This application uses the Rous sarcoma virus (RSV) as a model to study regulation of RNA processing. RSV primary transcripts are spliced by the host RNA splicing machinery, but this process is controlled since the majority (75%) of the RNA remain unspliced to serve as the message for the gag-pol proteins and as genomic RNA for progeny virions. Viral proteins are not required for the normal processing indicating that the cis elements are in the RNA and host-derived trans-acting factors are responsible for the ratio of spliced to unspliced RNA. This study characterizes these cis and trans-acting factors. One control element has been identified, the Negative regulator of splicing (NRS), which is located in gag and is distinctive in that it is not located near any of the splice sites, yet suppresses both the env and src 3 splice sites (ss). Two subregions of the NRS have been identified; an upstream purine-rich region that binds essential splicing factors called SR proteins and an unknown factor (p55), and a downstream sequence that is required for binding of U11 snRNP, an RNP particle that recognizes the 5 ss in a new class of rare introns (AT-AC introns). U1 and U2, abundant snRNPs of the major splicing pathway also interact with the NRS RNA.
Four specific aims are proposed. The first uses in vivo and in vitro approaches to further characterize the cis elements and trans-acting factors which govern NRS splicing inhibition.
The second aim tests a hypothesis that the U11 snRNP, when bound to the NRS interacts nonproductively with the major pathway splicing factors present at the authentic 3 ss, excluding pairing with the authentic 5 ss.
The third aim studies an element which suppresses src splicing, named the SSS.
The final aim i nvestigates the basis of the position dependence of the NRS and SSS in splicing inhibition. This may be effected by splice site competition or the position relative to the 5' cap structure.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
1R01CA078709-01
Application #
2686129
Study Section
Virology Study Section (VR)
Program Officer
Cole, John S
Project Start
1998-08-14
Project End
2003-04-30
Budget Start
1998-08-14
Budget End
1999-04-30
Support Year
1
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Medical College of Wisconsin
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
073134603
City
Milwaukee
State
WI
Country
United States
Zip Code
53226
Hudson, Stephen W; McNally, Lisa M; McNally, Mark T (2016) Evidence that a threshold of serine/arginine-rich (SR) proteins recruits CFIm to promote rous sarcoma virus mRNA 3' end formation. Virology 498:181-191
Hudson, Stephen W; McNally, Mark T (2011) Juxtaposition of two distant, serine-arginine-rich protein-binding elements is required for optimal polyadenylation in Rous sarcoma virus. J Virol 85:11351-60
Van Dusen, Courtney M; Yee, Lily; McNally, Lisa M et al. (2010) A glycine-rich domain of hnRNP H/F promotes nucleocytoplasmic shuttling and nuclear import through an interaction with transportin 1. Mol Cell Biol 30:2552-62
Maciolek, Nicole L; McNally, Mark T (2008) Characterization of Rous sarcoma virus polyadenylation site use in vitro. Virology 374:468-76
McNally, Mark T (2008) RNA processing control in avian retroviruses. Front Biosci 13:3869-83
Maciolek, Nicole L; McNally, Mark T (2007) Serine/arginine-rich proteins contribute to negative regulator of splicing element-stimulated polyadenylation in rous sarcoma virus. J Virol 81:11208-17
Sun, Hai-Yuan; McNally, Mark T; Jackson, Vaughn E et al. (2006) Urea-nuclease treatment of concentrated retrovirions preserves viral RNA and removes polymerase chain reaction-amplifiable cellular RNA and DNA. J Virol Methods 137:304-8
McNally, Lisa M; Yee, Lily; McNally, Mark T (2006) Heterogeneous nuclear ribonucleoprotein H is required for optimal U11 small nuclear ribonucleoprotein binding to a retroviral RNA-processing control element: implications for U12-dependent RNA splicing. J Biol Chem 281:2478-88
Cochrane, Alan W; McNally, Mark T; Mouland, Andrew J (2006) The retrovirus RNA trafficking granule: from birth to maturity. Retrovirology 3:18
Maciolek, Nicole L; Alward, Wallace L M; Murray, Jeffrey C et al. (2006) Analysis of RNA splicing defects in PITX2 mutants supports a gene dosage model of Axenfeld-Rieger syndrome. BMC Med Genet 7:59

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