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.

Upstream open reading frames (uORFs) play important roles in regulating the main coding DNA sequences (CDSs) via translational repression. Despite their prevalence in the genomes, uORFs are overall discriminated against by natural selection. However, it remains unclear why in the genomes there are so many uORFs more conserved than expected under the assumption of neutral evolution. Here, we generated genome-wide maps of translational efficiency (TE) at the codon level throughout the life cycle of Drosophila melanogaster. We identified 35,735 uORFs that were expressed, and 32,224 (90.2%) of them showed evidence of ribosome occupancy during Drosophila development. The ribosome occupancy of uORFs is determined by genomic features, such as optimized sequence contexts around their start codons, a shorter distance to CDSs, and higher coding potentials. Our population genomic analysis suggests the segregating mutations that create or disrupt uORFs are overall deleterious in D. melanogaster. However, we found for the first time that many (68.3% of) newly fixed uORFs that are associated with ribosomes in D. melanogaster are driven by positive Darwinian selection. Our findings also suggest that uORFs play a vital role in controlling the translational program in Drosophila. Moreover, we found that many uORFs are transcribed or translated in a developmental stage-, sex-, or tissue-specific manner, suggesting that selective transcription or translation of uORFs could potentially modulate the TE of the downstream CDSs during Drosophila development. Upstream open reading frames (uORFs) in the 5′ untranslated regions (UTRs) of messenger RNAs can potentially inhibit translation of the downstream regions that encode proteins by sequestering protein-making machinery the ribosome. Moreover, mutations that destroy existing uORFs or create new ones are known to cause human disease. Although mutations that create new uORFs are generally deleterious and are selected against, many uORFs are evolutionarily conserved across eukaryotic species. To resolve this dilemma, we used extensive mRNA-Seq and ribosome profiling to generate high-resolution genome-wide maps of ribosome occupancy and translational efficiency (TE) during the life cycle of the fruit fly D. melanogaster. This allowed us to identify the sequence features of uORFs that influence their ability to associate with ribosomes. We demonstrate for the first time that the majority of the newly fixed uORFs in D. melanogaster, especially the translated ones, are under positive Darwinian selection. We also show that uORFs exert widespread repressive effects on the translation of the downstream protein-coding region. We find that many uORFs are transcribed or translated in a developmental stage-, sex-, or tissue-specific manner. Our results suggest that during Drosophila development, changes in the TE of uORFs, as well as the inclusion/exclusion of uORFs, are frequently exploited to inversely influence the translation of the downstream protein-coding regions. Our study provides novel insights into the molecular mechanisms and functional consequences of uORF-mediated regulation.

Morphogen gradients, which provide positional information to cells in a developing tissue, could in principle adopt any nonuniform profile. To our knowledge, how the profile of a morphogen gradient affects positional precision has not been well studied experimentally. Here, we compare the positional precision provided by the Drosophila morphogenetic protein Bicoid (Bcd) in wild-type (wt) embryos with embryos lacking an interacting cofactor. The Bcd gradient in the latter case exhibits decreased positional precision around mid-embryo compared with its wt counterpart. The domain boundary of Hunchback (Hb), a target activated by Bcd, becomes more variable in mutant embryos. By considering embryo-to-embryo, internal, and measurement fluctuations, we dissect mathematically the relevant sources of fluctuations that contribute to the error in positional information. Using this approach, we show that the defect in Hb boundary positioning in mutant embryos is directly reflective of an altered Bcd gradient profile with increasing flatness toward mid-embryo. Furthermore, we find that noise in the Bcd input signal is dominated by internal fluctuations but, due to time and spatial averaging, the spatial precision of the Hb boundary is primarily affected by embryo-to-embryo variations. Our results demonstrate that the positional information provided by the wt Bcd gradient profile is highly precise and necessary for patterning precision.

Morphogen molecules form concentration gradients to provide spatial information to cells in a developing embryo. Precisely how cells decode such information to form patterns with sharp boundaries remains an open question. For example, it remains controversial whether the Drosophila morphogenetic protein Bicoid (Bcd) plays a transient or sustained role in activating its target genes to establish sharp expression boundaries during development. In this study, we describe a method to simultaneously detect Bcd and the nascent transcripts of its target genes in developing embryos. This method allows us to investigate the relationship between Bcd and the transcriptional status of individual copies of its target genes on distinct scales. We show that, on three scales analyzed concurrently—embryonic, nuclear and local, the actively-transcribing gene copies are associated with high Bcd concentrations. These results underscore the importance of Bcd as a sustained input for transcriptional decisions of individual copies of its target genes during development. We also show that the Bcd-dependent transcriptional decisions have a significantly higher noise than Bcd-dependent gene products, suggesting that, consistent with theoretical studies, time and/or space averaging reduces the noise of Bcd-activated transcriptional output. Finally, our analysis of an X-linked Bcd target gene reveals that Bcd-dependent transcription bursts at twice the frequency in males as in females, providing a mechanism for dosage compensation in early Drosophila embryos. Our study represents a first experimental uncovering of the actions of Bcd in controlling the actual transcriptional events while its positional information is decoded during development. It establishes a sustained role of Bcd in transcriptional decisions of individual copies of its target genes to generate sharp expression boundaries. It also provides an experimental evaluation of the effect of time and/or space averaging on Bcd-dependent transcriptional output, and establishes a dosage compensation mechanism in early Drosophila embryos.