MicroRNAs (miRNAs) are involved in many processes including development, proliferation, hematopoiesis, apoptosis, and human disease progression. Most miRNAs repress the translation of their target genes; however, surprisingly, recent evidence indicates that some miRNAs activate the translation of their targets. In C. elegans, fertility and germline maintenance are dependent on the germline RNA helicase, GLH-1, an integral and constitutive component of germline-specific P granules. According to miR-Base
(http://www.microrna.sanger.ac.uk/), two miRNAs, miR-67 and miR-83, are predicted to bind target sites in the 3'-UTR of
glh-1 mRNA, thereby potentially regulating the gene. Our laboratory has discovered that GLH-1 and the ribo-endonuclease Dicer-1 (DCR-1) are interdependent; these two proteins bind each other and when either is missing in the germline, the other is severely affected. We have also observed that DCR-1 protein levels increase in a matter of hours when worms are grown in JNK or proteosome inhibitors, implying DCR-1 may be targeted for degradation by the Jun-N-terminal like kinase, KGB-1 (one of only three JNKs in C. elegans), as is the case for GLH-1. Recently, microarray data from the Bass group (U Utah) indicated that
glh-1 and other germline mRNAs are down-regulated in a
dcr-1 null mutant. Therefore, we hypothesized that miR-83 and miR-67 could function to up-regulate the translation of
glh-1; thus GLH-1 levels would decrease when these miRNAs are absent. Deletion mutants were recently generated for all the known C. elegans miRNA genes; however, these mutants have not yet been extensively characterized. We obtained miR-67 and miR-83 mutant strains and generated a miR-67; miR-83 double mutant. Our preliminary results indicate that GLH-1 protein levels are indeed decreased in the miR-83 mutant and are further decreased in the double. In addition, miR-83 mutants exhibit an abnormal bifurcated distal gonad similar to that seen with loss of
glh-1 by RNA interference. Based on our findings, we propose that miRNAs are activating the translation of
glh-1 in order to recruit it to the miRNA pathway where GLH-1 associates with DCR-1 and directs the RISC machinery to appropriate mRNA targets. These interactions could occur in the P granules or in P-body-like germline cytoplasmic foci. In addition to determining whether DCR-1 regulates
glh-1 through miR67 and miR83, we are also focusing on identifying the mRNAs and miRNAs present in the GLH-1 complex.