MicroRNAs (miRNAs) are endogenous ∼23 nt RNAs that play important gene-regulatory roles in animals and plants by pairing to the mRNAs of protein-coding genes to direct their posttranscriptional repression. This review outlines the current understanding of miRNA target recognition in animals and discusses the widespread impact of miRNAs on both the expression and evolution of protein-coding genes.

MicroRNAs: Target Recognition and Regulatory Functions

The mTOR Complex 1 (mTORC1) protein kinase promotes growth and is the target of rapamycin, a clinically useful drug that also prolongs lifespan in model organisms A persistent mystery is why the phosphorylation of many bona fide mTORC1 substrates is resistant to rapamycin We find that the in vitro kinase activity of mTORC1 toward peptides encompassing established phosphorylation sites varies widely and correlates strongly with the resistance of the sites to rapamycin as well as to nutrient and growth factor starvation within cells Slight modifications of the sites were sufficient to alter mTORC1 activity toward them in vitro and to cause concomitant changes within cells in their sensitivity to rapamycin and starvation Thus, the intrinsic capacity of a phosphorylation site to serve as an mTORC1 substrate, a property we call substrate quality, is a major determinant of its sensitivity to modulators of the pathway Our results reveal a mechanism through which mTORC1 effectors can respond differentially to the same signals

mTORC1 Phosphorylation Sites Encode Their Sensitivity to Starvation and Rapamycin

Deep analysis of next-generation sequencing data unveils numerous small non-coding RNAs with distinct functions. Recently, fragments derived from tRNA, named as tRNA-derived small RNA (tsRNA), have attracted broad attention. There are mainly two types of tsRNAs, including tRNA-derived stress-induced RNA (tiRNA) and tRNA-derived fragment (tRF), which differ in the cleavage position of the precursor or mature tRNA transcript. Emerging evidence has shown that tsRNAs are not merely tRNA degradation debris but have been recognized to play regulatory roles in many specific physiological and pathological processes. In this review, we summarize the biogeneses of various tsRNAs, present the emerging concepts regarding functions and mechanisms of action of tsRNAs, highlight the potential application of tsRNAs in human diseases, and put forward the current problems and future research directions.

tRNA-Derived Small RNA: A Novel Regulatory Small Non-Coding RNA

MicroRNAs (miRNAs) are endogenous, small non-coding RNAs known to regulate expression of protein-coding genes. A large proportion of miRNAs are highly conserved, localized as clusters in the genome, transcribed together from physically adjacent miRNAs and show similar expression profiles. Since a single miRNA can target multiple genes and miRNA clusters contain multiple miRNAs, it is important to understand their regulation, effects and various biological functions. Like protein-coding genes, miRNA clusters are also regulated by genetic and epigenetic events. These clusters can potentially regulate every aspect of cellular function including growth, proliferation, differentiation, development, metabolism, infection, immunity, cell death, organellar biogenesis, messenger signalling, DNA repair and self-renewal, among others. Dysregulation of miRNA clusters leading to altered biological functions is key to the pathogenesis of many diseases including carcinogenesis. Here, we review recent advances in miRNA cluster research and discuss their regulation and biological functions in pathological conditions.

Clustered miRNAs and their role in biological functions and diseases

Traditional annotation of protein-encoding genes relied on assumptions, such as one open reading frame (ORF) encodes one protein and minimal lengths for translated proteins. With the serendipitous discoveries of translated ORFs encoded upstream and downstream of annotated ORFs, from alternative start sites nested within annotated ORFs and from RNAs previously considered noncoding, it is becoming clear that these initial assumptions are incorrect. The findings have led to the realization that genetic information is more densely coded and that the proteome is more complex than previously anticipated. As such, interest in the identification and characterization of the previously ignored 'dark proteome' is increasing, though we note that research in eukaryotes and bacteria has largely progressed in isolation. To bridge this gap and illustrate exciting findings emerging from studies of the dark proteome, we highlight recent advances in both eukaryotic and bacterial cells. We discuss progress in the detection of alternative ORFs as well as in the understanding of functions and the regulation of their expression and posit questions for future work.

Alternative ORFs and small ORFs: shedding light on the dark proteome.

MicroRNAs (miRNAs) are endogenously expressed small noncoding RNAs. The genomic locations of animal miRNAs are significantly clustered in discrete loci. We found duplication and de novo formation were important mechanisms to create miRNA clusters and the clustered miRNAs tend to be evolutionarily conserved. We proposed a "functional co-adaptation" model to explain how clustering helps newly emerged miRNAs survive and develop functions. We presented evidence that abundance of miRNAs in the same clusters were highly correlated and those miRNAs exerted cooperative repressive effects on target genes in human tissues. By transfecting miRNAs into human and fly cells and extensively profiling the transcriptome alteration with deep-sequencing, we further demonstrated the functional co-adaptation between new and old miRNAs in the miR-17-92 cluster. Our population genomic analysis suggest that positive Darwinian selection might be the driving force underlying the formation and evolution of miRNA clustering. Our model provided novel insights into mechanisms and evolutionary significance of miRNA clustering.

/pdf/micrornas-in-the-same-clusters-evolve-to-coordinately-wk29v3mo19.pdf

microRNAs in the Same Clusters Evolve to Coordinately Regulate Functionally Related Genes

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.

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

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.

/pdf/genome-wide-maps-of-ribosomal-occupancy-provide-insights-3gi52oqnmm.pdf

Genome-wide maps of ribosomal occupancy provide insights into adaptive evolution and regulatory roles of uORFs during Drosophila development

The repertoire of miRNAs has considerably expanded during metazoan evolution, and duplication is an important mechanism for generating new functional miRNAs. However, relatively little is known about the functional divergence between paralogous miRNAs and the possible coevolution between duplicated miRNAs and the genomic contexts. By systematically examining small RNA expression profiles across various human tissues and interrogating the publicly available miRNA:mRNA pairing chimeras, we found that changes in expression patterns and targeting preferences are widespread for duplicated miRNAs in vertebrates. Both the empirical interactions and target predictions suggest that evolutionarily conserved homo-seed duplicated miRNAs pair with significantly higher numbers of target sites compared to the single-copy miRNAs. Our birth-and-death evolutionary analysis revealed that the new target sites of miRNAs experienced frequent gains and losses during function development. Our results suggest that a newly emerged target site has a higher probability to be functional and maintained by natural selection if it is paired to a seed shared by multiple paralogous miRNAs rather than being paired to a single-copy miRNA. We experimentally verified the divergence in target repression between two paralogous miRNAs by transfecting let-7a and let-7b mimics into kidney-derived cell lines of four mammalian species and measuring the resulting transcriptome alterations by extensive high-throughput sequencing. Our results also suggest that the gains and losses of let-7 target sites might be associated with the evolution of repressiveness of let-7 across mammalian species.

/pdf/microrna-duplication-accelerates-the-recruitment-of-new-25xa3wlijp.pdf

Junjie Luo

Papers

microRNAs in the Same Clusters Evolve to Coordinately Regulate Functionally Related Genes

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

Genome-wide maps of ribosomal occupancy provide insights into adaptive evolution and regulatory roles of uORFs during Drosophila development

MicroRNA duplication accelerates the recruitment of new targets during vertebrate evolution.