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Molecular and Cellular Biology, July 2009, p. 3941-3952, Vol. 29, No. 14
0270-7306/09/$08.00+0     doi:10.1128/MCB.00384-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

A Targeted Bypass Screen Identifies Ynl187p, Prp42p, Snu71p, and Cbp80p for Stable U1 snRNP/Pre-mRNA Interaction{triangledown}

Rosemary Hage,1,{dagger} Luh Tung,2,3,{dagger} Hansen Du,4,{ddagger} Leah Stands,1,§ Michael Rosbash,4 and Tien-Hsien Chang1,2,3*

Department of Molecular Genetics, The Ohio State University, Columbus, Ohio 43210,1 Molecular, Cellular, and Developmental Biology Program, The Ohio State University, Columbus, Ohio 43210,2 Genomics Research Center, Academia Sinica, Taipei, Taiwan,3 Howard Hughes Medical Institute and Department of Biology, Brandeis University, Waltham, Massachusetts 024544

Received 25 March 2009/ Returned for modification 22 April 2009/ Accepted 7 May 2009

To understand how DEXD/H-box proteins recognize and interact with their cellular substrates, we have been studying Prp28p, a DEXD/H-box splicing factor required for switching the U1 snRNP with the U6 snRNP at the precursor mRNA (pre-mRNA) 5' splice site. We previously demonstrated that the requirement for Prp28p can be eliminated by mutations that alter either the U1 snRNA or the U1C protein, suggesting that both are targets of Prp28p. Inspired by this finding, we designed a bypass genetic screen to specifically search for additional, novel targets of Prp28p. The screen identified Prp42p, Snu71p, and Cbp80p, all known components of commitment complexes, as well as Ynl187p, a protein of uncertain function. To examine the role of Ynl187p in splicing, we carried out extensive genetic and biochemical analysis, including chromatin immunoprecipitation. Our data suggest that Ynl187p acts in concert with U1C and Cbp80p to help stabilize the U1 snRNP-5' splice site interaction. These findings are discussed in the context of DEXD/H-box proteins and their role in vivo as well as the potential need for more integral U1-snRNP proteins in governing the fungal 5' splice site RNA-RNA interaction compared to the number of U1 snRNP proteins needed by metazoans.

* Corresponding author. Mailing address: Department of Molecular Genetics, The Ohio State University, 484 West 12th Ave., Columbus, OH 43210. Phone: (614) 688-8678. Fax: (614) 292-4466. E-mail: chang.108{at}osu.edu

{triangledown} Published ahead of print on 18 May 2009.

{dagger} R.H. and L.T. contributed equally to this study.

{ddagger} Present address: Gerontology Research Center, National Institute on Aging, LCMB, Box 12, 5600 Nathan Shock Drive, Baltimore, MD 21224-6825.

§ Present address: Department of Biology, Virginia Military Institute, Lexington, VA 24450.

Molecular and Cellular Biology, July 2009, p. 3941-3952, Vol. 29, No. 14
0270-7306/09/$08.00+0     doi:10.1128/MCB.00384-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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