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)

Global Analyses of the Effect of Different Cellular Contexts on MicroRNA Targeting

MicroRNA (miRNA) regulation clearly impacts animal development, but the extent to which development—with its resulting diversity of cellular contexts—impacts miRNA regulation is unclear. Here, we compared cohorts of genes repressed by the same miRNAs in different cell lines and tissues and found that target repertoires were largely unaffected, with secondary effects explaining most of the differential responses detected. Outliers resulting from differential direct targeting were often attributable to alternative 3′ UTR isoform usage that modulated the presence of miRNA sites. More inclusive examination of alternative 3′ UTR isoforms revealed that they influence ~10% of predicted targets when comparing any two cell types. Indeed, considering alternative 3′ UTR isoform usage improved prediction of targeting efficacy significantly beyond the improvements observed when considering constitutive isoform usage. Thus, although miRNA targeting is remarkably consistent in different cell types, considering the 3′ UTR landscape helps predict targeting efficacy and explain differential regulation that is observed.Korea (South). Ministry of Education, Science and Technology (MEST) (National Research Foundation of Korea. NRF-2013R1A1A1010185)National Institutes of Health (U.S.) (Grant RO1 GM067031)National Institutes of Health (U.S.) (Grant K99 GM102319)National Science Foundation (U.S.). Graduate Research Fellowship Progra

High-throughput sequencing of RNA 5'- and 3'-termini yields insights into viral and vertebrate gene expression

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2015.Cataloged from PDF version of thesis.Includes bibliographical references.Next-generation sequencing techniques are unparalleled in their resolution and dynamic range, but are limited by read depletion at transcript ends. Protocols that specifically target these ends overcome this limitation and enable the study of biological phenomena that would otherwise prove refractory to RNA-Seq. Here, we use two such techniques to study heterogeneous sequences at the 5' ends of influenza transcripts and alternative polyadenylation at the 3' ends of vertebrate transcripts. The 5' ends of influenza mRNAs include heterogeneous sequences derived from host RNAs. In a process termed cap snatching, the viral polymerase cleaves host RNAs ~10-13 nucleotides downstream of their caps and uses the resulting fragments to prime viral transcription. High-throughput 5' rapid amplification of cDNA ends resulted in 54 million chimeric reads containing host-derived leaders. These sequences provided evidence for stuttering during transcription initiation and an influence of the viral template on the extent of realignment. Accounting for realignment suggested a common preference by the polymerase irrespective of the viral template, and suggested that a single base pair is sufficient to prime transcription. Mapping trimmed leaders to annotated transcription start sites (TSSs) revealed that the most abundant leaders correspond to small nuclear RNAs, consistent with cap snatching of nascent transcripts. The 3' ends of mRNAs are generally appended with a poly(A) tail, but alternative polyadenylation sites may vary depending on cellular context. 3P-Seq is a method that specifically captures alternative polyadenylation sites without relying on oligo(dT) priming, which may cause artifacts. Applying 3P-Seq to eukaryotic model organisms improved their gene annotations and provided insight into targeting by microRNAs, a class of ~21-23 nucleotide RNAs that mediate mRNA destabilization. The isoform ratios of transcripts containing miR-155 sites were predictive of the extent to which these transcripts would respond to miR-155 transfection. Conversely, knocking out miR-22 in mice specifically upregulated isoforms containing miR-22 sites, suggesting that microRNAs reciprocally affect the 3'-UTR landscape. Lastly, analysis of other datasets derived from zebrafish embryos revealed broad lengthening of 3'-UTR isoforms during development and noncanonical polyadenylation during the maternal-to-zygotic transition.by David N.P. Koppstein.Ph. D