FACILITATING MECHANISTIC STUDIES OF PRE-MRNA SPLICING VIA A NOVEL TARGETED SEQUENCING APPROACH
Most eukaryotic genes have their protein coding sequences interrupted by non-coding introns, which must be removed from nascent pre-mRNAs by the spliceosome to generate a translatable mRNA. The past few decades have been marked by a significant increase in our appreciation for the central role that splicing plays in regulating eukaryotic gene expression. Nevertheless, the mechanisms by which this process is normally regulated, and can be mis-regulated with pathological consequences, remain poorly understood. Next-generation sequencing (NGS) technologies have had a profound effect on our understanding of pre-mRNA splicing. Yet in spite of the power presented by this approach, it is less widely appreciated that the depth of sequencing necessary to quantitatively detect many splicing isoforms is significantly higher than most RNA-Seq experiments generate. Here I present the development and implementation of a novel sequencing method designed to harness the quantitative power of sequencing while focusing it on user-selected splice junctions of interest through targeting for introns at the reverse transcription step. This approach can dramatically enrich the fraction of reads in each sequencing experiment that are informative about splicing status, and in doing so enable a significant increase in the precision with which changes in splicing can be detected, all while decreasing experimental costs.
intron; lariat intermediate; Biology; splicing; Sequencing; spliceosome; Biogeochemistry
Pleiss, Jeffrey A.
Alani, Eric E.; Ke, Ailong
Biochemistry, Molecular and Cell Biology
Ph. D., Biochemistry, Molecular and Cell Biology
Doctor of Philosophy
dissertation or thesis