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Regulation of U1 snRNP / 5' splice site interactions during pre-mRNA splicing in saccharomyces cerevisiae

Stands, Leah Rae
http://rave.ohiolink.edu/etdc/view?acc_num=osu1056395043

Abstract Details

2003, Doctor of Philosophy, Ohio State University, Molecular Genetics.
Intron removal is a complex process carried out by a dynamic cellular machine termed the spliceosome. To form a functional spliceosome, five small nuclear ribonucleoprotein particles (snRNPs) sequentially bind to a precursor mRNA (pre-mRNA) and then rearrange via an ordered series of RNA-RNA interactions. Initially, the 5’ splice site (SS) of the pre-mRNA is recognized by base pairing with the U1 snRNA. This interaction must be disrupted to allow the U6 snRNA to base pair with the 5’ SS. Prp28p, a putative RNA helicase, is required for the switch of U1 for U6 at the 5’ SS and may promote this switch by destabilize the U1/5’ SS RNA duplex. Mutant versions of the U1 snRNP-specific U1-C protein can eliminate the requirement for the normally essential Prp28p. Mutant U1-C proteins may weaken the U1/5’ SS interaction, thus eliminating the need for Prp28p to destabilize the RNA duplex. Therefore Prp28p may not only disrupt an RNA duplex, but also counteract the stabilizing effect of the U1-C protein. Presented here are studies aimed at understanding the complex target of Prp28p and its function in splicing. In addition to continuing analysis of alterations in the U1-C protein that allow bypass of Prp28p, I have identified a specific domain within the U5 snRNP protein Prp8p that may assist Prp28p in displacing U1 from the 5’ SS. Alterations in two additional U1 snRNP proteins, Prp42p and Snu71p, were also found to bypass Prp28p, indicating that these proteins may also be part of the target of Prp28p. Alterations in Sm proteins known to disrupt the U1/5’ SS association cannot bypass Prp28p, indicating that the bypass abilities of U1-C, Prp42p and Snu71p are specific and that these proteins likely represent true targets of Prp28p. I present a model in which U1-C, Prp42p, and Snu71p facilitate the U1/5’ SS interaction while Prp28p and Prp8p work antagonistically to disrupt this association. My data also contribute to a shift in the way RNA helicase function is viewed. These enzymes may not target only RNA duplexes, but instead may function as RNPases, altering both protein and RNA contacts within RNP complexes. Intron removal is a complex process carried out by a dynamic cellular machine termed the spliceosome. To form a functional spliceosome, five small nuclear ribonucleoprotein particles (snRNPs) sequentially bind to a precursor mRNA (pre-mRNA) and then rearrange via an ordered series of RNA-RNA interactions. Initially, the 5’ splice site (SS) of the pre-mRNA is recognized by base pairing with the U1 snRNA. This interaction must be disrupted to allow the U6 snRNA to base pair with the 5’ SS. Prp28p, a putative RNA helicase, is required for the switch of U1 for U6 at the 5’ SS and may promote this switch by destabilize the U1/5’ SS RNA duplex. Mutant versions of the U1 snRNP-specific U1-C protein can eliminate the requirement for the normally essential Prp28p. Mutant U1-C proteins may weaken the U1/5’ SS interaction, thus eliminating the need for Prp28p to destabilize the RNA duplex. Therefore Prp28p may not only disrupt an RNA duplex, but also counteract the stabilizing effect of the U1-C protein. Presented here are studies aimed at understanding the complex target of Prp28p and its function in splicing. In addition to continuing analysis of alterations in the U1-C protein that allow bypass of Prp28p, I have identified a specific domain within the U5 snRNP protein Prp8p that may assist Prp28p in displacing U1 from the 5’ SS. Alterations in two additional U1 snRNP proteins, Prp42p and Snu71p, were also found to bypass Prp28p, indicating that these proteins may also be part of the target of Prp28p. Alterations in Sm proteins known to disrupt the U1/5’ SS association cannot bypass Prp28p, indicating that the bypass abilities of U1-C, Prp42p and Snu71p are specific and that these proteins likely represent true targets of Prp28p. I present a model in which U1-C, Prp42p, and Snu71p facilitate the U1/5’ SS interaction while Prp28p and Prp8p work antagonistically to disrupt this association. My data also contribute to a shift in the way RNA helicase function is viewed. These enzymes may not target only RNA duplexes, but instead may function as RNPases, altering both protein and RNA contacts within RNP complexes.
Tien-Hsien Chang (Advisor)
pre-mRNA splicing; snRNPs

Recommended Citations

Citations

  • Stands, L. R. (2003). Regulation of U1 snRNP / 5' splice site interactions during pre-mRNA splicing in saccharomyces cerevisiae [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1056395043

    APA Style (7th edition)

  • Stands, Leah. Regulation of U1 snRNP / 5' splice site interactions during pre-mRNA splicing in saccharomyces cerevisiae. 2003. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1056395043.

    MLA Style (8th edition)

  • Stands, Leah. "Regulation of U1 snRNP / 5' splice site interactions during pre-mRNA splicing in saccharomyces cerevisiae." Doctoral dissertation, Ohio State University, 2003. http://rave.ohiolink.edu/etdc/view?acc_num=osu1056395043

    Chicago Manual of Style (17th edition)

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This open access ETD is published by The Ohio State University and OhioLINK.