Candidate Name: Tiffany Barwell
Program: Biology
Committee Chairs: Dr. Kausik Chakrabarti
Committee Members: Dr. Pinku Mukherjee, Dr. Christine Richardson, Dr. Adam Reitzel, Dr. Didier Dreau, Dr. Eva Ge
Abstract:

Pre-mRNA splicing is the process by which nascent mRNAs join the protein coding sequences, exons, to create mature mRNAs while getting rid of noncoding sequences, known as introns. During splicing, introns are spliced out forming a ‘lariat’- like structure. These lariat RNAs are quickly degraded by RNA debranching enzyme 1 (DBR1), leading to their turnover. Loss of DBR1 gene is embryonically lethal in humans. Thus, DBR1 is an essential protein in mammalian cells, as it is the only debranching enzyme that hydrolyzes 2’-5’ bonds during pre-mRNA processing, to promote efficient pre- mRNA splicing. Our aim to knockdown DBR1 with siRNAs in HEK293 cells resulted in significant accumulation of aberrant splicing products. To further understand if DBR1 is involved in functional coupling between pre-mRNA splicing and the mRNA quality control mechanism, nonsense-mediated decay (NMD) which degrades transcripts containing premature termination codons (PTCs) to prevent translation of unnecessary or aberrant transcripts, we asked if DBR1 can be copurified with NMD factors from soluble mammalian cell extracts. Indeed DBR1, in an RNase sensitive manner, coimmunoprecipitated NMD factors UPF1 and CASC3 suggesting its overlapping role in the mRNA surveillance pathway. Further, in-depth characterization of DBR1 interactome identified several essential proteins involved in stress granule (SG) assembly and enrichment of specific transcripts in DBR1 depleted cells that are part of the SG transcriptome. These results demonstrate involvement of DBR1, potentially as a sensor for aberrantly spliced transcripts and targeting those to a translationally repressed cellular storage foci in mammalian cells.