
There are at least six types of tsRNA, which differ in the cleavage position of the mature or precursor tRNA transcript 1 . Stress-induced 30-to 40-nucleotide (nt) tRNA halves are generated by angiogenin [1] [2] [3] and affect cell proliferation, apoptosis, and epigenetic inheritance [4] [5] [6] [7] . 5′ tRNA halves also inhibit global translation 8, 9 . There is growing evidence that some of the shorter 5′ and 3′ ends of mature tRNAderived 18-to 22-nt type I tsRNAs (5′ and 3′ tsRNAs), and type II tsRNAs derived from the 3′ ends of precursor tRNAs, have important roles in cellular homeostasis 7 and in the regulation of transposition 10, 11 ; it remains unclear whether they can function as microRNAs (miRNAs) [12] [13] [14] . Because type I tsRNAs are derived from mature tRNAs, their sequences are more highly conserved between species. During our study of tsRNAs, we discovered a functional role for the 22-nt LeuCAG3′ tsRNA in regulating ribosome biogenesis.
LeuCAG3′ tsRNA is essential for cell viability
We identified and selected tsRNAs from expression screens in HeLa and HCT-116 cells and used locked nucleic acid or DNA-mixed antisense oligonucleotides (LNA/ASO mixmers) 15, 16 to inhibit various transcripts (Fig. 1a) . Blocking the LeuCAG3′ tsRNA with a complementary mixmer LNA significantly reduced cell viability (P = 0.036 in HeLa cells, P = 0.007 in HCT-116 cells), as determined by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) cell proliferation 17 and counting assays (Fig. 1a, b and Extended Data Fig. 1a-d ). Various controls (LNAs containing scrambled sequence, 2-nt mismatches, complementarity to other regions of the Leu-tRNA or directed against other 3′ tsRNAs) did not affect cell viability (Fig. 1a, b and Extended Data Fig. 1a-d) . Similar results were obtained using an LNA/DNA gapmer antisense oligonucleotide (ASO), in which central nucleotides are replaced with LNAs. These induce RNase H-mediated cleavage of the target RNA 15 (Extended Data Fig. 1e ). These effects were not due to loss of the mature tRNA because there was no LNA binding or degradation of the intact mature LeuCAG-tRNA (Extended Data Fig. 1f) , and no significant reduction in global protein synthesis (Extended Data Fig. 1g-i) . Finally, we replaced 13 Leu CUG codons with Leu non-CUG (CUC or CUU) codons from the Renilla gene and left the firefly luciferase gene unmodi fied (Supplementary Table 1 ) in the psicheck2 expression plasmid. Substitution of the Leu codons did not affect Renilla luciferase expression in the presence or absence of the LNA (Fig. 1c) . Thus, the anti-Leu3′ tsLNA did not affect mature LeuCUG-tRNA function.
LeuCAG3′ tsRNA is essential for cell viability
Standard deep sequencing methods cannot accurately quantify tsRNA species owing to abundant post-transcriptional tRNA modifications 18, 19 (Extended Data Fig. 1j ). Northern blot analyses demonstrated that the major isoform of the LeuCAG3′ tsRNA was 22 nt rather than 18 nt (Extended Data Fig. 1k ). In contrast to the effect of tsRNA knockdown, transfection of a synthetic 22-nt LeuCAG3′ tsRNA into cells increased cell viability (Fig. 1d) . The specificity of this outcome was supported by the lack of a growth response to two different controls or to a 27-nt 3′ end of LeuCAG-tRNA containing an additional 5 nt of LeuCAG-tRNA sequence.
LeuCAG3′ tsRNA is essential for cell viability
Three separate assays 20 established that cells in which LeuCAG3′ tsRNA is inhibited undergo apoptosis. Three days after transfection, Annexin V and propidium iodide staining and flow cytometry analysis ( Fig. 2a and Extended Data Fig. 2a) showed that the percentages of LeuCAG3′ tsRNA-inhibited HeLa cells undergoing early and late apoptosis were 21.5 ± 4.6% and 24.5 ± 2.7%, respectively, compared to 5.7 ± 1.4% and 4.6 ± 1.2% in control cells (Fig. 2a) . Similar results were obtained in HCT-116 cells (Extended Data Fig. 2b ). Induction of apoptosis was confirmed in both HeLa and HCT-116 cells by terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labelling (TUNEL; Fig. 2b and Extended Data Fig. 2c ) and poly(ADP-ribose) polymerase (PARP) cleavage (Extended Data Fig. 2d , e).
LeuCAG3′ tsRNA inhibition suppresses tumour growth
Hydrodynamic transfection of 125 μ g of anti-Leu3′ tsLNA into normal mice to achieve a rapid and high concentration of the oligonucleotide in liver hepatocytes did not affect serum alanine aminotransferase (ALT) levels, a sensitive marker for liver toxicity (Extended Data  Fig. 2f ). Because the LeuCAG3′ tsRNA was highly expressed in mouse liver tumours compared to normal liver (Extended Data Fig. 2g ), we studied the potential antitumour effect of an anti-Leu3′ tsLNA in an orthotopic patient-derived hepatocellular carcinoma (HCC) xenograft (PDX) model. Anti-Leu3′ tsLNA administered during a 4-week period significantly reduced tumour growth (Extended Data Fig. 2h , P = 0.0018, P = 0.0027) and final xenograft volumes ( Fig. 2c and Extended Data Fig. 2i , P = 0.0025, P = 0.0002), compared to mice treated with saline or control LNA, respectively. LeuCAG3′ tsRNA, but not mature tRNA, was nearly completely inhibited by anti-Leu3′ tsLNA treatment (Extended Data Fig. 2j ). No overall toxicity was observed (the body weights of treated mice remained stable; Extended Data Fig. 2k) . A TUNEL assay demonstrated substantial apoptosis in treated xenograft tissues (Fig. 2d) , consistent with our observations in cultured cells. These results show that inhibition of LeuCAG3′ tsRNA induces apoptosis in tumour but not normal liver cells in vivo.
LeuCAG3′ tsRNA inhibition impairs ribosome biogenesis
Although a recent study suggested that 3′ tsRNAs function like miRNAs 13 , our experiments detected no such function for the Leu3′ tsRNA (Extended Data Fig. 3a-c) . In addition, RNA sequencing (RNA-seq) profiles showed no global changes in mRNA transcription from cells in which LeuCAG3′ tsRNA was inhibited (Extended Data Fig. 3d) .
LeuCAG3′ tsRNA inhibition impairs ribosome biogenesis
To investigate a possible relationship between the LeuCAG3′ tsRNA and translation, we quantified the number of polysomal and ribo somal subunits in different conditions by sucrose gradient fractionation. Although the number of polysome fractions remained unchanged in cells in which LeuCAG3′ tsRNA was inhibited, compared to control cells, the abundance of 40S and 80S ribosomal complexes was substantially decreased, and the relative abundance of 60S subunits increased (Fig. 3a) . Puromycin dissociation of 80S and polysomal ribosomes into free 40S and 60S subunits 21 revealed that the cells in which LeuCAG3′ tsRNA was inhibited had 58.1 ± 1.1% and 90.7 ± 5.7% as many 40S and 60S subunits, respectively, as control cells (Fig. 3b) . Consistent with the decrease in 40S ribosomal subunits, 18S mature rRNA abundance decreased to 66 ± 6.2% of control levels in cells in which LeuCAG3′ tsRNA was inhibited (Fig. 3c) . Inhibition of other 5′ and 3′ tsRNAs did not affect ribosome profiles (Extended Data Fig. 4a ).
LeuCAG3′ tsRNA inhibition impairs ribosome biogenesis
To test whether rRNA transcription and/or processing were affected by LeuCAG3′ tsRNA inhibition, we examined the abundance of different pre-and mature rRNAs by northern quantification in three cell lines ( Fig. 3d and Extended Data Fig. 4b, c) . After inhibition of LeuCAG3′ tsRNA, the 30S pre-rRNA accumulated to 205 ± 16.7% of control levels, whereas the 21S and 18S-E pre-rRNA signals decreased to 39.8% and 59.4% of control, respectively (Fig. 3d and Supplementary  Table 3 ). However, the 32S pre-rRNA and the 45S primary transcript signals increased only slightly. Together, these results suggest that the LeuCAG3′ tsRNA did not significantly affect either 28S mature rRNA processing or the abundance of the 45S primary transcript. Instead, a processing step resulting in the removal of the 5′ external transcribed spacer (ETS) from the 30S intermediate was impaired, with a subsequent reduction in 18S rRNA.
LeuCAG3′ tsRNA inhibition impairs ribosome biogenesis
As the LeuCAG3′ tsRNA and anti-Leu3′ tsLNA do not bind the rRNA precursors, such binding could not explain the rRNA processing changes (Extended Data Fig. 5a, b) . Notably, the accumulation of the 30S intermediate and the reduction in 21S and 18S-E pre-rRNAs and 18S mature rRNA are known consequences of reduced expression of specific small ribosomal proteins (RPS) such as RPS6, RPS7, RPS13, RPS16, RPS24 and RPS28 (ref. 22) . We therefore reduced the concentration of these proteins or of the large ribosomal protein RPL7, or of XRN2 or c-MYC, which are important for rRNA biogenesis 23 , using small inhibitory RNAs (siRNAs; Fig. 3d ). Depletion of RPS6, RPS13 or RPS28, but not of the other proteins, phenocopied the 5′ ETS processing defect induced by the anti-Leu3′ tsLNA, suggesting a possible functional relationship between these proteins and the Leu3′ tsLNA inhibition-mediated defect (Fig. 3d) . Using western blotting, we measured the abundance of several ribosomal proteins known to affect 5′ ETS1-mediated rRNA processing (Fig. 4a ) after a reduction in LeuCAG3′ tsRNA. All the RPL proteins were excluded because they are required for maturation of 28S rRNA but not 18S rRNA 22 . Of the RPs evaluated, only RPS28 was substantially downregulated relative to control levels in cells in which LeuCAG3′ tsRNA was inhibited (Fig. 4a) , and the nuclear-cytoplasm ratio of RPS6 and RPS28 was not altered (Extended Data Fig. 6a ). RPS28 protein levels were also reduced in anti-Leu3′ tsLNA-treated PDX liver tumours (Extended Data Fig. 6b, c) . The reduction in the expression of some ribosomal proteins (for example, RPS13, RPS19 and RPL11) or other defects in rRNA biogenesis induce apoptosis 24, 25 . A reduction in RPS28 also resulted in apoptosis ( Fig. 4b and Extended Data Fig. 6d ). We were able to substantially rescue the tsRNA knockdown phenotype by co-transfection of the anti-Leu3′ tsLNA and an RPS28 expression plasmid ( Fig. 4c and Extended Data Fig. 6e-h ) that increased protein levels about threefold (compared to an enhanced green fluorescence protein (EGFP) control plasmid). Specifically, the increase in RPS28 expression in antiLeu3′ tsLNA-treated cells increased cell viability from 45% to 76% of that of control cells ( Fig. 4c and Extended Data Fig. 6e ) and reduced the accumulation of the 30S rRNA precursor from 164.2 ± 16.1% to 127.0 ± 16.2%, resulting in the recovery of the 18S mature rRNA from 67% to 93% of control levels (Extended Data Fig. 6f-h ). Collectively, these results suggest that the phenotypic effect of LeuCAG3′ tsRNA knockdown is the result of reduced RPS28 expression, which in turn impairs 18S rRNA maturation and ultimately induces apoptosis. Because the inhibition of LeuCAG3′ tsRNA reduced RPS28 protein abundance ( Fig. 4a and Extended Data Fig. 6a-c) and not RPS28 mRNA levels in HeLa cells and the PDX HCC model (Fig. 4d and Extended Data Fig. 6i) , we studied the polysomal sedimentation of various ribosomal protein and control mRNAs ( Fig. 4e and Extended Data Fig. 7) . Inhibition of the LeuCAG3′ tsRNA selectively shifted the RPS28 mRNA peak association from 3-4 ribosomes (fraction 10) to 2 ribosomes (fraction 8); GAPDH and other control mRNAs were not significantly shifted ( Fig. 4e and Extended Data Fig. 7 ). These results suggest that the tsRNA has a role in the translation of RPS28 mRNA.
LeuCAG3′ tsRNA enhances translation by binding mRNA
We established that there was a direct physical interaction between LeuCAG3′ tsRNA and RPS28 mRNA using a modified cross-linking chromatin isolation by RNA purification assay 26, 27 (Extended Data  Fig. 8a ). We identified two potential LeuCAG3′ tsRNA binding sites in the RPS28 mRNA using RNAhybrid 28 (Extended Data Fig. 8b ). mRNA secondary structure prediction programs (RNAfold, mfold, and RNAstructure) [29] [30] [31] and minimal free energy calculations implied that target1 (in the 3′ untranslated region (UTR)) forms a duplex with a roughly 22-nt region that overlaps the translation initiation site (anti-target1), whereas target2 (in the coding sequence) is a hairpin structure (Fig. 5a ). We hypothesized that the tsRNA binds RPS28 mRNA to enhance translation, perhaps by unfolding the mRNA secondary structure through competition with folding of target sites (Extended Data Fig. 8c ). We constructed expression plasmids containing either wild-type RPS28 mRNA or variant RPS28 mRNA sequences that maintained the same protein coding sequence but disrupted the tsRNA-RPS28 mRNA interaction in order to negate the effect of LeuCAG3′ tsRNA on mRNA translation (Fig. 5a ). We co-transfected Article reSeArcH each plasmid with either the anti-Leu3′ tsLNA or control LNA in HeLa cells. RPS28 mRNAs harbouring mutants in the anti-target1 (A) or target2 (C) regions lost their ability to respond to altered cellular levels of the tsRNA. By contrast, RPS28 mRNAs containing similar nucleotide changes outside the target sites (B and D) responded in the same manner as the wild-type mRNA to the varied concentration of the tsRNA ( Fig. 5b and Extended Data Fig. 8d ). Similar results were obtained using C-terminal Flag-tagged versions of the same wild-type and mutant RPS28 expression plasmids that allowed us to distinguish the newly synthesized RPS28 protein from total RPS28 protein in the whole cellular pool ( Fig. 5b and Extended Data Fig. 8e, f) .
LeuCAG3′ tsRNA enhances translation by binding mRNA
To corroborate the genetic complementation data, we synthesized the variant RPS28 mRNAs and tested them for translation in a cellfree rabbit reticulocyte system with or without the addition of the synthetic tsRNA. Synthetic LeuCAG3′ tsRNA enhanced translation of the wild-type RPS28 mRNA but not the firefly luciferase and Xenopus elongation factor1 (Xef1) mRNAs (Extended Data Fig. 8g-i) . Notably, mRNA translation from mutants A and C was not enhanced relative to the wild-type or mutant B mRNA in the presence of the LeuCAG3′ tsRNA ( Fig. 5c and Extended Data Fig. 8j ). To ensure that LeuCAG3′ tsRNA enhances RPS28 mRNA translation by base-pairing with the target2 site, we used the modified target2 sequence (mutant C) RPS28 mRNA for in vitro translation but added a sequence-modified tsRNA mimic designed to be nearly complementary to the modified target2 site (mutant C). This compensatory tsRNA enhanced the translation of mutant C but not wild-type RPS28 mRNA ( Fig. 5d and Extended Data Fig. 8k ). These experiments strongly support the idea that binding of LeuCAG3′ tsRNA affects RPS28 translation through base-pairing interactions at target sites within the mRNA.
LeuCAG3′ tsRNA target prediction
To determine whether LeuCAG3′ tsRNA can modulate the translation of other mRNAs, we first selected mRNAs in which the coding sequence (CDS) and flanking 30 bp were nearly complementary to LeuCAG3′ tsRNA on the basis of minimal free energy 28 (Extended Data Fig. 9a ). Target sites contained in a UTR might affect translation by forming secondary structures with distal coding sequences. However, because current algorithms cannot accurately predict the secondary structure of longer mRNAs 32 , we excluded potential target sites in UTR regions fully outside of the CDS by more than 30 nt. Notably, the mRNAs for ribosomal proteins RPS9, RPS14 and RPS15 contained prospective target sites in their CDSs (Extended Data Fig. 9b, c) . To estimate whether the target sites were in a duplexed RNA structure, we analysed the secondary structure of the target site using in vivo click selective 2′ -hydroxyl acylation and profiling (icSHAPE) data in living cells 33 . This showed that the target1, anti-target1 and target2 sites in RPS28 mRNA primarily exist in a double-stranded state (Extended Data Fig. 9d) , as predicted by RNAfold (Fig. 5a ) and other prediction programs. The icSHAPE result also showed that the predicted RPS15 mRNA target is double-stranded (Extended Data Fig. 9d ). RPS15 is involved in a later step of 18S ribosomal RNA processing, and its depletion does not affect mature 18S rRNA abundance 22 . The target sites in the RPS9 and RPS14 mRNAs were primarily located in single-stranded regions (Extended Data Fig. 9d ). We hypothesized that translation of the RPS15 mRNA but not the RPS9 or RPS14 mRNAs would be influenced by the concentration of LeuCAG3′ tsRNA.
LeuCAG3′ tsRNA target prediction
The presence of LeuCAG3′ tsRNA enhanced RPS15 mRNA translation in HeLa cells and the PDX HCC tumour model (Extended Data Fig. 10a-e) . By contrast, the levels of RPS9 and RPS14 protein were not significantly different after ASO/LNA-mediated reduction of the tsRNA (Extended Data Fig. 10f, g ). Plasmids expressing either wild-type or target variant RPS9, RPS14 and RPS15 mRNA sequences (similar to target variant RPS28 mRNA) were co-transfected with either anti-Leu3′ tsLNA or control LNA into HeLa cells (Extended Data Fig. 10f-i) . The RPS15 target-site mutant mRNA lost its responsiveness to tsRNA inhibition (Extended Data Fig. 10h, i) . However, translation of wildtype and target-site RPS9 and RPS14 mRNAs remained unchanged regardless of the tsRNA concentration (Extended Data Fig. 10f, g ).
LeuCAG3′ tsRNA target prediction
In vitro translation assays with RPS15 mRNA corroborated these genetic complementation data by showing that LeuCAG3′ tsRNA . sicontrol, control siRNA; siRPS28, siRNA targeting RPS28. c, Overexpression of RPS28 protein increases cell viability. After co-transfection of the designated LNA and plasmids, cell viability was determined (n = 6 independent experiments). d, Inhibition of LeuCAG3′ tsRNA does not alter RPS28 mRNA levels, determined by PCR with reverse transcription (RT-PCR) (n = 3 independent experiments). e, Inhibition of LeuCAG3′ tsRNA alters sedimentation of the RPS28 mRNA within the sucrose gradient. Northern analysis was performed on each gradient fraction. The amount of the specific mRNA for each fraction was normalized to the sum of the mRNA signal across all gradient fractions (RPS28, n = 4; GAPDH, n = 3 independent experiments). x axis, fraction number. The diagram above each fraction shows the number of ribosomes associated with mRNA based on a gradient profile (Fig. 3a) . Data shown as mean ± s.d.; indicated P value by two-tailed t-test (b-e). For gel source data, see Supplementary Fig. 1. enhanced translation from the wild-type and non-target mutant RPS15 mRNAs, whereas no change was observed with the RPS15 target-site mutant mRNA (Extended Data Fig. 10j, k) . We propose that LeuCAG3′ tsRNA is involved in unfolding mRNA target sites that have duplexed secondary structures, thereby allowing enhanced translation.
LeuCAG3′ tsRNA target prediction
The specific tsRNA studied here fine-tunes the production of at least two ribosomal proteins and ultimately the number of ribosomes in proliferating cells. Decreasing the production of individual ribosomal proteins did not have a major effect on global protein synthesis (Extended Data Fig. 1g, h) , consistent with previous studies 34, 35 . Our studies show how a non-coding RNA positively regulates mRNA translation. Further investigation is required to establish how the 3′ tsRNA is generated and regulated in normal and disease states, the mechanism of target site recognition, and the process of translational regulation. The presence of hundreds of different tsRNAs suggests that they have a biological role in cells, perhaps in similar or different post-transcriptional gene regulatory pathways that might involve altering RNA secondary structures. The relatively high concentration of LeuCAG3′ tsRNA in tumours and immortalized cells, and the induction of apoptosis in such cells when the tsRNA is reduced, warrants additional evaluation as a potential therapeutic target in treating cancer.
LeuCAG3′ tsRNA target prediction
Online Content Methods, along with any additional Extended Data display items and Source Data, are available in the online version of the paper; references unique to these sections appear only in the online paper. Fig. 8b ). Black and grey, coding and untranslated sequences, respectively. Large font black AUG located in anti-target1 region indicates the translation initiation codon. b, A potential binding site is required for the tsRNA to regulate RPS28 protein production. The relative protein levels were determined as described in Extended Data Fig. 8d , f (n = 4 independent experiments). x axis indicates the RPS28 construct (Fig. 5a, Extended Data Fig. 8e and Supplementary Table 8) . Article reSeArcH Fig. 3a, b and Extended Data Fig. 1g, h, and assisted Fig. 2c and Extended Data Fig. 2h, i, 
Article reSeArcH

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Cell culture and transfection. HeLa, HCT-116 and HEK293T cells were purchased from the American Type Culture Collection (ATCC) and were grown in Dulbecco's modified Eagle's medium (DMEM; GIBCO-BRL) with 2 mM l-glutamine and 10% heat-inactivated fetal bovine serum with antibiotics. All cells were negative for mycoplasma. 30-60 nM LNA mixmers, 10-30 nM LNA gapmers, 100 nM synthetic tsRNAs, and/or plasmids were transfected using Lipofectamine 2000 (Life Technologies) according to the manufacturer's instructions. LNA oligos (Supplementary Table 4 /SzJ (Nod-SCID-Gamma; NSG) mice (20-25 g body weight) (Jackson Laboratory). Tumour development was monitored daily. Once the subcutaneous xenograft reached 1 cm in diameter, it was removed, cut into 2-mm 3 pieces and surgically implanted into the left lobe of the liver of 6-week-old NSG mice. Tumour growth was monitored by bioluminescence imaging using the Xenogen IVIS in vivo imaging system (Caliper Life Sciences) until the tumours were established (usually one week after tumour implantation). Mice with similar tumour bioluminescence signals were selected and randomized into three groups (n = 10 for each group) for intraperitoneal injection with saline (control), control LNA (0.6 mg/kg), or anti-Leu3′ ts LNA (0.6 mg/kg) every 2-3 days. The LNAs used for injection were phosphorothioate modified. Two mice in the saline and one mouse in the control group died at an early time point prior to any measurements. The saline group had three males and five females. The control group had five males and four females. The anti-Leu3′ ts group had six males and four females. Tumour growth was monitored weekly by bioluminescence imaging, and growth curves were plotted using average bioluminescence within each group. Body weight was also measured every 2-3 days. After 4 weeks of treatment, the mice were killed and the tumours and normal livers removed. Tumour size was measured with digital calipers and tumour volume was calculated using the formula π /6 × larger diameter × [smaller diameter] 2 . Liver and tumour tissues were fixed in formalin and embedded with paraffin. ALT measurements. BALB/cJ mice (6-to 8-weeks old) were randomized into groups (n = 2-3 each). The LNAs used for injection were phosphorothioate modified. After hydrodynamic injection of saline, 125 μ g control LNA or antiLeu3′ tsLNA 37, 38 , mouse serum was collected by centrifugation of whole clotted blood obtained by retro-orbital bleeding on the indicated day (Extended Data  Fig. 2f ). ALT levels were measured using an ALT kinetic measurement kit (Teco Diagnostics). Plasmid constructs. Complementary sense and antisense primers used for cloning were synthesized by IDT (Supplementary Table 5 ). The dimers were cloned into the 3′ UTR or 5′ UTR of the pGL3 control luciferase plasmid using XbaI and EcoRI or HindIII and NcoI enzymes, respectively. The Renilla gene from the psiCHECK-2 plasmid (Promega), for which all the CUG codons were replaced by CUU or CUC codons, was synthesized by Life Technologies. The full-length RPS9 gene was amplified with primers (5′ -CTCTTTCTCAGTGACCGGG-3′ and 5′ -AGCGCTGATCCTGTTTATTTG-3′ ). The full-length RPS14 gene was amplified with primers (5′ -CTCCGCCCCCTCCCACTCTC-3′ and 5′ -TTTTTGAAACAGTTTACATGAAGGC-3′ ). The full-length RPS15 gene was amplified with primers (5′ -GGCAGTCTCGCGATAACTGC-3′ and 5′ -AGTCATGTGCGCCTTTATTAG-3′ ). The full-length RPS28 gene was amplified with primers (5′ -CTCTCCGCCAGACCGCCGC-3′ and 5′ -TTTTTTTTTTTTTTTTTTTTTAACTTGAAACACAAACGCTT-3′ ). The full-length RPS9, RPS14, RPS15 and RPS28 protein sequences were cloned into the pcDNA3.3 plasmid. C-terminal Flag-tagged RPS28 sequences were generated using site-directed mutagenesis. The CMV promoter in C-terminal Flag-tagged RPS28 plasmid was replaced by the human PGK promoter. Site-directed mutagenesis was performed with the QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene) to generate point mutations or deletions in the recombinant RPS9, RPS14, RPS15 and RPS28 genes (primers in Supplementary Table 9 ). All plasmid clones were confirmed by DNA sequencing.
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Dual-luciferase reporter assay. Both pGL3 and pRL plasmid DNA, or psiCHECK-2 plasmid DNA, were co-transfected with 60-100 nM LNA into HeLa or HCT-116 cells in 24-well plates. Firefly luciferase and Renilla luciferase activities were measured 24 h after transfection using Promega's dual-luciferase kit protocol and luminescence was detected using a Modulus Microplate Luminometer (Turner BioSystems). Firefly or Renilla luciferase activity from pGL3 plasmid or psiCHECK-2 plasmid were normalized to the co-transfected Renilla activity from pRL or firefly activity from psiCHECK-2, respectively. The values obtained in the anti-Leu3′ tsLNA co-transfected cells were normalized to the control co-transfected cells. Each independent experiment was performed in triplicate. RNA isolation and northern blotting. Total RNA was isolated with TRIZOL reagent (Life Technologies) according to the manufacturer's instructions. Total RNA was resolved by electrophoresis on a 15% (w/v) polyacrylamide gel with 7 M urea for detection of small RNAs (< 200 bp) or on a 0.9% agarose denaturating gel for detection of large RNAs (> 200 bp). The RNA was transferred onto a Hybond-N+ nylon membrane (Amersham). 32 P-labelled oligonucleotides or amplified cDNA probes were hybridized to the membrane in PerfectHyb Plus hybridization buffer (Sigma). All antisense oligonucleotides for northern probes are listed in Supplementary Table 4. All oligonucleotides for the generation of cDNAs as northern probes are listed in Supplementary Table 6 . Measurement of cell proliferation. Cell proliferation was measured with a CellTiter 96 nonradioactive cell proliferation assay kit (MTS assay; Promega) according to the manufacturer's instructions. Each independent experiment was performed in triplicate. Apoptosis assay. The cell apoptosis assay was performed by measuring translocation of membrane phospholipid phosphatidylserine using an Annexin V-FITC apoptosis detection kit (BD Pharmingen) according to the manufacturer's instructions. Cells were analysed using a FACScalibur instrument using FlowJo software (Tree Star). For the TUNEL assay, the apoptotic responses were identified 24 h post-transfection using Invitrogen's Click-iT TUNEL Alexa Fluor 594 Imaging Assay kit according to the manufacturer's instructions. For the TUNEL staining of paraffin-embedded 6-μ m tissue sections, the ApopTag Peroxidase In situ Apoptosis Detection Kit (EMD Millipore) was used according to the manufacturer's instructions. Western blotting. Twenty-four hours after transfection, cell lysates were prepared using 1× cell lysis buffer (Cell Signaling) with 1 mM PMSF (Cell Signaling). 6-10 μ g of protein lysate was run on a 4-20% SDS PAGE and transferred to Hybond-P or nitrocellulose membrane (GE Healthcare). The membrane was incubated for 20 min at room temperature in a 4% BSA (Omnipur) solution, washed, and incubated overnight at 4 °C with one of the antibodies listed below. After washing and incubation for 1 h at room temperature with secondary antibody, the protein signal was detected using a Pierce ECL2 substrate (Thermoscientific) or Odyssey CLx imaging system (LI-COR Biosciences) according to the manufacturer's instructions. Antibodies for immunoblotting were as follows: anti-PARP rabbit monoclonal Ab (clone 46D11; Cell Signaling), anti-cleaved PARP (Asp214) rabbit monoclonal Ab (clone D64E10; Cell Signaling), anti-GAPDH-peroxidase mouse Ab (Sigma), anti-RPS13 rabbit polyclonal Ab (Abnova), anti-RPS7, RPS10, RPS16, RPS19, RPS24, and RPS28, XRN2 Ab (Abcam), anti-RPS9, RPS14, and RPS15 Ab (Aviva), anti-RPL7, 13a, 26, and XRN2 Ab (Abcam), anti-RPS6 mouse monoclonal Ab, anti-Flag Ab (Cell Signaling). High-throughput large RNA sequencing. Twenty-four hours post-transfection, total RNA was isolated using the TRIZOL reagent. Residual DNA contamination and ribosomal sequences were removed from total RNA using TURBO DNA-free Kit (Life Technologies) and Ribo-Zero Gold kit (Epicentre), respectively, according to the manufacturer's instructions. A total of 150-200 ng of purified RNA was subjected to strand-specific RNA-seq using the ScriptSeq v2 mRNA-SEQ library preparation kit (Epicentre), which uses a random-hexamer method of cDNA synthesis, according to the manufacturer's instructions. Fiftybase pair paired-end reads were generated on an Illumina HiSeq 2000 machine yielding a total of 18-40-million paired-end reads. Sequences were mapped to the human hg19 genome using TopHat version 1.4.1 with the following parameters: -r 180,-mate-std-dev 130 -library-type fr-secondstrand 39 . FPKM values were calculated using the CuffDiff program version 1.3.0 40,41 using refGene transcripts as an input file for comparison of gene expression levels. Sequences have been deposited in the NCBI Gene Expression Omnibus (accession number GSE54878). Real-time PCR. 500 ng total RNA was reverse transcribed with the superscript II RT kit (Life Technologies) and subjected to gene expression analyses with gene-specific TaqMan probes (4326317E for GAPDH, Hs04195024_g1 for RPS6, Hs01652370_gH for RPS10, HS01358643_g1 for RPS15, either HS02597256_g1 or HS02597258_g1 for RPS28). Real-time PCR was performed on a CFX384 RealTime system (Bio-Rad). Each independent experiment was performed in triplicate. All mRNA levels were normalized to GAPDH mRNA.
Article reSeArcH
Polysome gradient and RNA preparation. Polysome gradient and RNA preparation were performed as described previously 42 with modifications. Twenty-four hours post-transfection, cells were treated with 100 μ g/ml cycloheximide (SigmaAldrich) for 3 min, and cells were lysed in buffer containing 15 mM Tris-HCl (pH 7.5), 150 mM KCl, 5 mM MgCl 2 , 500 U/ml RNasin (Promega), and 1% Triton X-100. The cleared lysates were loaded onto 10-50% sucrose gradients (15 mM Tris-HCl (pH 7.5), 150 mM KCl, 5 mM MgCl 2 , 20 U/ml SUPERaseIn (Life Technologies), and 100 μ g/ml cycloheximide with a 60% sucrose cushion. Gradients were centrifuged at 35,000 r.p.m. at 4 °C for 2 h 45 m and fractionated with a Teledyne Isco Foxy R1 Retriever/UA-6 detector system. The gradient fractions were sequentially treated for 30 min at 37 °C with 0.5 mg/ml proteinase K (New England Biolabs) in the presence of 5 mM EDTA. RNAs were extracted with an equal volume of phenol-chloroform-isoamylalcohol (25:24:1; Life Technologies) and re-extracted with chloroform, and ethanol precipitation was performed. Puromycin gradients. Puromycin gradients were performed as described previously 42 with modifications. Twenty-four hours post-transfection, cells were lysed in buffer containing 15 mM Tris-HCl (pH 7.5), 500 mM KCl, 2 mM MgCl 2 , 2 mM puromycin, 500 U/ml RNasin (Promega), 20 U/ml SUPERaseIn (Life Technologies), and 1% Triton X-100. Following incubation on ice for 15 min, 80S ribosome subunits were separated at 37 °C for 10 min. Following centrifugation, cleared lysates were loaded onto 10-50% sucrose (500 mM KCl, 15 mM Tris-HCl (pH 7.5), 2 mM MgCl 2 , and 20 U/ml SUPERaseIn (Life Technologies)) with a 60% sucrose cushion. Gradients were centrifuged at 35,000 r.p.m. at 4 °C for 2 h 45 min. Measurement of global protein synthesis. Twenty-four hours post-transfection, cells were washed twice with PBS and grown in DMEM without cystine and methionine (DMEM -Cys-Met) (Life Technologies) for 30 min at 37 °C. The medium was removed and DMEM -Cys-Met plus 100 μ Ci protein labelling mix (Perkin Elmer) was added for 10 min at 37 °C. Cells were washed twice with cold PBS, harvested, and lysed with RIPA buffer. Equal amounts of protein were resolved on 4-12% SDS-PAGE and stained with Coomassie brilliant blue, the gel was dried, and the incorporated radioactivity was scanned using a PMI (Personal Molecular Imager). A global protein synthesis assay using a Click-iT AHA (l-azidohomoalanine) Alexa Fluor 488 (Life Technologies) assay in HeLa cells was performed 24 h post-transfection according to the manufacturer's instructions. Immunoprecipitation. Antibodies were incubated with protein A/G UltraLink Resin (Thermo Scientific) overnight at 4 °C. Cells were lysed with IP buffer (25 mM Tris pH7.5, 150 mM KCl, 0.5% NP40, 0.02 mM EDTA) containing RNasin plus (Promega) and cOmplete Protease Inhibitor Cocktail Tablets (Roche) and incubated with prepared protein A/G conjugated antibodies for 2 h at 4 °C. All samples were washed with IP buffer three times. Immunoprecipitated RNAs were extracted using TRIZOL (Life Technologies) according to the manufacturer's instructions. Immunoprecipitated proteins were separated from protein A/G UltraLink resin by adding RIPA buffer and boiling for 5 min. Antibodies for immunoprecipitation were as follows: anti-Ago1 monoclonal Ab (clone 1F2; Wako), anti-Ago2 monoclonal Ab (clone 2D4; Wako), and anti-Ago3 monoclonal Ab (clone 1C12; Wako). Modified cross-linking chromatin isolation by RNA purification (ChIRP) assay. The modified ChIRP assay was performed as described earlier 26, 27 with slight modifications. After transfection of the synthetic LeuCAG3′ tsRNA into cells, RNA cross-linking was performed and the RPS28 or GAPDH mRNAs were pulled down with 4 and 14 biotinylated tiling oligonucleotides (Supplementary Table 7) , respectively. Specifically, after transfection of 6 nM of synthetic LeuCAG3′ tsRNA, 293T cells were cross-linked with 3.7% formaldehyde and sonicated with a Qsonica sonicator with the following settings: 15 s pulse with 15 s between each pulse; 75% ampl for 1 min at 4 °C. The cleared lysate was then hybridized with 14 and 4 biotinylated tiling oligos against GAPDH and RPS28 mRNAs, respectively (sequences in Supplementary Table 7) . After a 4-h hybridization, the streptavidin beads were hybridized together for 1 h, washed six times, and subjected to phenol-chloroform extraction. Extracted RNA was used for RT-PCR to detect GAPDH and RPS28, and resolved on a 15% denaturing gel to detect the LeuCAG3′ tsRNA. In vitro translation. Each mRNA was synthesized using T7 MEGAscript (Life Technologies). In vitro translation using the Flexi Rabbit Reticulocyte Lysate System (Promega) was performed according to the manufacturer's instructions with minor modifications. In brief, 100 ng luciferase mRNA or 60 ng of both RPS28 mRNA and Xef1 mRNA were mixed in 15 μ l translation reaction containing 70% reticulocyte lysate. The indicated amounts or 14 pmol of synthetic LeuCAG3′ ts RNA or control RNAs were added to the reaction and incubated for 30-45 min at 30 °C. Firefly luciferase activity was measured with a Luciferase Assay System (Promega). RPS28 and Xef1 products were resolved on 4-12% SDS PAGE, exposed with a phosphor image screen, and quantified. mRNA structures and structure probing of tsRNA targets. The m.f.e. (minimal free energy) between the LeuCAG3′ tsRNA and binding site in each mRNA was predicted using a RNA-hybrid program (https://bibiserv2.cebitec.uni-bielefeld. de/rnahybrid). The RPS28 mRNA secondary structure was predicted by RNAfold (http://rna.tbi.univie.ac.at) and we retrieved icSHAPE data from a previous study, allowing us to experimentally predict each mRNA structure 33 . This approach allowed us to probe the nucleotide reactivity (that is, single-strandedness) in HEK293T cells. While this study was performed in whole treated cells, we also obtained cytoplasmic RNA structure data from an unpublished study that separated and measured RNA structures in different cellular fractions with icSHAPE. Statistical information. No statistical methods were used to predetermine sample size. All data are presented as mean ± s.d. Data were analysed with two-tailed Student's t-test in PRISM 6.0. A P value of 0.05 or lower was considered significant. Data availability. All data generated or analysed during this study are available from the authors and/or have been included in this published article and online Supplementary Information. Data from RNA-seq have been deposited in the NCBI Gene Expression Omnibus (accession number GSE54878). There was no specific statistical method to determine sample size. However, the experimental conditions were carried out in more than triplicate and with at least one and usually 2 independent set of experiments. The exception was a negative result.
Data exclusions
Describe any data exclusions. 1. No animals were excluded from evaluation after assignment to the treatment groups. Tumors were implanted in 46 mice. The animals were imaged for luciferase activity 3 to 4 days later to make sure the tumors were implanted into the host liver. Animals that were deemed or had questionable tumor implantation were excluded prior to treatment. Animals were randomly assigned to 3 groups to closely match the luciferase activities and the groups randomly assigned. 2. For target site prediction, target sites contained in an untranslated region (UTR) might affect mRNA translation by forming secondary structures with distal coding sequences. However, because current algorithms cannot accurately predict the secondary structure of longer mRNAs we excluded potential target sites in UTR regions fully outside of the CDS by >30 nucleotides.
Replication
Describe whether the experimental findings were reliably reproduced.
Replication
All attempts at replication were successfull.
Randomization
Describe how samples/organisms/participants were allocated into experimental groups.
Randomization
Tumors were implanted in 46 mice. The animals were imaged for luciferase activity 3 to 4 days later to make sure the tumors were implanted into the host liver. Animals were randomly assigned to 3 groups to closely match the luciferase activities and the groups randomly assigned.
Blinding
Describe whether the investigators were blinded to group allocation during data collection and/or analysis.
Blinding
None of the experiments were truly blinded. The randomization of animals for the tumor studies is described in the methods.
Blinding
For all figures and tables that use statistical methods, confirm that the following items are present in relevant figure legends (or in the Methods section if additional space is needed).
Blinding
n/a Confirmed
Blinding
The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement (animals, litters, cultures, etc.)
Blinding
A description of how samples were collected, noting whether measurements were taken from distinct samples or whether the same sample was measured repeatedly A statement indicating how many times each experiment was replicated
Blinding
The statistical test(s) used and whether they are one-or two-sided (note: only common tests should be described solely by name; more complex techniques should be described in the Methods section)
Blinding
A description of any assumptions or corrections, such as an adjustment for multiple comparisons
Blinding
The test results (e.g. P values) given as exact values whenever possible and with confidence intervals noted A clear description of statistics including central tendency (e.g. median, mean) and variation (e.g. standard deviation, interquartile range)
Clearly defined error bars
See the web collection on statistics for biologists for further resources and guidance.
Software
Policy information about availability of computer code
Software
Describe the software used to analyze the data in this study.
Software
RNAseq data are analyzed using CuffDiff and TopHat. All graphs are draw using Prism. Flow Cytometry data are analyzed using FlowJo. mRNA and tsRNA interaction is predicited by RNAhybrid. Secondary structure of target mRNAs were predicited by RNAfold, mfold, and RNAstructure.
Software
For manuscripts utilizing custom algorithms or software that are central to the paper but not yet described in the published literature, software must be made available to editors and reviewers upon request. We strongly encourage code deposition in a community repository (e.g. GitHub). Nature Methods guidance for providing algorithms and software for publication provides further information on this topic.
Materials and reagents
Policy information about availability of materials
Materials availability
Indicate whether there are restrictions on availability of unique materials or if these materials are only available for distribution by a for-profit company.
Materials availability
No unique materials were used.
Antibodies
Describe the antibodies used and how they were validated for use in the system under study (i.e. assay and species).
Antibodies
Briefly, single tumor cells labeled with luciferase gene were suspended in BEME medium containing 50% Matrix Matrigel, and then subcutaneously injected into 4-8 weeks old NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (Nod-SCID-Gamma; NSG) mice (Jackson Laboratory; Bar Harbor, MA) (20-25 g body weight). Once the subcutaneous xenograft reached 1 cm in diameter, it was removed and cut into 2 mm3 pieces and surgically implanted into the left lobe of the liver of another group of 6 weeks old NSG mice. Saline group has 3 males and 5 females. con group has 5 males and 4 females. Anti-Leu3'ts group has 6 males and 4 females.
Antibodies
Policy information about studies involving human research participants
Description of human research participants
Describe the covariate-relevant population characteristics of the human research participants.
Description of human research participants
The primary human liver tumor used for the xenotransplant studies was ontained from a 62 year old male at the time of surgical resection

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