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Access Type

WSU Access

Date of Award

January 2020

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Biological Sciences

First Advisor

Dr. Athar Ansari

Abstract

Rat1 is a 5′→3′ exoribonuclease in budding yeast belonging to the XRN-family of nucleases. It is a highly conserved protein with homologs being present in fission yeast, flies, worms, mice and humans. Rat1 and its homolog in metazoan have been shown to function in multiple facets of RNA metabolism. In this study, we report a novel role of Rat1 in splicing of pre-mRNA in budding yeast. In the absence of the functional Rat1 in the nucleus, an increase in the level of unspliced transcripts was observed in yeast cells. Strand-specific TRO analysis revealed that the accumulation of unspliced transcripts upon nuclear depletion of Rat1 was not due to stabilization of intron-containing transcripts. The unspliced transcripts were capped at their 5ʹ end, suggesting that Rat1 nuclear RNA surveillance function is not responsible for the phenotype. Furthermore, altered elongation rate of RNAPII in Rat1 mutant was not linked to the accumulation of unspliced transcripts. Inhibition of termination by inactivation of Rna14, a component of CF1A complex as well as deletion of Rat1 termination complex components, Rai1 and Rtt103, did not affect the level of unspliced transcripts. These results strongly suggest a novel role of Rat1 in splicing, which is independent of its termination function. The interaction of Rat1 with the splicing competent introns, and its association with spliceosomal components; Clf1, Isy1, Yju2, Sub2 and Prp43 suggest that accumulation of unspliced transcripts in the Rat1 mutant is due to a direct role of Rat1 in splicing and not due to lack of degradation of unspliced transcripts in rat1 mutant. Lastly, the recruitment of Prp2 splicing factor on the intron was compromised in the Rat1 mutant suggesting that Rat1 is a critical factor in the dynamics of spliceosome assembly in budding yeast.

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