Browsing by keyword "Splicing"
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Deep sequencing of pre-translational mRNPs reveals hidden flux through evolutionarily conserved AS-NMD pathwaysDeep sequencing of mRNAs (RNA-Seq) is now the preferred method for transcriptome-wide quantification of gene expression. Yet many mRNA isoforms, such as those eliminated by nonsense-mediated decay (NMD), are inherently unstable. Thus a significant drawback of steady-state RNA-Seq is that it provides marginal information on the flux through alternative splicing pathways. Measurement of such flux necessitates capture of newly made species prior to mRNA decay. One means to capture nascent mRNAs is affinity purifying either the exon junction complex (EJC) or activated spliceosomes. Late-stage spliceosomes deposit the EJC upstream of exon-exon junctions, where it remains associated until the first round of translation. As most mRNA decay pathways are translation-dependent, these EJC- or spliceosome-associated, pre-translational mRNAs should provide an accurate record of the initial population of alternate mRNA isoforms. Previous work has analyzed the protein composition and structure of pre- translational mRNPs in detail. While in the Moore lab, my project has focused on exploring the diversity of mRNA isoforms contained within these complexes. As expected, known NMD isoforms are more highly represented in pre-translational mRNPs than in RNA-Seq libraries. To investigate whether pre-translational mRNPs contain novel mRNA isoforms, we created a bioinformatics pipeline that identified thousands of previously unannotated splicing events. Though many can be attributed to “splicing noise”, others are evolutionarily-conserved events that produce new AS-NMD isoforms likely involved in maintenance of protein homeostasis. Several of these occur in genes whose overexpression has been linked to poor cancer prognosis.
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Intron and Small RNA Localization in Mammalian NeuronsRNA molecules are diverse in form and function. They include messenger RNAs (mRNAs) that are templates for proteins, splice products such as introns that can generate functional noncoding RNAs, and a slew of smaller RNAs such as transfer RNAs (tRNAs) that help decode mRNAs into proteins. RNAs can show distinct patterns of subcellular localization that play an important role in protein localization. However, RNA distribution in cells is incompletely understood, with prior studies focusing primarily on RNAs that are long (>200 nucleotides), fully processed, and polyadenylated. We examined the distribution of RNAs in neurons. Neuronal compartments can be separated by long distances and play distinct roles, raising the possibility that RNA localization is especially overt and functionally meaningful in these cells. In our exploration, we physically dissected projections from cell bodies of neurons from the rat brain and sequenced total RNA. We describe two main findings. First, we identified excised introns that are enriched in neuronal projections and confirmed their localization by single- molecule fluorescence in situ hybridization. These are a previously unknown set of circular RNAs in neuronal projections: tailless lariats that possess a non- canonical C branchpoint. Second, we observed a highly abundant population of small (20-150 nucleotide) RNAs in neuronal projections, most of which are tRNAs. For both circular introns and tRNAs, we did not observe known RNA localization signals. Thus, many types of RNA, if sufficiently stable, appear free to diffuse to distant locations, their localization perhaps aided by the movement of large organelles in the confines of neuronal projections. Our survey of RNA molecules across subcellular compartments provides a foundation for investigating the function of these molecules and the mechanisms that localize them.
