Cotranscriptional Processing of Histone pre-mRNA Public Deposited

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  • March 20, 2019
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  • Sullivan, Kelly Daniel
    • Affiliation: School of Medicine, Department of Biochemistry and Biophysics
Abstract
  • Metazoan histone mRNAs are unique in that they lack introns and poly(A) tails; thus these mRNAs require a single endonucleolytic cleavage for maturation. Cleavage of histone mRNA is guided by two cis-elements, a stemloop (SL) 5' of the cleavage site and a 3' purine-rich histone downstream element (HDE). Binding of the stemloop binding protein to the SL and basepairing of the U7 snRNA component of the U7 snRNP recruit a cleavage factor that contains the endonuclease CPSF73 which cleaves the pre-mRNA. I show here that a subset of poly(A) factors are required for histone pre-mRNA processing in Drosophila melanogaster. I present evidence that CPSF73, CPSF100, and Symplekin are required for histone pre-mRNA processing and that they form a stable complex. RNAi depletion of these factors results in misprocessing and polyadenylation of endogenous histone H2A mRNA. Knockdown of CPSF73, CPSF100 or Symplekin results in codepletion of the other factors. Coimmunoprecipitation experiments show that these proteins interact with one another and with the histone specific factors SLBP and Lsm11. I present chromatin immunoprecipitation data demonstrating that CPSF73 and Symplekin associate with both histone and poly(A) genes at similar levels in vivo, but that CstF50, a poly(A) specific factor associates with poly(A) genes at a much higher level. Experiments combining RNAi and ChIP reveal that knockdown of SLBP results in recruitment of additional components of the poly(A) apparatus (CstF50) to histone genes. I also present ChIP data which show that knockdown of factors (CPSF160, CstF64) that codeplete proteins required for both poly(A) and histone pre-mRNA processing (CPSF73, Symplekin) specifically deplete the pool of these factors associated with poly(A) genes, consistent with two distinct processing complexes. I also present evidence in mammalian cells that CPSF73 is the exonuclease which degrades the 5' downstream cleavage product potentially resulting in transcription termination. Finally, I describe a role for the histone pre-mRNA processing factor SLBP in nuclear export of the mature mRNA. A detailed molecular analysis of the histone mRNA in cells with extremely low levels of SLBP reveals that the major lesion in histone mRNA metabolism is retention of mature, properly processed mRNA in the nucleus.
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Advisor
  • Marzluff, William
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