Examination of RNA Helicase A Function in Small Regulatory RNA Pathways of the Caenorhabditis elegans Germline

Date of Award

2012

Document Type

Thesis

Degree Name

Bachelors

Department

Natural Sciences

First Advisor

Walstrom, Katherine

Keywords

c. elegans, RNA Interference, RNA Helicase A

Area of Concentration

Biochemistry

Abstract

The discovery of RNA interference (RNAi) has lead to sweeping changes in the current understanding of gene expression regulation. However, many necessary RNAi components, in addition to their functional significance, still remain to be fully elucidated. The continued exploration of RNAi components is critical for greater scientific knowledge and for use of RNAi as a potential therapeutic. ERI-1, an exonuclease, represents a protein necessary for RNAi of the Caenorhabditis elegans germline. RNA helicase A (RHA-1) currently presents as a protein with undetermined functions within C. elegans RNAi. RHA-1 has been previously linked to control of transcriptional processes within the germline of C. elegans. Upon mutation of either ERI-1 or RHA-1, worms present with a temperature sensitive sterility phenotype, thus supporting a possible connection between these proteins. The work of this thesis examined changes in mRNA transcript expression of 11 genes of C. elegans, where genes were either known small RNA targets, germline-enriched, or selected for use as a control. Gene expression changes were observed through the use of quantitative PCR (qPCR). All RNA was purified from and quantified within 4 unique worm strains: wild-type N2, eri-1 mutants, rha-1 mutants and eri-1;rha-1 mutants. As ERI-1 is a known component of various germline-specific small regulatory RNA pathways of C. elegans, namely the 26G endo-siRNA pathway, eri-1 mutants were used to probe the possible inclusion of RHA-1 within these regulatory pathways. Experimental results display modest support of this hypothesis. Emphasis is placed on spermatogenesis-expressed genes F18C5.4, C36H8.1, K08D10.7 and Y54G11.A.6, all which present over expression upon RHA-1 knockout, in addition to literature supported over expression in eri-1 mutants. Additionally, experimental results also support a possible role of RHA-1 in the expression of C. elegans transposon Tc1. Here, loss of RHA-1 results in under expression of Tc1, an occurrence consistent with previous work showing that rha-1 mutations can suppress Tc1 excision. Therefore, RHA-1 could hold a currently unknown function in the expression of Tc1.

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