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Richard J. Balzer
The College of New Jersey
Thesis Advisor: Michael Henry, Ph.D.
Cell and Molecular Biology Program
Science Center, Room 290
Friday, May 9, 2008
Proper pre-mRNA processing is necessary to insure the timely production of mature transcripts competent for translation. These steps include 5’ end capping, 3’ end termination/polyadenylation, and intron splicing. Furthermore, alternative or regulated splicing can be applied to genes that are transcribed, but whose products may be deleterious or unnecessary to the cell. A quality control checkpoint at the nuclear pore that prohibits the export of intron-containing transcripts to the cytoplasm has been described. In this work, I provide evidence that the Snu56 and Npl3 proteins are required for alternative pre-mRNA splicing and nuclear retention, respectively.
In the yeast S. cerevisiae, positive splicing regulation occurs during meiosis in which diploid cells divide to form haploid gametes. The Mer1 protein recruits the U1 snRNP to specific pre-mRNAs permitting spliceosomal assembly and intron removal. The mature transcripts are required for meiotic progression, and subsequently, sporulation. I have identified a novel allele (snu56-2) of the essential U1 snRNP protein Snu56p that exhibits a sporulation defect. Using a CUP1 reporter assay and reverse transcriptase PCR, I demonstrate that this allele specifically impairs Mer1p-activated splicing. This is not a reflection of a generally deficient spliceosome as these cells splice vegetative transcripts efficiently. Furthermore, Snu56p depletion in vivo does not significantly impact mitotic splicing. Thus, its splicing function appears limited to Mer1p-activated meiosis-specific splicing. Two hybrid studies indicate that Snu56p interacts with the other two U1 snRNP factors (Mer1p and Nam8p) required for this process. Interestingly, these two proteins do not interact suggesting that Snu56p links pre-mRNA bound Mer1p to Nam8p in the U1 snRNP. This work demonstrates that the Snu56 protein is required for splicing only during meiosis.
Few proteins have been linked to the nuclear retention of unspliced transcripts and its mechanism remains unclear. In the remainder of this thesis, I provide evidence that the mRNA transporter Npl3p is required for nuclear pre-mRNA retention. We utilize a LacZ reporter assay to show that specific npl3 alleles permit cytoplasmic pre-mRNA accumulation. These alleles belong to a single phenotypic class with respect to Npl3p localization and nuclear mRNA accumulation. Mutants of two additional mRNA exporters, Hrp1p and Nab2p, do not exhibit pre-mRNA leakage to the cytoplasm. This latter result was unexpected, as it is known that Nab2p interacts with a component of the nuclear pore quality control machinery. Two-hybrid studies identified an interaction between Npl3p and Nab2p that is strengthened 15-fold by an npl3 leakage allele. I propose a model in which a strong Npl3p-Nab2p interaction signals complete mRNA processing and promotes transit through the nuclear pore.