A transfer-RNA-derived small RNA regulates ribosome biogenesis

Abstract

Transfer-RNA-derived small RNAs (tsRNAs; also called tRNA-derived fragments) are an abundant class of small non-coding RNAs whose biological roles are not well understood. Here we show that inhibition of a specific tsRNA, LeuCAG3'tsRNA, induces apoptosis in rapidly dividing cells in vitro and in a patient-derived orthotopic hepatocellular carcinoma model in mice. This tsRNA binds at least two ribosomal protein mRNAs (RPS28 and RPS15) to enhance their translation. A decrease in translation of RPS28 mRNA blocks pre-18S ribosomal RNA processing, resulting in a reduction in the number of 40S ribosomal subunits. These data establish a post-transcriptional mechanism that can fine-tune gene expression during different physiological states and provide a potential new target for treating cancer.

Extended Data Figure 1. 22-nt LeuCAG3′tsRNA is involved in cell viability

a, b, Inhibition of LeuCAG3′tsRNA impairs HCT-116 cell viability. 3 days post-transfection, a MTS assay was performed (n=3 independent experiments). Each LNA is perfectly complementary to colored region on the tRNA diagram above each bar. Blue and red asterisk mark, different 2-nt mismatches. c, d, Inhibition of the LeuCAG3′tsRNA decreased the number of viable HeLa (c) and HCT-116 cells (d) (n=3 independent experiments). The cell number at each day was normalized to the day zero value. e, Cleavage of LeuCAG3′tsRNA impairs HeLa cell viability. 3 days post-transfection, a MTS assay was performed as in (a) (n=3 independent experiments). f, Northern analysis of the LeuCAG3′tsRNA and mature Leu-tRNA post-transfection. To quantify the tsRNA and mature tRNA level correctly, transferred blots were cut at the 40–50 nt position and hybridized with the same probe to detect tRNA (top) and tsRNA (bottom) separately (n=2 independent experiments). U6 snRNA, the loading control. g, 24 h post-transfection, global protein synthesis assay using [³⁵S]-methionine metabolic labeling in HeLa cells (n=3 independent experiments). h, 24 h post-transfection, global protein synthesis detection on gels using [³⁵S]-methionine metabolic labeling in HeLa cells. Coomassie brilliant blue (left) as a loading control; gels were scanned to measure incorporated radioactivity (right) (n=2 independent experiments). Each number multiplied by 10⁵, the number of cells on 6-well culture dishes 24 h prior to transfection. i, Global protein synthesis assay using a Click-iT AHA (L-Azidohomoalanine) Alexa Fluor 488 assay in HeLa cells was performed 24 h post-transfection. The nucleus was stained with DAPI, blue color. Protein synthesis was measured with AHA, green color. Merge represents the DAPI and AHA merged images (n=2 independent experiments). Un, untreated cells; mock, transfection without LNA; CHX, cycloheximide-treated positive control. j, The abundance of sequencing reads aligned to LeuCAG-tRNA from HeLa cells. This analysis was generated from tRFdb (http://genome.bioch.virginia.edu/trfdb/search.php). X-axis, a position on LeuCAG mature tRNA. Blue line, the 18 and 22 nt of LeuCAG 3′tsRNAs. k, The LeuCAG3′tsRNA major isoform is 22 nt. A northern hybridization was performed. The 18-nt isoform was not detected (n=2 independent experiments). Mean is indicated. Error bar, s.d.; indicated p-value by two-tailed t-test (a–e).

Extended Data Fig. 2. Inhibition of LeuCAG3′tsRNA induces apoptosis in vitro and inhibits the growth of hepatocellular carcinoma (HCC) patient-derived xenograft

a, A representative result of the apoptosis assay. Apoptosis in HeLa cells was measured using Annexin V-FITC and PI staining at 24, 48 and 72 h post-transfection. The percentage of cells was shown in each gate. Q1 (Healthy cells), stained with neither Annexin V nor PI; Q2 (early apoptotic cells), positive with Annexin V; Q3 (late apoptotic cells), positive with Annexin V and PI; Q4 (dead cells), positive with PI. The average cell population of the apoptosis assay is provided in Fig. 2a (n=3 independent experiments). b, Inhibition of the LeuCAG3′tsRNA results in increased apoptosis in HCT-116 cells. The apoptosis assay was done as in (a) (control at 1 d, n=2; all other samples, n=3 independent experiments). c, Inhibition of LeuCAG3′tsRNA causes DNA fragmentation in HCT-116 cells. A TUNEL assay was performed 24 h post-transfection. DNase I is a positive control. TUNEL-positive cells are stained red. Merge is DAPI and TUNEL merged staining (n=2 independent experiments). d, e, Inhibition (mixmer LNA) (d) and cleavage (gapmer LNA) (e) of LeuCAG3′tsRNA causes PARP protein cleavage. Western blot analysis was performed 24 h post transfection. Anti-PARP Ab detects both full-length (116 kD) and cleaved (89 kD) PARP protein. ACTB is the loading control (n=2 independent experiments). f, For liver toxicity, 125 µg of each LNA (tsRNA sequence is the same in human and mouse) was injected into C57BL/6J mice by hydrodynamic tail vein injection (saline group, n=2; other group, n=3 independent mice). g, The LeuCAG3’tsRNA is highly expressed from mouse models of HCC generated from conditional TKO (Rb^lox/lox; p130^lox/lox; p107^−/−) adult mice. Normal liver was harvested from C57/BL6 mice. Northern hybridization was performed as in Extended Data Fig. 1f (n=2 independent experiments). h, Over the 4-week study period, mice containing an orthotopic human xenotransplanted hepatocellular carcinoma were given intraperitoneal injections of: saline, con, and anti-Leu3′tsLNA. The luciferase signal as a marker of tumor growth was monitored weekly (saline group, n=8; con group, n=9; Anti-Leu3′ts group, n=10 independent mice). i, After the 4week injection period, all mice were sacrificed and tumors harvested. j, Anti-Leu3′tsLNA inhibits LeuCAG3′tsRNA in vivo. Northern hybridization was performed with total RNAs from saline or anti-Leu3′tsLNA injected mice (n=2 independent experiments). k, Body weight curve of individual mice bearing HCC xenografts during the 4-week experiment. (saline group, n=8; con group, n=9; Anti-Leu3′ts group, n=10 independent mice). Mean is indicated. Error bar, s.d.; indicated p-value by two-tailed t-test (b, h); p-value (b), early apoptosis population. For gel source data, see Supplementary Figure 1.

Extended Data Figure 3. LeuCAG3′tsRNA does not have transgene silencing activity

a, LeuCAG3′tsRNA does not repress luciferase gene expression containing perfect complementary target sites in its 3′ UTR or 5′ UTR. A luciferase plasmid (x-axis) was co-transfected with con or anti-Leu3′tsLNA (n=3 independent experiments). The normalization protocol is described in Methods. Scramble, scrambled sequences in 3′ UTR; LeuCAG3′tsPM in 3′ UTR, two copies of the perfect complementary sequence of the LeuCAG3′tsRNA in 3′ UTR; LeuCAG3’tsPM in 5′ UTR, two copies of the perfect complementary sequence of the LeuCAG3′tsRNA in 5′ UTR; Let-7 PM is a positive control, a single copy of perfect complementary sequences of the Let-7 miRNA in 3′ UTR. b, AspGTC3′tsRNA or SerGCT3′tsRNA does not repress luciferase gene expression in a construct that contains two copies of the corresponding perfect complementary target site in its 3′ UTR. A luciferase assay was performed as in (a) (n=3 independent experiments). X-axis, target sites in the 3′ UTR. c, The tsRNAs are not associated with Ago proteins. Endogenous Ago1, 2, and 3 proteins were immunoprecipitated by the indicated Ab and the associated RNAs were subjected to northern blotting. The closed triangle in each northern blot indicates the detected tsRNA. IgG, control. Let-7 is a positive control (n=2 independent experiments). d, LeuCAG3′tsRNA does not affect global gene expression in HeLa and HCT-116 cells. Scatter plots comparing gene expression (log₂ (FPKM+1)) of two RNA-Seq datasets from samples 24 h post-transfection (Supplementary Table 2). The Pearson correlation coefficient is indicated by the r value in each plot (n=1). Mean is indicated. Error bar, s.d. For gel source data, see Supplementary Figure 1.

Extended Data Figure 4. LeuCAG3′tsRNA is a non-coding RNA required for ribosome biogenesis

a, LNAs directed against 5′ end of the LeuCAG-tRNA, Ser3′tsRNA (3′ end of the SerGCT-tRNA), and Met3′tsRNA (3′ end of the MetCAT-tRNA) does not change the ribosome/polysomal profiles. 24 h post-transfection cytoplasmic lysates from HeLa cells were treated with cycloheximide and separated on 10−50% sucrose gradients. The polysomal profile was analyzed as in Fig. 3a (n=2 independent experiments). b, Pre-rRNA processing pathways in human cells based on prior studies^, is shown. The 45S primary transcript (pre-45S) is processed and categorized as: 5′external transcribed spacers (5′ETS), mature 18S rRNA, internal transcribed spacer 1 (ITS1), mature 5.8S rRNA, internal transcribed spacer 2 (ITS2), mature 28S rRNA, and 3′external transcribed spacers (3′ETS). There are two alternative processing pathways. Inhibition of LeuCAG3′tsRNA inhibits processing from the 30S intermediate to 21S intermediate form depicted in pathway B. Arrowhead and number indicate cleavage sites. c, Inhibition of the LeuCAG3′tsRNA suppressed 5′ETS processing in 18S rRNA biogenesis in 293T and HCT-116 cells. Northern hybridization was performed with total RNA from HCT-116 and 293T cells 24 h post-transfection. The ITS1 probe detects the 45S primary transcript and intermediate forms of the mature 18S rRNA including 41S, 30S, 21S, and 18S-E pre-rRNAs. The 5′ETS probe detects the 45S primary transcript and 30S intermediate form of the mature 18S rRNA. Each number, multiplied by 10⁴, on top of image represents the number of cells plated on 6-well culture dishes the day prior to transfection (n=2 independent experiments). For gel source data, see Supplementary Figure 1.

Extended Data Figure 5. LeuCAG3′tsRNA and anti-Leu3′tsLNA do not affect 18S rRNA biogenesis through binding to 45S pre-rRNA

a, Schematic picture showing putative binding sites of the LeuCAG3′tsRNA and anti-Leu3′tsLNA on the 45S primary transcript (45S pre-rRNA). To identify the tsRNA binding sites in the 45S pre-rRNA, we used the RNAhybrid program and 18- and 22-nt sequences from the 3′ end of LeuCAG-tRNA. The resulting five putative binding sites were positioned in the 5′ETS, 1 site in ITS1, 1 site in ITS2, 3 sites in 28S rRNA, and 1 site in the 3′ETS. The putative LeuCAG3′tsRNA binding site is indicated as a black bar. The putative binding site of anti-Leu3′tsLNA is indicated as a red bar. b, LeuCAG3′tsRNA and anti-Leu3′tsLNA do not bind to 45S pre-rRNA. To inhibit the interaction between LeuCAG3′tsRNA (or anti-Leu3′tsLNA) and 45S pre-rRNA, each LNA design was based on the sequence shown in (a) and transfected into HeLa cells for 24 h prior to RNA extraction and northern hybridization (n=2 independent experiments). The sequences of each LNA are listed on Supplementary Table 4. For gel source data, see Supplementary Figure 1.

Extended Data Figure 6. Inhibition of LeuCAG3′tsRNA decreases RPS28 protein level, inducing apoptosis

a, Inhibition of the LeuCAG3’tsRNA does not change the nuclear-cytoplasmic subcellular localization of RPS6 and RPS28 proteins in HeLa cells. Western blotting was performed 24 h post transfection. (n=2 independent experiments). Total, total extracts; C, cytoplasm; N, nucleus. b and c, RPS28 protein levels were down-regulated in anti-Leu3′tsLNA-treated HCC samples isolated from the orthotopic PDX. b, Total protein extracts from tumors (Extended Data Fig 2i) were subjected to western blotting (n=4 independent experiments). c, Quantification of the RPS28 protein level (n=4 independent experiments). d, A decrease in RPS28 protein level induces apoptosis in HeLa cells. Western blotting was performed 24 h post transfection of indicated siRNA (n=2 independent experiments). GAPDH, the loading control. e, RPS28 overexpression in HeLa cells for Fig. 4c and Extended Data Fig. 6e–h. The number below the image represents the relative RPS28 protein level normalized to GAPDH (n=2 independent experiments). f–h, Overexpression of RPS28 restores 18S rRNA processing. After co-transfection with the indicated LNAs and plasmids in HeLa cells for 24 h, northern blots using probes complementary to the 18S rRNA precursor, 18S and 28S rRNA as in (Fig. 3e) are shown. f, A representative northern result (n=3 independent experiments). g, Relative abundance of 30S pre-rRNA (n=3 independent experiments). h, Relative abundance of 18S rRNA normalized to 28S rRNA. Each value is normalized to that of con-EGFP transfected cells, which was set at 100 (n=3 independent experiments). i, RPS28 mRNA levels were unchanged in anti-Leu3′tsLNA treated HCC samples isolated from an orthotopic patient derived xenograft. RT- PCR was performed with total RNA from tumors (Fig. 2d). Each mRNA level was normalized to GAPDH mRNA (con and saline groups, n=4; Anti-Leu3′ts LNA group, n=5). Mean is indicated. Error bar, s.d.; indicated p-value by two-tailed t-test (c, g, h). For gel source data, see Supplementary Figure 1.

Extended Data Figure 7. Inhibition of LeuCAG3′tsRNA specifically alters sedimentation of RPS28 mRNA

Total RNA from each sucrose gradient fraction in Fig. 3a was extracted (left), and a northern analysis performed. The indicated bp provided to the left of the labeled gene name indicates the size of the coding sequences. The polysome profile is the same as shown in Fig. 3a. Relative distribution of mRNA populations across the gradient (right). Each amount of the specific mRNA for each gradient fraction was normalized using the sum of the mRNA signal across all gradient fractions. X-axis is the gradient fraction number; y-axis is % of mRNA abundance (n=2 independent experiments). For gel source data, see Supplementary Figure 1.

Extended Data Figure 8. LeuCAG3′tsRNA regulates RPS28 mRNA translation

a, LeuCAG3′tsRNA is associated with the RPS28 mRNA. RPS28 or GAPDH mRNAs were pulled down with tiling oligos. The enrichment of each mRNA was measured by RT-PCR (left). The associated LeuCAG3′tsRNA was detected by northern hybridization (middle). The relative percentage of associated LeuCAG3′tsRNA is shown. The LeuCAG3′tsRNA was enriched 26-times (right) in the RPS28 versus GAPDH mRNA pulldown after normalization (left). b, The two putative LeuCAG3′tsRNA binding sites in the RPS28 mRNA. Target-site 1 (nt 255 to 279) in the 3′ UTR. Target-site 2 in the coding sequence (CDS) has two possible predicted configurations (target 2a (nt 108–134) and target2b (nt 117–134). The target2 mutant (labeled C in Fig. 5a and Extended Data Fig. 8e) alters both predicted target 2a and 2b conformations. c, Proposed model of translational regulation. The 3′tsRNA binds to the RPS28 mRNA and disrupts the secondary structure resulting in enhanced mRNA translation. Protein X, unknown protein(s). d, RPS28 protein levels are affected by the LeuCAG3′tsRNA concentration when the target sites remain unchanged. A representative western result after co-transfection of LNAs (con and Anti-Leu3′ts) and the RPS28 mutant plasmids. The relative RPS28 protein level was calculated after normalization to GAPDH (Fig. 5b). Con, control; Anti, anti-Leu3′tsLNA; wt, wild-type construct; other mutant constructs (Fig. 5a and Supplementary Table 8). e, Schematic of C-terminal flag-tagged RPS28 secondary structure with putative LeuCAG3′tsRNA binding sites. Red, the altered sequences in each mutant; blue, the putative LeuCAG3′tsRNA binding sites in the RPS28 mRNA; black and grey, the coding and non-protein coding sequences, respectively. Black bold, C-terminal flag tag sequences. f, The C-terminal flag-tagged RPS28 protein level is affected by LeuCAG3′tsRNA concentrations when the target sites remain unchanged. A representative western result after co-transfection of LNAs (con and Anti-Leu3′ts) and plasmids (flag-RPS28 and flag-EGFP). The relative RPS28 signal is shown in Fig. 5b and calculated after normalization to co-transfected flag tagged EGFP. g, Uncapped firefly luciferase mRNA was translated in rabbit reticulocyte lysate (RRL) with the indicated amounts of synthetic LeuCAG3′tsRNA (Leu). h, Synthetic LeuCAG3′tsRNA increases RPS28 mRNA translation in vitro. Xef1 (control) and RPS28 mRNAs were translated with a synthetic LeuCAG3′tsRNA in vitro. mock, no mRNA; (-), no control and LeuCAG3′tsRNA; con1, con2, and con3, three different control RNA. i, The relative RPS28 translation product was normalized to Xef1 from (h). j, Leu3′tsRNA does not affect translation of RPS28 in vitro when the putative binding sites are altered. Xef1 and RPS28 wt or mutant mRNAs were translated in RRL. The normalized RPS28 protein level is shown in Fig. 5c. k, RPS28 target2 mutant C translation was enhanced by a compensatory tsRNA mimic (tsRNA(comp)) nearly complementary to sequence-modified target2 site sequences from mutant C. Xef1 and RPS28 wt or mutant C mRNAs were translated with the compensatory tsRNA mimic (tsRNA(comp)). Normalized quantification of the RPS28 translation products is shown in Fig. 5d. Each western and IVT figure was cropped from a single image (gel source data, Supplementary Fig. 1). Mean is indicated. Error bar, s.d.; p-value by two-tailed t-test (a, i). For a,d,f,g-j (n=3) and k (n=4) independent experiments, respectively.

Extended Data Figure 9. Double-strandedness of LeuCAG3′tsRNA target sites

a, Schematic prediction of LeuCAG3′tsRNA binding sites. The target sites of the LeuCAG3′tsRNA in the coding sequences (CDSs) and flanking 30 bp of each mRNA were predicted using RNAhybrid based on the m.f.e. Secondary structures of target sites that were predicted to have a binding site based on the low m.f.e. were analyzed by icSHAPE. b, c Thermodynamics of the putative LeuCAG3′tsRNA binding sites in the RPS15 (b), RPS9 and RPS14 (c) mRNAs. Indicated numbers on each diagram represents the 5′ end and 3′ end position on each mRNA, respectively. d, The icSHAPE data track of LeuCAG3′tsRNA binding sites and 20 nt of flanking regions contained within the RPS28, RPS15, RPS9, and RPS14 mRNAs. The icSHAPE data are scaled from 0 (no reactivity; double-strandedness) to 1 (maximum reactivity; single-strandedness). Red box represents a target site. The complete icSHAPE data for each mRNA are in Supplementary Table 10.

Extended Data Figure 10. The tsRNA affects RPS15 but not RPS9 and RPS14 protein levels

a, LeuCAG3′tsRNA inhibition decreases the RPS15 protein concentration. Protein levels were determined by western blot (n=3 independent experiments). The number under the image is the relative RPS15 protein level (anti-Leu3′tsLNA) normalized to control (con). b, RPS15 protein levels were down-regulated in anti-Leu3′tsLNA-treated HCC orthotopic PDX. Western blotting was performed with PDX tumors (Extended Data Fig 2i) (n=4 independent mice). c, Quantification of the RPS15 protein level from (b) (n=4 independent mice). d, Inhibition of LeuCAG3′tsRNA does not alter RPS15 mRNA levels. RT-PCR was performed (n=3 independent experiments). e, RPS15 mRNA levels were unchanged in anti-Leu3′tsLNA treated HCC orthotopic PDX. RT-PCR was performed with total RNA from tumors (Extended Data Fig. 2i) (n=4 independent mice). f, g, The RPS9 and RPS14 wt and target site mutant protein levels are not affected by LeuCAG3′tsRNA concentrations. A representative western result after co-transfection of LNAs (con and Anti-Leu3′ts) and RPS9 wt or target mutant plasmids (f), and RPS14 wt or target mutant plasmids (g) (each n=2 independent experiments). Target, modified target site mutant. h, The RPS15 protein level is affected by LeuCAG3′tsRNA concentrations when the target site is not altered. Western blotting was performed as in (f) (n=4 independent experiments). Non target, modified non-target site mutant. i, The normalized RPS15 protein level from (h) was calculated as in Extended Data Fig. 8d (wt and target group, n=6; non-target group, n=4 independent experiments). j, Leu3′tsRNA does not affect translation of RPS15 mRNA in vitro when the target site is altered. Xef1 and RPS15 wt or mutant mRNAs were translated in RRL as in Fig. 5c (n=4 independent experiments). k, The normalized RPS15 protein level from (j) (n=4 independent experiments). Each gel figure was cropped from a single image. Normalization of RT-PCR result is described in the Methods. Mean is indicated. Error bar, s.d.; indicated p-value by two-tailed t-test (c, i, k). For gel source data, see Supplementary Figure 1. The mutant constructs are listed in Supplementary Table 8.

Figure 1. LeuCAG3′tsRNA is required for cell viability

a, b, Inhibition of the LeuCAG3′tsRNA impairs HeLa cell viability. 3 days post-transfection, a MTS assay was performed (Anti-Leu3′ts(LNA), n=4; others, n=3 independent experiments). The indicated mixmer LNA is perfectly complementary to the darkened portion of the tRNA above the bar graph. Different asterisk marks in (b) depict different 2-nt mismatches; con, control mixmer LNA. c, Inhibition of the LeuCAG3′tsRNA does not affect the function of the mature LeuCAG-tRNA. A Luciferase assay was performed 24 h after co-transfection of the designated LNA and luciferase plasmid. The CUG plasmid has the unmodified Renilla and firefly luciferase gene. The CUC/CUU plasmid contains unmodified firefly and modified Renilla gene where 13 CUG codons were replaced with CUC or CUU codons (n=3 independent experiments) (Supplementary Table 1). Normalization is described in the Methods. d, The 22-nt synthetic LeuCAG3′tsRNA enhances cell viability. The MTS assay was performed as in (a) (27-nt 3′ end of LeuCAG-tRNA, n=6; others, n=3 independent experiments). Con1 and con2, different scrambled sequences; 22-nt LeuCAG3′tsRNA and 27-nt 3′ end of LeuCAG-tRNA sequences, 22- and 27-nt sequences from the 3′ end of mature LeuCAG-tRNA. Mean is indicated. Error bar, s.d.; indicated p-value by two-tailed t-test (a, b, c, d).

Figure 2. Inhibition of LeuCAG3′tsRNA induces apoptosis and inhibits the growth of hepatocellular carcinoma (HCC) patient-derived xenograft

a, Apoptotic HeLa cell populations were determined using Annexin V-FITC and PI staining every 24 h post-transfection (each at 1 day, n=3; Anti-Leu3′tsLNA at 3 day, n=5; others, n=4 biological replicates) (Extended Data Fig. 2a). b, TUNEL assay in HeLa cells, 24 h post transfection (n=2 independent experiments). DNase I, positive control. DAPI staining, nuclei. c, Patient-derived orthotopic HCC tumor volume. Administration of the Anti-Leu3′tsLNA or control materials (saline or con LNA) were performed by IP injections. After the 4-week injection period, all mice were sacrificed and tumors harvested (Extended Data Fig. 2i) and measured (saline, n=8; con, n=9; Anti-Leu3′ts, n=10 independent tumors). d, Representative TUNEL assay. Apoptotic cells are stained with a brown-colored nucleus (n=2 independent experiments). Mean is indicated. Error bar, s.d.; indicated p-value by two-tailed t-test (a, d); p-value (a), apoptosis population.

Figure 3. LeuCAG3′tsRNA is required for ribosome biogenesis

a, b, Inhibition of LeuCAG3′tsRNA decreases the amount of 40S ribosomal subunits. (a) Polysome profiles (n=3 independent experiments). (b) Ribosomal subunit profiles after puromycin-mediated dissociation (n=3 independent experiments). c, Inhibition of LeuCAG3′tsRNA decreases steady-state levels of 18S rRNA. Northern hybridization was performed in HeLa cells 24 h post-transfection (n=3 independent experiments). d, Inhibition of LeuCAG3′tsRNA suppressed removal of 5′ETS during 18S rRNA biogenesis. Northern hybridization was performed as in (c). Intermediate forms of mature 18S rRNA (30S, 21S, and 18S-E pre-rRNA) and mature 28S and 5.8S rRNAs (32S and 12S) were detected by the ITS1 and ITS2 probes, respectively. 30S pre-rRNA is also detected by the 5′ETS1 probe (n=3 independent experiments). The rRNA processing pathway is described in Extended Data Fig. 4b. Each normalized pre-rRNA level is in Supplementary Table 3. For gel source data, see Supplementary Figure 1.

Figure 4. Inhibition of LeuCAG3′tsRNA down-regulates RPS28 mRNA translation

a, Western blotting of RPs from LeuCAG3′tsRNA-inhibited HeLa cells (n=3 independent experiments). GAPDH, loading control. The number below each image, relative protein level from Anti-Leu3′tsLNA-treated cells normalized to control. Con, control; Anti, Anti-Leu3′tsLNA. b, Decreased RPS28 protein concentrations impair HeLa cell viability, determined as in Fig. 1a (n=4 independent experiments). 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 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, the number of ribosomes associated with mRNA based on a gradient profile (Fig. 3a). Mean is indicated. Error bar, s.d.; indicated p-value by two-tailed t-test (b–e). For gel source data, see Supplementary Figure 1.

Figure 5. LeuCAG3′tsRNA is required for efficient translation of RPS28 through base-pairing with its mRNA

a, Schematic of the RPS28 mRNA secondary structure predicted by RNAfold. Red, modified nucleotide. Blue, potential binding sites of the LeuCAG3′tsRNA (Extended Data Fig. 8b). Black and grey, coding and untranslated sequences, respectively. 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). c, The Leu3′tsRNA does not affect translation of RPS28 mRNA in vitro when the potential binding sites are modified. Xef1 and RPS28 wt or mutant mRNAs were translated in RRL (Extended Data Fig. 8j) (n=3 independent experiments). (-), absence of control and LeuCAG3′tsRNA. d, RPS28 target2 mutant C translation was enhanced by a compensatory tsRNA mimic (tsRNA(comp)) complementary to the altered target2 sequence. In vitro translation was performed as in (c) (Extended Data Fig. 8k) (n=4 independent experiments). Relative in vitro translated RPS28 level was normalized to the Xef1 level (c, d). Mean is indicated. Error bar, s.d.; indicated p-value by two-tailed t-test (b–d).