Characterization of Factors Required for 3’ End Processing of Histone pre-mRNAs Public Deposited

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  • March 19, 2019
Creator
  • Sabath, Ivan
    • Affiliation: School of Medicine, Department of Biochemistry and Biophysics
Abstract
  • Metazoan replication dependent histone mRNAs form their 3' end by endonucleolytic cleavage of the pre-mRNA transcript without the subsequent addition of a poly(A) tail. These are the only cellular mRNAs that are not polyadenylated but instead end in a highly conserved stem-loop. Analogous to the poly(A) tail of canonical mRNAs, the terminal 3' stem-loop of histone mRNAs is a crucial regulatory feature that provides an interface for mechanisms coupling the mRNA to cell-cycle regulation, stability, and translation. Histone pre-mRNA 3' end processing requires the U7 snRNP, which interacts with a purine rich sequence called the histone downstream element (HDE) located about 10 nts 3' of the cleavage site. The U7 snRNP is composed of a small 60 nucleotide snRNA bound with a U7 specific heptameric ring resembling that of splicesomal snRNPs except that SmD1 and SmD2 are replaced by Lsm10 and Lsm11, respectively. Unlike other Lsm proteins, Lsm11 has an extended N-terminal domain, which interacts with FLASH. The interaction between the N-terminus of Lsm11 and FLASH is essential for processing. My research shows that the native U7 snRNP has a larger, more complex, composite structure than previously described. The Lsm11/FLASH complex in the U7 snRNP stably associates with a subset of polyadenylation factors termed the histone pre-mRNA cleavage complex (HCC). This composite U7 snRNP can be isolated from mammalian nuclear extracts in a single step biochemical purification. Mammalian HCC consists of all six subunits of CPSF in addition to symplekin and CstF64. Association of the U7 snRNP with the HCC is absolutely dependent on a highly conserved LDLY motif in FLASH. I define the region of Lsm11 required for binding FLASH and identify the region of Lsm11 in the context of an Lsm11/FLASH complex necessary to recruit the HCC. Additionally, my work identified the Drosophila HCC, which has a similar composition to the mammalian HCC but lacks two CPSF subunits, Fip1 and CPSF30. Using extracts prepared from Drosophila cultured cells with FLASH knocked down by RNA interference, I also determined the critical elements in FLASH necessary to support histone pre-mRNA processing in vitro.
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  • In Copyright
Advisor
  • Matera, Gregory
  • Xiong, Yue
  • Strahl, Brian
  • Fried, Howard
  • Marzluff, William
  • Dominski, Zbigniew
Degree
  • Doctor of Philosophy
Degree granting institution
  • University of North Carolina at Chapel Hill Graduate School
Graduation year
  • 2014
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  • Chapel Hill, NC
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