Cross-regulation and interaction between eukaryotic gene regulatory processes
Author(s)Spies, Noah (Noah Walter Benjamin)
Massachusetts Institute of Technology. Dept. of Biology.
David P Bartel and Christopher B Burge.
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Regulation of genes is fundamental to all living processes and can be exerted at many sequential steps. We studied several eukaryotic gene regulatory mechanisms with an emphasis on understanding the interplay between regulatory processes on a genome-wide scale. Gene splicing involves the joining of exonic RNA stretches from within a precursor messenger RNA (mRNA). Splicing typically occurs co-transcriptionally as the pre-mRNA is being produced from the DNA. We explored the relationship between the chromatin state of the gene-encoding DNA and the splicing machinery. We found a marked enrichment for nucleosomes at exonic DNA in human T cells, as compared to surrounding introns, an effect mostly explained by the biased nucleotide content of exons. The use of nucleosome positioning information improved splicing simulation models, suggesting nucleosome positioning may help determine cellular splicing patterns. Additionally, we found several histone marks enriched or depleted at exons compared to the background nucleosome levels, indicative of a histone code for splicing. These results connect the chromatin regulation and mRNA splicing processes in a genome-wide fashion. Another pre-mRNA processing step is cleavage and polyadenylation, which determines the 30 end of the mature mRNA. We found that 3P-Seq was able to quantify the levels of 30 end isoforms, in addition to the method's previous use for annotating mRNA 30 ends. Using 3P-Seq and a transcriptional shutoff experiment in mouse fibroblasts, we investigated the e?effect of nuclear alternative 30 end formation on mRNA stability, typically regulated in the cytoplasm. In genes with multiple, tandem 30 untranslated regions (30 UTRs) produced by alternative cleavage and polyadenylation, we found the shorter UTRs were significantly more stable in general than the longer isoforms. This di?difference was in part explained by the loss of cis-regulatory motifs, such as microRNA targets and PUF-binding sites, between the proximal and distal isoforms. Finally, we characterized the small interfering RNAs (siRNAs) produced from heterochromatic, silenced genomic regions in fission yeast. We observed a considerable bias for siRNAs with a 5' U, and used this bias to infer patterns of siRNA biogenesis. Furthermore, comparisons with between wild-type and the Cid14 non-canonical poly(A) polymerase mutant demonstrated that the exosome, the nuclear surveillance and processing complex, is required for RNA homeostasis. In the absence of a fully functional exosome complex, siRNAs are produced to normal exosome targets, including ribosomal and transfer RNAs, indicating these processes may compete for substrates and underscoring the interconnectedness of gene regulatory systems.
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2012.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Cataloged from student submitted PDF version of thesis.Includes bibliographical references.
DepartmentMassachusetts Institute of Technology. Department of Biology
Massachusetts Institute of Technology