Investigating the role of germline RNA helicase-1 in Caenorhabditis elegans

Abstract

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] The germline specific RNA helicase GLH-1, a constitutive P-granule component, and DCR-1, the riboendonuclease responsible for processing small RNAs in the miRNA pathway, have been shown to be interdependent in the C. elegans germline. The work described here is based on the hypothesis that if GLH-1 functions with DCR-1 in the miRNA pathway in the C. elegans germline, then when GLH-1 is missing or reduced, there will be a difference in miRNA processing and/or abundance in glh-1 mutants as compared to wild type worms. We utilized deep sequencing on an Illumina platform to analyze differences in miRNA abundance between glh-1(gk100) mutant and wild type worms. In addition, to ascertain whether any miRNAs are germline specific, we also analyzed differences in miRNA expression between glp-4(bn2) worms cultured at both 15oC and 26oC. Results of deep sequencing and qRT-PCR indicated that in general miRNA abundance is moderately decreased in glh-1(gk100) mutants, and this reduction is significant in mutants cultured at 26oC. Our results also indicated that 11 of the 153 miRNAs identified to date in C. elegans are germline-enhanced, including miR-38, miR-40, miR-228, and miR-244, and may be germline-specific.