P granules, ribonucleoprotein (RNP) complexes specific to the cytoplasmic side of the nuclear pores of C. elegans germ cells, are implicated in post-transcriptional control of maternally-transcribed mRNAs. Here we show a relationship in C. elegans of Dicer, the riboendonuclease processing enzyme of the RNA interference and microRNA pathways, with GLH-1, a germline-specific RNA helicase and a constitutive component of P granules. Based on results from GST-pull-downs and immunoprecipitations, GLH-1 binds DCR-1 and this binding does not require RNA. Both GLH-1 protein and glh-1 mRNA levels are reduced in the dcr-1(ok247) null mutant background; conversely, a reduction of DCR-1 protein is observed in the glh-1(gk100) deletion strain. Thus, in the C. elegans germline, DCR-1 and GLH-1 are interdependent. In addition, evidence indicates DCR-1 protein levels, like those of GLH-1, are likely regulated by the Jun N-terminal kinase (JNK), KGB-1. In adult germ cells, DCR-1 is found in uniformly-distributed, small puncta both throughout the cytoplasm and the nucleus, on the inner side of nuclear pores, and associated with P granules. In arrested oocytes, GLH-1 and DCR-1 re-localize to cytoplasmic and cortically-distributed RNP granules and are necessary to recruit other components to these complexes. We predict the GLH-1/DCR-1 complex may function in the transport, deposition, or regulation of maternally-transcribed mRNAs and their associated miRNAs.
The research presented in this dissertation expands the basic scientific knowledge of the function of the C. elegans germline protein GLH-1 in germline development. GLH-1 is found in P granules, which are ribonucleoprotein complexes specific to the cytoplasmic side of the nuclear pores of C. elegans germ cells. P granules are implicated in post-transcriptional control of maternally-transcribed mRNAs, their function remains elusive. Through genetic studies our laboratory has shown that GLH-1 is essential for fertility; however, the biochemical function of the GLH complex is still unknown. With immunoprecipitations and GST-pull-downs, we found that GLH-1 and the riboendonuclease Dicer, which is the key dicing enzyme in RNA interference, bind one another in complex. Our findings go on to show the regulation of Dicer, evidence indicates Dicer protein levels, like those of GLH-1, are regulated by proteosomal degradation and are much increased when the Jun N-terminal kinase KGB-1 is missing in the kgb-1(um3) null. This study is the first to report the localization of Dicer in the adult C. elegans germline and demonstrates that Dicer is located throughout the cytoplasm as well as at the inner nuclear pores of the germ cell nuclei, in close opposition to GLH-1. Under stress conditions in oocytes GLH-1 and DCR-1 both relocate and recruit other components to large cytoplasmic RNP granules. The research presented here indicates that the GLH-1/DCR-1 complex may function in the transport, deposition, or regulation of maternally-transcribed mRNAs perhaps with their associated miRNAs.
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