Poly(A)-tail profiling reveals an embryonic switch in translational control

Poly(A) tails enhance the stability and translation of most eukaryotic messenger RNAs, but difficulties in globally measuring poly(A)-tail lengths have impeded greater understanding of poly(A)-tail function. Here we describe poly(A)-tail length profiling by sequencing (PAL-seq) and apply it to measure tail lengths of millions of individual RNAs isolated from yeasts, cell lines, Arabidopsis thaliana leaves, mouse liver, and zebrafish and frog embryos. Poly(A)-tail lengths were conserved between orthologous mRNAs, with mRNAs encoding ribosomal proteins and other ‘housekeeping’ proteins tending to have shorter tails. As expected, tail lengths were coupled to translational efficiencies in early zebrafish and frog embryos. However, this strong coupling diminished at gastrulation and was absent in non-embryonic samples, indicating a rapid developmental switch in the nature of translational control. This switch complements an earlier switch to zygotic transcriptional control and explains why the predominant effect of microRNA-mediated deadenylation concurrently shifts from translational repression to mRNA destabilization.National Institutes of Health (U.S.) (Grant GM067031)National Institutes of Health (U.S.) (Medical Scientist Training Program Fellowship T32GM007753

Extensive alternative polyadenylation during zebrafish development

The post-transcriptional fate of messenger RNAs (mRNAs) is largely dictated by their 3′ untranslated regions (3′ UTRs), which are defined by cleavage and polyadenylation (CPA) of pre-mRNAs. We used poly(A)-position profiling by sequencing (3P-seq) to map poly(A) sites at eight developmental stages and tissues in the zebrafish. Analysis of over 60 million 3P-seq reads substantially increased and improved existing 3′ UTR annotations, resulting in confidently identified 3′ UTRs for >79% of the annotated protein-coding genes in zebrafish. mRNAs from most zebrafish genes undergo alternative CPA, with those from more than a thousand genes using different dominant 3′ UTRs at different stages. These included one of the poly(A) polymerase genes, for which alternative CPA reinforces its repression in the ovary. 3′ UTRs tend to be shortest in the ovaries and longest in the brain. Isoforms with some of the shortest 3′ UTRs are highly expressed in the ovary, yet absent in the maternally contributed RNAs of the embryo, perhaps because their 3′ UTRs are too short to accommodate a uridine-rich motif required for stability of the maternal mRNA. At 2 h post-fertilization, thousands of unique poly(A) sites appear at locations lacking a typical polyadenylation signal, which suggests a wave of widespread cytoplasmic polyadenylation of mRNA degradation intermediates. Our insights into the identities, formation, and evolution of zebrafish 3′ UTRs provide a resource for studying gene regulation during vertebrate development.National Institutes of Health (U.S.) (Grant GM067031)

The influence of microRNAs and poly(A) tail length on endogenous mRNA–protein complexes

Background: All mRNAs are bound in vivo by proteins to form mRNA-protein complexes (mRNPs), but changes in the composition of mRNPs during posttranscriptional regulation remain largely unexplored. Here, we have analyzed, on a transcriptome-wide scale, how microRNA-mediated repression modulates the associations of the core mRNP components eIF4E, eIF4G, and PABP and of the decay factor DDX6 in human cells. Results: Despite the transient nature of repressed intermediates, we detect significant changes in mRNP composition, marked by dissociation of eIF4G and PABP, and by recruitment of DDX6. Furthermore, although poly(A)-tail length has been considered critical in post-transcriptional regulation, differences in steady-state tail length explain little of the variation in either PABP association or mRNP organization more generally. Instead, relative occupancy of core components correlates best with gene expression. Conclusions: These results indicate that posttranscriptional regulatory factors, such as microRNAs, influence the associations of PABP and other core factors, and do so without substantially affecting steady-state tail length.National Institutes of Health (U.S.) (Grant K99GM102319)National Institutes of Health (U.S.) (Grant T32GM007753)National Institutes of Health (U.S.) (Grant R01GM067031)National Institutes of Health (U.S.) (Grant R35GM118135)Natural Sciences and Engineering Research Council of Canada (Discovery Grant

Function and regulation of poly(A)-tail length

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, June 2014.Cataloged from PDF version of thesis. "May 2014." Vita.Includes bibliographical references.Poly(A) tails are found at the 3' ends of nearly all eukaryotic messenger RNAs (mRNAs) and long non-coding RNAs. The presence of a poly(A) tail promotes translation and inhibits decay of an mRNA, with both effects mediated through poly(A)-binding protein. However, an understanding of the relationship between the length of a poly(A) tail and these aspects of mRNA metabolism has been limited, primarily because of the lack of a technology that provides high-resolution poly(A)-tail length measurements in a global manner. This dissertation describes a new, high-throughput-sequencing-based method (PAL-seq) that measures the tails of individual mRNA molecules by coupling a fluorescence-based readout of poly(A)-tail length with sequencing of the poly(A)-proximal region. Using PAL-seq, we have found that poly(A)-tail lengths exhibit a notably poor correlation with translational efficiency (as measured by ribosome profiling) across genes in nearly all systems we have examined. In contrast, early zebrafish and Xenopus laevis embryos display a striking correlation (Spearman R > 0.6) that disappears at gastrulation. This developmental uncoupling of tail length and translational efficiency explains the different outcomes of microRNA (miRNA)-mediated poly(A)-tail shortening in zebrafish embryos before and after gastrulation, with translational repression being the predominant effect before and mRNA destabilization after. We have also observed that poly(A)-tail lengths do not correlate positively with mRNA half-lives in mammalian cells, and that miRNAs do not promote any apparent tail shortening in this setting. Since these results could be explained by differences in deadenylation rates, we performed a kinetic analysis in which we captured newly-made mRNAs of different age ranges. The deadenylation rates that we calculated after measuring tails over time correlated strongly with mRNA half-lives (Spearman R < -0.6), reinforcing the notion that tail shortening leads to mRNA downregulation. When we repeated the timecourse with prior overexpression of a miRNA, we found that miRNAmediated tail shortening was generally modest, but of a magnitude not significantly different from that expected given the accompanying decreases in mRNA stability.by Alexander O. Subtelny.Ph. D

Predicting microRNA targeting efficacy in Drosophila

Background: MicroRNAs (miRNAs) are short regulatory RNAs that derive from hairpin precursors. Important for understanding the functional roles of miRNAs is the ability to predict the messenger RNA (mRNA) targets most responsive to each miRNA. Progress towards developing quantitative models of miRNA targeting in Drosophila and other invertebrate species has lagged behind that of mammals due to the paucity of datasets measuring the effects of miRNAs on mRNA levels. Results: We acquired datasets suitable for the quantitative study of miRNA targeting in Drosophila. Analyses of these data expanded the types of regulatory sites known to be effective in flies, expanded the mRNA regions with detectable targeting to include 5′ untranslated regions, and identified features of site context that correlate with targeting efficacy in fly cells. Updated evolutionary analyses evaluated the probability of conserved targeting for each predicted site and indicated that more than a third of the Drosophila genes are preferentially conserved targets of miRNAs. Based on these results, a quantitative model was developed to predict targeting efficacy in insects. This model performed better than existing models, and it drives the most recent version, v7, of TargetScanFly. Conclusions: Our evolutionary and functional analyses expand the known scope of miRNA targeting in flies and other insects. The existence of a quantitative model that has been developed and trained using Drosophila data will provide a valuable resource for placing miRNAs into gene regulatory networks of this important experimental organism. Keywords: Non-coding RNAs, miRNA target prediction, Post-transcriptional gene regulationNational Science Foundation (U.S.). Graduate Research Fellowship ProgramNational Institutes of Health (U.S.) (T32GM007753)National Institutes of Health (U.S.) (GM067031)National Institutes of Health (U.S.) (GM118135