Year of Award

2020

Document Type

Dissertation

Degree Type

Doctor of Philosophy (PhD)

Degree Name

Cellular, Molecular and Microbial Biology

Other Degree Name/Area of Focus

Cellular and Developmental Biology

Department or School/College

Division of Biological Sciences

Committee Chair

Jesse Hay

Commitee Members

Ekaterina Voronina, Brent Ryckman, Bruce Bowler, Mark Grimes, Jesse Hay

Keywords

dynein light chain 1, LC8, P granules, Proximity Ligation Assay, RNA binding proteins, RNA granules

Publisher

University of Montana

Subject Categories

Cell Biology | Developmental Biology

Abstract

Gene regulation is essential for ensuring maintenance, proliferation, and proper development of a cell. RNA binding proteins (RBPs) regulate gene expression by targeting and binding mRNAs to control their translation and often localize to cytoplasmic assemblies of protein and RNA called RNA granules to facilitate post-transcriptional mRNA regulation. Using C. elegans as a model organism, we report on the function of dynein light chain 1 (DLC-1), a subunit of the dynein motor complex, in post-transcriptional mRNA regulation in the gonad. Previous work suggests that DLC-1 is an RBP cofactor that functions independent of the dynein motor. It is unknown how widespread this regulatory role for DLC-1 may be or what direct interactions between DLC-1 and RBPs make mRNA regulation possible. The work presented in this dissertation suggests that DLC-1 is an important contributor to post-transcriptional mRNA regulation as well as RNA granule assembly. First, we used RNA immunoprecipitation coupled with high throughput sequencing (RIP-seq) to identify the mRNAs associated with DLC-1 through its interaction with RBPs. We found that DLC-1 is involved in post-transcriptional regulation of the oogenic transcriptome and demonstrated that DLC-1-associated transcripts depend on DLC-1 for regulation of their expression in the germline. From this work we identified the RBP OMA-1 as a new interactor of DLC-1 by an in vitro pulldown. Furthermore, we developed a protocol for application of in situ Proximity Ligation Assay (PLA) for use in C. elegans to probe for protein-protein interactions across developmental stages. This allowed us to detect formation of DLC-1/OMA-1 complexes in the gonad. Finally, we used a bioinformatic scan to find additional C. elegans RBPs that might interact with DLC-1. Using in vitro pulldowns, we verified predicted direct interactions between DLC-1 and 4 core RBP components of P granules, which are a subtype of RNA granule. Knockdown or knockout of dlc-1 disrupts embryonic P granule assembly, suggesting that DLC-1 has an important role in this process. As a whole, this work expands upon the alternate and emerging functions of dynein light chains and suggests that cofactors like DLC-1 play critical roles in promoting mRNA regulation.

Chapter 4 Supplemental Table 1.xlsx (236 kB)
Chapter 4 Supplemental Table 1

Available for download on Thursday, July 01, 2021

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