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, 46 (10), 5250-5268

Drosophila tsRNAs Preferentially Suppress General Translation Machinery via Antisense Pairing and Participate in Cellular Starvation Response

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Drosophila tsRNAs Preferentially Suppress General Translation Machinery via Antisense Pairing and Participate in Cellular Starvation Response

Shiqi Luo et al. Nucleic Acids Res.

Abstract

Transfer RNA-derived small RNAs (tsRNAs) are an emerging class of small RNAs, yet their regulatory roles have not been well understood. Here we studied the molecular mechanisms and consequences of tsRNA-mediated regulation in Drosophila. By analyzing 495 public small RNA libraries, we demonstrate that most tsRNAs are conserved, prevalent and abundant in Drosophila. By carrying out mRNA sequencing and ribosome profiling of S2 cells transfected with single-stranded tsRNA mimics and mocks, we show that tsRNAs recognize target mRNAs through conserved complementary sequence matching and suppress target genes by translational inhibition. The target prediction suggests that tsRNAs preferentially suppress translation of the key components of the general translation machinery, which explains how tsRNAs inhibit the global mRNA translation. Serum starvation experiments confirm tsRNAs participate in cellular starvation responses by preferential targeting the ribosomal proteins and translational initiation or elongation factors. Knock-down of AGO2 in S2 cells under normal and starved conditions reveals a dependence of the tsRNA-mediated regulation on AGO2. We also validated the repressive effects of representative tsRNAs on cellular global translation and specific targets with luciferase reporter assays. Our study suggests the tsRNA-mediated regulation might be crucial for the energy homeostasis and the metabolic adaptation in the cellular systems.

Figures

Figure 1.
Figure 1.
tsRNAs are conserved, abundant and prevalent in Drosophila. (A) Boxplots of the ratios of tsRNA reads to miRNA reads in individual developmental categories (embryos, larvae, pupae, heads, bodies, testes and ovaries, each represented by the initial uppercase letter) and S2 cells of Drosophila melanogaster, based on a total of 147 small RNA libraries that were prepared without any specific isolation step or treatments. In the boxplots throughout the manuscript, the inside bands represent the median values, the bottom and top of the boxes represent the first and third quantiles and the whiskers extend to the most extreme data points which are no more than 1.5 interquartile range. Also shown are the average ratios of tsRNA reads to all genome-mapped reads excluding rRNAs, snRNAs and snoRNAs (red line). (B) The normalized abundances of individual 20–22 nt tsRNA species (N = 1725 with RPM > 1) that were shared in D. melanogaster (x-axis) and Drosophila virilis (y-axis), measured as reads per million (RPM). (C) The percentage of short tsRNAs (20–23 nt) out of the total tsRNAs (20–29 nt) in the small RNA libraries of different developmental categories. For each developmental stage/tissue/cell-line (defined as in A), the mean and s.d. of the percentage were given. (D) The distributions of tsRNA lengths in AGO1, AGO2, AGO3, AUB and PIWI-IP libraries. The AGO1 and AGO2-IP results were extracted from two sets of studies in S2 cells (GSM280087, GSM280088; SRR768820, SRR768821); the AGO3, AUB and PIWI-IP results were extracted from two sets of studies in female ovaries (SRR060648, SRR060649, SRR060651; SRR2147101, SRR2147102, SRR2147103). The black lines represent the small RNA reads mapped to the primary miRNAs; the red lines represent the small RNA reads mapped to tRNAs; the blue lines represent the 23–29 nt small RNA reads mapped to the whole genome excluding miscRNAs, miRNAs and tRNAs. (E) The relative coverage of tsRNAs at each nucleotide position on the mature tRNAs in each library (N = 495).
Figure 2.
Figure 2.
Transfections of tsRNA mimics into S2 cells repress global translational activities. (A) Schematic representations of the structures of three tsRNAs tsRNAAspGUC (T3), tsRNAGluCUC (T6) and tsRNALysUUU (T10) in the mature tRNAs. tsRNAs are in red and the anti-codons in blue. (B) Absorbance profiles (at a UV wavelength of 254 nm) of RNAs partitioned using 10–45% sucrose gradients from S2 cells transfected without any RNA sequence (Mock, black), with a negative-control small RNA (ss-NC, orange), with tsRNA T3 (red), T6 (green) or T10 (blue). For each profile, both the absorbance and the position were normalized according to the monosome peak. Dashed lines indicate the boundaries of the monosome and polysome partitions for a measurement of the overall translational activity. (C) The ratios of the aggregate intensities within polysome partition (P) to the monosome partition (M) normalized by the median value of the control experiments. Error bars represent one standard deviation computed from three repeated experiments. Asterisks indicate significant different P/M ratios between transfection and mock: *P < 0.05; **P < 0.01; ***P < 0.001. (D) Scatter plots of the Ribo-seq read counts against the mRNA-seq read counts of individual genes in T3 transfection experiments. Blue circles represent genes that have >50 mRNA-seq reads and >50 Ribo-seq reads.
Figure 3.
Figure 3.
tsRNAs repress translation of target mRNAs via antisense pairing. (A) Cumulative distribution of changes in translational efficiency (TE) for all the genes. The x-axis is the log2(FCTE) in S2 cells transfected with the tsRNA mimic (T3, T6, T10 from left to right) versus transfection with ss-NC. The genes with 7-mer sites conserved between Drosophila melanogaster and Drosophila virilis and antisense paired to any part of a tsRNA are defined as tsRNA target genes. Red: genes with one conserved tsRNA target site, Green: genes with two conserved tsRNA target sites, Blue: genes with more than two conserved tsRNA target sites, Gray: genes without any target site of the transfected tsRNA in the entire mRNA. The number of genes in each category is indicated in parentheses. The asterisks indicate a significant difference in TE FC compared with the ss-NC control group. ***P < 0.001. (B) Repression efficiency of tsRNA is independent of the location of the tsRNA target sites in a mRNA transcript. The y-axis is the log2(FCTE) (comparing T3, T6 and T10 transfections with ss-NC from left to right). The x-axis is the location of the tsRNA mimic target sites in a mRNA transcript. ‘No sites’: mRNAs without conserved target sites for that tsRNA; ‘5′UTR’: mRNAs with conserved target sites in the 5′ UTRs, ‘CDS’: mRNAs with conserved target sites in CDSes, ‘3′UTR’: mRNAs with conserved target sites in the 3′ UTRs. The number of genes in each category is given in the parentheses. *P < 0.05; N.S. not statistically significant. (C) A scheme showing a tsRNA antisense pairing to evolutionarily conserved 7-mer target sites (in red) in different locations of a mRNA.
Figure 4.
Figure 4.
tsRNAs mediated translational repression in an AGO2-dependent manner. (A) GO analysis of the top 600 target genes of the AGO2-bound tsRNAs. The ontology related to translation is in red. (B) The target site density of AGO2-bound tsRNAs on RpS9 mRNA. The y-axis is the -log2 (reads of AGO2-bound tsRNAs) for each target site. (C) The log2(FC) for TEs in the AGO2 knock-down versus ds-NC transfected S2 cells that were cultured in normal condition. The targets of tsRNAs: mRNAs with conserved target sites of AGO2-bound tsRNAs with the density >1.5 per kb of mRNA (N = 351); ‘No sites": the remaining mRNAs expressed in S2 cells; RP/IEFs: the RPs or translational IEFs that were also targets of tsRNAs; non-RP/IEFs: the targets of tsRNAs that are not RPs or translational IEFs. The number of genes in each category is given in the parentheses. ***P < 0.001. (D) The density of tsRNA target sites in a mRNA (x-axis) is positively correlated with the extent of translational de-repression (y-axis) after AGO2 was knocked down. The top 351 targets of the AGO2-bound tsRNAs were used in this analysis. RP/IEFs were shown in red (N = 48). The Spearman’s correlation and P-value were shown.
Figure 5.
Figure 5.
tsRNAs participate in Drosophila cellular starvation response. (A) The log2 (FC) of tsRNA abundance from 5′, middle and 3′ end of each tRNA after serum starvation. Each tRNA type was denoted by the amino acid and anti-codon. (B) The changes in tsRNA abundances are different in tsRNAs from 5′, middle and 3′ ends of tRNAs. Upregulation under serum starvation is shown in blue, and downregulation under serum starvation is shown in red. The y-axis is the percent of upregulated/downregulated tsRNAs in the three types of tsRNAs. The upregulated tsRNAs were defined as tsRNAs with FC > 1.5 after serum starvation. The downregulated tsRNAs were defined as those tsRNAs with a FC < 0.66 after serum starvation. (C) The abundance of tsRNAs (RPM) in normal and starved S2 cells. tsRNA not bound by AGO1 nor AGO2 is in gray, tsRNA only bound by AGO2 is in red, tsRNA only bound by AGO1 is in black, tsRNA both bound by AGO1 and AGO2 is in orange. The tsRNA/AGO binding information was taken from previously published IP-seq results from Czech et al. (46). (D) Western blotting of AGO2 in the four different conditions of S2 cells. The four conditions of S2 cells: cultured under normal condition, serum starvation, AGO2 knockdown while serum deprived, and AGO2 knockdown. Error bars represent one standard deviation computed from two repeated experiments.
Figure 6.
Figure 6.
tsRNAs repress translation of target genes in cellular starvation response. (A) mRNAs with a higher density of target sites by the upregulated AGO2-bound tsRNAs (UpSites score, x-axis) show stronger translational repression (y-axis) under cellular starvation. The genes with UpSites score of zero (N = 2765) were grouped together, and the remaining 3746 genes were evenly divided into 38 groups based on increasing UpSites scores. The Spearman’s correlation and P-value were shown. (B) Genes targeted by the upregulated AGO2-bound tsRNAs (UpSites score > 3 per kb, N = 408) have decreased TEs in the starved S2 cells. ‘RP/IEFs’: RPs or translational IEFs; ‘backgrounds’: the remaining genes (UpSites score < 3 per kb, N = 6103) expressed in S2 cells. ***P < 0.001. (C) A significant negative correlation between FCTE (log2 scaled, y-axis) and the UpSites score (x-axis) for the 5′TOP genes under serum starvation (N = 115). (D) The change of TEs (y-axis) for the highest scoring target genes of upregulated tsRNAs in the starved S2 cells (UpSites > 3, and 5′TOP genes were not included, N = 376) under normal (left) and AGO2 knock-down condition (right panel). ***P < 0.001; N.S. not significant.
Figure 7.
Figure 7.
A model describing how tsRNAs suppress translation of specific targets and global mRNAs. (A) An overview of the experimental treatments of S2 cells and sequencing workflow in the study. (B) tsRNAs preferentially target RPs and IEFs via antisense pairing to regulate the global translational activities. (1) AGO2 bound the tsRNAs cleaved from tRNAs, and those tsRNAs specifically bind to the mRNAs with partial complementarity and inhibit their translation. (2) Translation of some RPs and IEFs is repressed by tsRNAs, which in turn suppresses the global translational activities. Under starvation, translational repression is also regulated by mTOR pathway via 5′TOP genes, and some of the tsRNA targets are overlapping with the 5′TOP genes (in green).
Figure 8.
Figure 8.
Verifying the repressive effects of tsRNAs on global translational activities and specific target sites with dual luciferase reporter assay. (A) Co-transfection of tsRNA mimics and the psiCHECK-2 plasmids into S2 cells reduced the activities of firefly luciferase. The color key shows different final concentrations of tsRNA mimics. The tsRNA cocktail was made by mixing equal amount of the nine tsRNA mimics of the same concentrations. The luciferase activity of S2 cells transfected with the tsRNA mimics were normalized by those transfected with ss-NC at the same concentration (three replicates were performed for each assay). (B) Higher concentrations of tsRNA cocktail cause lower firefly (red) and Renilla (blue) luciferase activities on the psiCHECK-2 plasmid. ***P < 0.001 (three replicates were performed for each assay). (C) The base pairing between tsRNA T16 and the predicted targeting sites in RpL8 and PpL27 mRNAs. The targeting sites conserved between Drosophila melanogaster and Drosophila virilis were underlined. The flanking 5 bp on both sides of the target sites (highlighted in cyan) were included in the synthesized targeting sites. (D and E) Relative luciferase activities in S2 cells co-transfected with T16 and the psiCHECK-2 plasmids containing the target site (RpL8, D; RpL27, E) are significantly lower than those co-transfected with ss-NC and the same plasmid. As negative controls, no significant difference in the relative luciferase activities was observed in S2 cells when the empty psiCHECK-2 plasmid (WT) was co-transfected with T16 or with ss-NC (*P < 0.05, **P < 0.01, five replicates were performed).

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