Investigating the expression and function of RNA-binding protein FUBL-1 in C. elegans

Abstract: RNA interference (RNAi) is an important mechanism of gene silencing in Caenorhabditis elegans, in which short RNAs direct sequence-specific silencing of gene expression mediated by Argonaute proteins. RNAi in C. elegans can be sorted into exogenous RNAi, where the short RNAs originate from foreign RNA sequences, and endogenous RNAi, where the short RNAs originate from RNA sequences in the genome. One endogenous RNAi pathway is the ERGO-1 pathway, which is active in the germline and in embryos. Mutants deficient in the ERGO-1 pathway show an increased response to exogenous RNAi, which is thought to be due to competition between exogenous and endogenous silencing pathways.  FUBL-1 is an RNA-binding protein found in C. elegans, which has three predicted functional isoforms: isoforms a, b and c. Prior research by the Hinas group has indicated that FUBL-1 may play a role in the ERGO-1 pathway of RNAi. FUBL-1 deletion mutants show an increased response to exogenous RNAi similar to ERGO-1 mutants, and also an upregulation of ERGO-1 target genes. They have also found that FUBL-1 is broadly expressed in somatic tissues, but germline expression has not been confirmed. The function of the three different isoforms has not yet been examined. The aim of this study was to further investigate the function of FUBL-1 by assessing the function of the different isoforms through RT-qPCR of C. elegans with mutations affecting the different isoforms, to use immunofluorescence staining to see whether FUBL-1 is expressed in the germline, and to identify FUBL-1 RNA targets through CLIP-seq. Preliminary RT-qPCR results indicated that the upregulation of ERGO-1 targets is less in isoform mutants than in FUBL-1 deletion mutants, indicating partial redundancy of the isoforms. Immunofluorescence staining showed that FUBL-1 is expressed in the germline nuclei. Growth protocols have been optimized and crosslinking has been performed on worms, but the full CLIP-seq protocol has not been performed yet.