultrahyb ( ambion ) , and signals detected using a fuji fla-5100 phosphorimager or by autoradiography . raw data were preprocessed using the fastx toolkit , specifically the fastx_clipper to remove 3 sequencing adapters , fastq_quality_trimmer to trim low - quality positions from the 3 end of reads , fastq_quality_filter to remove reads without a high - quality score throughout , and fastx_artifacts_filter to remove homopolymeric sequencing artifacts . most of the 5 linkers used to prepare crac libraries contain a random 3 nt sequence , which enabled pcr duplicates ( reads amplified from a single cdna ) to be removed by collapsing identical sequences . the 5 linkers also contain a barcode , enabling samples to be multiplexed for sequencing . following preprocessing , we separated reads by barcode then mapped them to the yeast genome ( sgd v64 ) using novoalign , and we refer to mapped reads as  hits .  to remove pcr duplicates that were not collapsed during preprocessing due to sequencing errors or differential trimming at the 3 end by fastx_quality_trimmer , we collapsed any reads with the same random 3 nt tag in their 5 linker and with 5 ends mapping to the same genomic coordinate . we downloaded gene annotations from ensembl ( ef4.68 ) , and supplemented them with the coordinates of utrs , cuts and suts ( xu et  al . , 2009 ) , additional antisense and intergenic lncrnas ( granovskaia et  al . , 2010 ; yassour et  al . , 2010 ) and xrn1-sensitive unstable transcripts ( van dijk et  al . , 2011 ) . to count hits for each genomic feature we used the pycrac package developed by sander granneman ( source code and documentation available from https://bitbucket.org/sgrann/pycrac ) . briefly , mapped reads from the novoalign output file are corrected for the presence of insertions , deletions or substitutions , then the corrected reads overlapping each genomic feature counted . we included flanking regions around mrnas of up to 50 nt at the 5 end and 300 nt at the 3 end to catch hits falling outside of misannotated features . hit table  for each sample . to assess the similarity between samples we calculated the spearman rank correlation coefficient between pairs of hit tables , considering all mrnas , cuts , suts , trnas , snrnas , snornas and rrnas detected in at least two data sets . to examine the distribution of hits across the length of individual genomic features we used the pycrac package to count the number of mapped reads overlapping each nucleotide along the feature of interest . to examine the distribution of hits for a particular protein across all members of a transcript class ( e.g. , cbc1 hits in mrnas ) , we used two related approaches , both performed on the most abundantly bound members of the transcript class . in the first approach , which provides an average binding profile across all transcripts , each transcript was divided into 100 bins of equal length , and 100 nt 5 and 3 flanking regions divided into 10 bins ( 120 bins in total ) . considering the first transcript , hits were counted for each nucleotide , then divided by the length of each bin to obtain hit densities . this was repeated for 1,000 transcripts , then each transcript normalized by linear scaling so that the densities for that transcript summed to 100 . we then averaged the 1,000 individual profiles , and the resulting plot reflects the typical hit distribution across all 1,000 transcripts , with the normalization step ensuring that each transcript contributes equally . we complemented this approach with an analysis in which 1,000 transcripts were sorted by length , hits counted at each position and scaled to the maximum value for each transcript , and the data plotted as a two dimensional heat map ( figure  5f ) . here , each row represents a transcript , and each column the absolute position from the aligned tsss . this enables the individual hit distributions of 1,000 transcripts to be displayed on one plot , without scaling by length . we used a similar approach to plot the distribution of crac hits or other transcriptome - wide data in 300 - 900 nt windows aligned to mrna transcription start and poly(a ) sites ( figure  5h ) , but in this case plotted the average rather than individual distributions . to search for sequence motifs , we used the pycrac package to calculate statistical overrepresentation scores for each possible k - mer according to the previously described algorithm ( wlotzka et  al . , high z scores indicate that a motif is significantly more abundant within hits than would be expected by chance , taking into account the sequence composition of the transcripts to which the hits map . to avoid detecting spurious motifs arising from sequencing artifacts or adapters that were not removed , we only used reads for which the 3 adaptor was detected . to restrict the analysis to encoded motifs we also excluded low - complexity reads ( with fewer than 7 nonmodal nucleotides , e.g. ,  gtccgaaaaaaaaa  would be excluded ) to avoid the artifactual detection of oligo(a ) motifs for reads with short non - genome - encoded oligo(a ) tails that can map to a - rich regions of the genome . having identified a motif , we then plotted the distribution of hits and deletions around all occurrences of that motif in the transcriptome using the pycrac package . using a novoalign file as input , this counts the number of mapped reads overlapping each nucleotide ( e.g. , 100 nt to  + 100 nt ) around all occurrences of a given motif , then sums the scores for each nucleotide . we used a similar strategy to plot hits around stop codons . to examine the presence of different length ( cnn)n repeats in different transcript classes , we counted the number of transcripts in which the longest cnn repeat was ( cnn)1 , ( cnn)2 , ( cnn)3 ,  ( cnn)n . we then plotted the proportion of transcripts for each value of n. to identify hits in spliced mrnas versus unspliced pre - mrnas we mapped reads to a library of spliced transcripts and another of unspliced transcripts , as previously described ( schneider et  al . , 2012 ) . we considered only reads mapping to intron - containing genes , and calculated the ratio of hits across exon - exon junctions to hits across intron - exon junctions . to classify transcripts by their rnp profiles ,  we extracted the number of hits for each mrna , cut and sut from cbc1 , mtr4 , nab2 , mex67 , ski2 and xrn1 hit tables . each data set was then normalized to hits per million hits in mrnas , cuts and suts , and transcripts rejected if they did not have at least 50 hits per million in two data sets . to remove transcripts for which data were not reproducible , we rejected transcripts with spearman rank correlation coefficients of  < 0.37 when comparing two replicate sets of observations for the 6 proteins tested . for each transcript , we then averaged replicate observations ( mean ) to reduce the influence of experimental variation upon the clustering analysis . to account for differences in transcript abundance this produced a set of 4,960 rnp profiles , which reflect the relative binding of a transcript to each of the six tested proteins . we also included pab1 in our analysis , but instead of total hits used a measure of peak sharpness to distinguish bona fide  pab1 interactions at the 3 end of transcripts ( sharp peak ) versus nonspecific interactions across transcript bodies ( broad  distribution ) . briefly , for each transcript the nucleotide with the greatest number of pab1 hits was identified , and the height of this binding peak divided by the maximum height when reads in a 400 nt window centered on this peak were placed  randomly . transcripts were then clustered by their rnp profiles ( including the pab1 score ) using cluster 3.0 ( k - medians , k  = 10 , euclidean distance ) . the data were displayed as a heat map , with all replicates ( rather than averages across two or three replicates ) shown . the third replicate data set for cbc1 , mtr4 , nab2 and mex67 was not used in the initial spearman rank filtering step , enabling us to verify that the data had not been over - fitted . for proteins not included in the clustering analysis ( e.g. , hek2 , tho2 and gbp2 ) , the amount of binding to transcripts in each cluster ( or a set of clusters ) was examined using box - and - whisker plots to summarize the distribution of transcript hit totals ( raw hit numbers ) . the stabilization of mrnas or suts in surveillance mutants ( gudipati et  al . , 2012 ) was examined for each cluster by plotting cumulative frequency distributions , as described in ( gudipati et  al . , 2012 ) . binding to ribosomes was examined using ribosome profiling data ( brar et  al . , 2012 ) briefly , the first 25 nt was extracted from each raw read , mapped to the yeast genome using novoalign , and the number of reads for each annotated mrna or sut counted using pycrac . go term analyses were performed using the sgd go term finder ( http://www.yeastgenome.org ) . to identify reads with non - genome - encoded oligo(a ) tails , we employed a pipeline developed by grzegorz kudla ( wlotzka et  al . , 2011 ) . we first selected reads where the 3 adaptor was identified and clipped in the data preprocessing steps . we then used  blastall to identify the region of the read mapping to the yeast genome . where the mapped region did not extend to the 3 end of the clipped read we selected reads where the non - genome - encoded portion contained two or more as , and fewer than one in five non - a residues . we then analyzed these reads in the same way described for total reads , to identify which transcripts they mapped to , where within transcripts they mapped , and the proportion of a - tailed reads in different transcript classes . the rna fragmentation step in the crac protocol preserves the length of a - tails , as rnase a and t1 only rarely cut after a residues , enabling the length distribution of a - tails to be analyzed for each sample . however , one limitation of this approach is that the adaptor , barcode and mapped portion of the read occupies 31 nt , and so long a - tails ( > 15 nt ) are underrepresented . to address this limitation , we removed the requirement for reads to contain a mapped region , and instead simply counted the number of a residues at the 3 end of each read . to avoid biases from different read length distributions in different data sets , we restricted this latter analysis to reads between 30 and 35 nt long ( after removal of the 3 adaptor ) . to precisely locate sites of 3 end formation on mrnas and suts , we selected all reads in the pab1 data set with non - genome - encoded oligo(a ) tails , and extracted the genomic coordinate of the last genome - encoded nucleotide . we defined these as poly(a ) ( pa ) sites , and for each gene selected the most frequently identified pa site (  major pa site  ) . to exclude adenosine - rich reads mapping with low confidence to genome - encoded oligo(a ) tracts , we removed low - complexity reads ( which we defined as reads with genome - encoded portions containing fewer than 8 nonmodal nucleotides ) . we also removed reads mapping within 200 nt of a tss , as these may correspond to upstream transcripts . we then plotted the frequency of motifs ( e.g. , uauaua ) or individual nucleotide bases ( i.e. , a , u , g or c ) across the genomic region flanking each major pa site .
summaryeukaryotic genomes generate a heterogeneous ensemble of mrnas and long noncoding rnas ( lncrnas ) . lncrnas and mrnas 
 are both transcribed by pol ii and acquire 5 caps and poly(a ) tails , but only mrnas are translated into proteins . to address how these classes are distinguished , we identified the transcriptome - wide targets of 13 rna processing , export , and turnover factors in budding yeast . comparing the maturation pathways of mrnas and lncrnas revealed that transcript fate is largely determined during 3 end formation . 
 most lncrnas are targeted for nuclear rna surveillance , but a subset with 3 cleavage and polyadenylation features resembling the mrna consensus can be exported to the cytoplasm . 
 the hrp1 and nab2 proteins act at this decision point , with dual roles in mrna cleavage / polyadenylation and lncrna surveillance . 
 our data also reveal the dynamic and heterogeneous nature of mrna maturation , and highlight a subset of  lncrna - like  mrnas regulated by the nuclear surveillance machinery .