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Nuclear accumulation of polyadenylated noncoding RNA leads to a breakdown in nuclear RNA homeostasis

  • Author / Creator
    Paul, Biplab
  • The biogenesis and processing of messenger RNAs (mRNAs) is central to the gene expression program. In eukaryotic cells, mRNAs must also be exported from nucleus to cytoplasm as part of the gene expression pathway. In this work, mutations in 1047 essential genes of Saccharomyces cerevisiae (S. cerevisiae) were screened for defects in mRNA export resulting in the identification of 26 mRNA export mutants. Single molecule fluorescent in situ hybridization (smFISH) experiments further showed that these mutants accumulated mRNAs within specific regions of the nucleus, which included: (1) transcripts enriched near nuclear pore complexes when components of the mRNA export apparatus were mutated, (2) build-up of mRNAs near transcription sites associated with mutations in genes required for 3′ end processing and chromosome segregation, and (3) mRNAs within the nucleolus when nucleocytoplasmic transport (e.g. srm1-ts), rRNA biogenesis (e.g. enp1-1), or RNA processing and surveillance (e.g. csl4-ph) were disrupted. These data demonstrate that alterations to RNA processing and overall nuclear function cause RNAs to stall, or be retained, at three distinct restriction points. This may reflect common failures in mRNA biogenesis and export, as well as, active mechanisms to hold mRNAs at discrete locations to protect the cell and fidelity of the gene expression when cells are dysfunctional. The nucleolus is mainly associated with noncoding RNA (ncRNA) processing, raising questions about the exact nature of the accumulated poly(A)-RNA material in csl4-ph, enp1-1, and srm1-ts strains. Follow up work using RNA-sequencing (RNA-seq) methods showed that these three mutants exhibited significant down regulation of protein-coding transcripts that are highly expressed under normal steady state growth conditions and exhibited increased levels of pervasive transcripts. Combined RNA-seq, Northern, and RNA binding data, further revealed that the errors in RNA biogenesis in csl4-ph and enp1-1 mutants led to the accumulation of polyadenylated ncRNA species. Loss of Csl4p or Enp1p was also found to result in a poly(A)-RNA binding protein, Nab2p, engaging in a protein interaction network that encompassed ncRNA processing factors. This included Nab2p becoming associated with proteins required for ribosomal RNA (rRNA) and small nucleolar RNA (snoRNA) processing, in contrast to the normal association of Nab2p with mRNA processing factors in a control strain. These data indicate that ncRNAs become stably polyadenylated in csl4-ph and enp1-1 mutants, which leads to the inappropriate association of Nab2p, and other mRNA processing factors, with ncRNAs. In line with this model, overexpression of another poly(A)-RNA binding protein, Pab1p, rescued the poly(A)-RNA accumulation phenotype and improved the growth of the enp1-1 strain at a semipermissive temperature. These findings provide evidence that polyadenylated ncRNAs can sequester mRNA biogenesis and export machinery, which precipitates a breakdown in nuclear homeostasis defined by high levels of nuclear poly(A)-RNA in the nucleolus. A set of events that can be mitigated by the overexpression of another poly(A)-RNA binding protein. Overall, these studies have identified essential genes that are required for the maintenance of the gene expression program and provided new insight into nuclear RNA homeostasis. A key finding is that mutations altering processes not directly linked to mRNA biogenesis or export (i.e. Enp1p) have the ability to disrupt gene expression through altering the abundance of functional RNA-binding proteins. Data that highlights the importance of nuclear surveillance and decay of aberrant RNA species in maintaining nuclear RNA homeostasis and proper cellular function.

  • Subjects / Keywords
  • Graduation date
    Spring 2020
  • Type of Item
    Thesis
  • Degree
    Doctor of Philosophy
  • DOI
    https://doi.org/10.7939/r3-ye2v-7132
  • License
    Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.