Drosophila tsRNAs preferentially suppress general translation machinery via antisense pairing and participate in cellular starvation response

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.

5’ tRNA halves are highly expressed in the primate hippocampus and sequence-specifically regulate gene expression

“…This demonstrates that a Argonaut-dependent regulation mechanism as proposed for tRFs previously (Haussecker et al 2010;Kuscu et al 2018) cannot fully explain the observed changes in gene expression. However, our results are in line with other studies identifying miRNA-untypical targeting (Deng et al 2015;Luo et al 2018). The mechanism seems to act at the post-transcriptional level, as transcripts with exon-junction spanning k-mer alignments were similarly often downregulated compared to transcripts with intra-exonic k-mer alignment (Supplemental_Fig_S3).…”

“…Indeed, studies could show that tRF-mediated transcript silencing is dependent on a 5' seed, which is complementary to target sites within the 3' UTR (Haussecker et al 2010;Kuscu et al 2018). Contrasting this, other studies observed sequence-specific gene silencing effects, where complementarity of the 5' seed was neglectable in favor for other tRF portions (Deng et al 2015;Luo et al 2018). Since Argonaut structure coerces the 5' end of small RNAs for target recognition (Boland et al 2011), the results of these studies suggest that tRFs interact with other effector proteins or act protein-independent to regulate genes.…”

“…Given the similar target regulation pattern, we suggest that 5' tRNA-fragments can regulate their targets via conserved mechanisms across different species, while the sequence stretch being most important for target regulation can vary for fragments from different parental tRNAs. Figure 7: Target pattern analysis of published RNA sequencing data of fly S2 cells, where the 20 nt long 5' tRF Glu-CTC, 5' tRFs Asp-GTC or Lys-TTT was overexpressed by tRF mimic transfection (Luo et al 2018). Plotted is the percentage of genes with (blue) or without (red) k-mer alignment that get downregulated.…”

“…So far, two superordinate concepts that aim explain how tRFs regulate gene expression have been identified. While some studies show that tRFs globally repress translation by inhibiting the assembly of the translation initiation machinery (Gebetsberger et al 2017;Guzzi et al 2018;Ivanov et al 2011;Keam et al 2017), others report a sequence-specific gene regulation (Deng et al 2015;Haussecker et al 2010;Kuscu et al 2018;Luo et al 2018). As tRFs were found to associate with Argonaut proteins, a miRNA-like gene regulation mechanism seems apparent (Kumar et al 2014;Kuscu et al 2018;Maute et al 2013).…”

Translational Control under Stress: Reshaping the Translatome

“…[110,111] Other data suggest that these tRNA fragments may be involved in translational regulation of certain transcripts by targeting their translation in a sequence-independent manner, although details are largely unknown. [112,113] Interestingly, in humans, fragments derived from tRNA Leu stimulate the expression of RPS28 in a sequence-dependent manner, [114] in contrast to many other examples where tRFs work as inhibitors of translation.…”