Poster abstracts

Poster number 38 submitted by Melanie Fox

Transcriptome-wide analysis of RNA processing defects in S. cerevisiae

Melanie J. Fox (Department of Biochemistry and Molecular Biology, IU School of Medicine), Hongyu Gao (Department of Medical and Molecular Genetics & Center for Computational Biology and Bioinformatics, IU School of Medicine), Yunlong Liu (Department of Medical and Molecular Genetics & Center for Computational Biology and Bioinformatics, IU School of Medicine), Amber L. Mosley (Department of Biochemistry and Molecular Biology & Center for Computational Biology and Bioinformatics, IU School of Medicine)

Abstract:
RNA Polymerase II (RNAPII) is the enzyme responsible for the transcription of messenger RNA (mRNA) and many small non-coding RNAs. While much is known about RNAPII transcription initiation and its role in expression regulation, eukaryotic termination mechanisms are still active areas of investigation. There are two described RNAPII termination mechanisms: most mRNAs are terminated by the polyadenylation dependent cleavage and polyadenylation complex and the Rat1 exonuclease while most noncoding RNAs and unstable transcripts are terminated by the Nrd1 complex and nuclear exosome. The nuclear exosome has an important role in RNA surveillance, processing, and degradation of a wide range of RNAs. The nuclear exosome is specifically required for 3’end trimming of small noncoding RNAs, and deletion of RRP6 causes accumulation of long, untrimmed transcripts (read-through transcription) as well as altered RNA expression levels. Disruptions in proteins that control RNAPII phosphorylation, such as Rtr1 and Ssu72, also cause read-through transcription and altered expression to different extents. Using RNA-sequencing data of total RNA isolated from S. cerevisiae, we are developing methods to identify read-through transcripts and altered expression levels genome-wide in rrp6Δ cells. This will allow us to identify novel targets of the nuclear exosome and gain a better understanding of termination pathway usage. Previous studies of RRP6 targets using titling arrays had limited dynamic range compared to RNA-sequencing, and we are able to detect a much larger difference between short, processed small noncoding RNAs which are extremely abundant and the less abundant read-through transcripts that accumulate when RRP6 is deleted. Read-through transcripts, even in exosome mutants, are a small fraction of the transcripts produced, and the depth of sequencing at these abundant snRNAs make them excellent targets for analysis. This study will allow us to optimize our analysis pipeline that can then be used to analyze the effects of disrupting other termination factors and proteins involved in RNAPII phosphorylation.

Keywords: RNA sequencing, exosome, 3 end processing