Making messenger RNA (mRNA), the primary event in expression of protein coding genes is central to the life of all cells and its corruption is a hallmark of cancer cells. mRNA synthesis requires transcription by RNA polymerase II (pol II) to produce a precursor that is matured by capping, splicing and cleavage/polyadenylation and packaged with RNA binding proteins into mRNP's for export to the cytoplasm. The template that is transcribed is made of chromatin: DNA wrapped around histone proteins in assemblies called nucleosomes. Transcription and pre- mRNA processing are not independent events;they are integrated with one another in a manner that depends on a specialized domain of RNA pol II called the CTD that acts as a landing pad for processing proteins. In fact transcripts made by RNA polymerases other than pol II or those made by pol II lacking the CTD are not processed correctly into mature mRNA. Our working hypothesis is that synthesis and processing of mRNA precursors is performed by an 'mRNA factory'complex which comprises pol II and processing factors held together by contacts with the CTD. This model is an example of a new way of thinking about a set proteins that were once thought to operate independently of one another rather than as a integrated team. We use genetic and biochemical approaches to ask how the 'mRNA factory'works to achieve efficient and accurate production of export-competent mRNP's coordinated synthesis and maturation of transcripts made by pol II. The objectives of this proposal are to 1. To determine the mechanism that permits cross-talk between pre-mRNA splicing and modification of chromatin structure. 2. To map patterns of pol II CTD phosphorylation at high resolution and determine how they related to co-transcriptional assembly of splicing and 3'end processing complexes in normal breast cells and breast cancer cells. 3. To determine how assembly of export competent RNP's is coordinated with processing of the mRNA by cleavage-polyadenylation.

Public Health Relevance

A major component of gene misexpression in cancer cells can be traced to abnormal patterns of pre-mRNA splicing and 3'processing by cleavage/polyadenylation. This work may help elucidate how the transcription and processing of mRNAs are regulated under normal conditions and how they become mis-regulated in cancer cells. We will use genome-wide analysis by ChIP-seq to compare patterns of pol II CTD phosphorylation and recruitment of processing factors to genes in normal breast cells and breast cancer cell lines. This study will help identify defects in the function of the 'mRNA factory" that may play a major role in corrupting splicing and cleavage/polyadenylation in cancer cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM058613-15
Application #
8399729
Study Section
Molecular Genetics C Study Section (MGC)
Program Officer
Bender, Michael T
Project Start
1999-01-01
Project End
2014-12-31
Budget Start
2013-01-01
Budget End
2013-12-31
Support Year
15
Fiscal Year
2013
Total Cost
$359,072
Indirect Cost
$122,647
Name
University of Colorado Denver
Department
Biochemistry
Type
Schools of Medicine
DUNS #
041096314
City
Aurora
State
CO
Country
United States
Zip Code
80045
Johnson, Sara A; Kim, Hyunmin; Erickson, Benjamin et al. (2011) The export factor Yra1 modulates mRNA 3' end processing. Nat Struct Mol Biol 18:1164-71
Kim, Soojin; Kim, Hyunmin; Fong, Nova et al. (2011) Pre-mRNA splicing is a determinant of histone H3K36 methylation. Proc Natl Acad Sci U S A 108:13564-9
Perales, Roberto; Zhang, Lian; Bentley, David (2011) Histone occupancy in vivo at the 601 nucleosome binding element is determined by transcriptional history. Mol Cell Biol 31:3485-96
Kim, Hyunmin; Erickson, Benjamin; Luo, Weifei et al. (2010) Gene-specific RNA polymerase II phosphorylation and the CTD code. Nat Struct Mol Biol 17:1279-86
Munoz, Manuel J; Perez Santangelo, M Soledad; Paronetto, Maria P et al. (2009) DNA damage regulates alternative splicing through inhibition of RNA polymerase II elongation. Cell 137:708-20
Johnson, Sara Ann; Cubberley, Gabrielle; Bentley, David L (2009) Cotranscriptional recruitment of the mRNA export factor Yra1 by direct interaction with the 3'end processing factor Pcf11. Mol Cell 33:215-26
Perales, Roberto; Bentley, David (2009) "Cotranscriptionality": the transcription elongation complex as a nexus for nuclear transactions. Mol Cell 36:178-91
Fong, Nova; Ohman, Marie; Bentley, David L (2009) Fast ribozyme cleavage releases transcripts from RNA polymerase II and aborts co-transcriptional pre-mRNA processing. Nat Struct Mol Biol 16:916-22
Glover-Cutter, Kira; Kim, Soojin; Espinosa, Joaquin et al. (2008) RNA polymerase II pauses and associates with pre-mRNA processing factors at both ends of genes. Nat Struct Mol Biol 15:71-8
Ryman, Kicki; Fong, Nova; Bratt, Eva et al. (2007) The C-terminal domain of RNA Pol II helps ensure that editing precedes splicing of the GluR-B transcript. RNA 13:1071-8

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