Identifying transcripts bound by FUBL-1 in C. elegans and the global transcriptomic effects following its knockout

In many eukaryotic organisms there is an epigenetic system known as RNA interference (RNAi). First discovered in the small nematode Caenorhabditis elegans it has shown to be of great importance in gene regulation. Fundamentally, the system utilises small RNAs as guides for the argonaute (AGO) proteins in the RNA-induced silencing complex (RISC) to target transcripts for translational inhibition or degradation. The type of small RNA used depends on the AGO. Certain AGOs bind micro (mi)RNAs, such as ALG-1, whilst other AGOs such as PRG-1 bind PIWI-interacting (pi)RNAs and ERGO-1 bind 26 nucleotides long RNAs with a preferred guanosine at the 5’ end (26G-RNAs). Some of these primary argonautes produce RNA fragments following target degradation, which in turn can be used by RNA-dependent RNA polymerases (RdRPs) to generate secondary 22 nucleotides long RNAs with a preferred guanosine at the 5’ end (22G-RNAs) in an amplification process. These are in turn used by the worm specific AGOs (WAGOs) to target the transcripts they were generated from. Through earlier work in the Hinas group, the Far Upstream Element Binding Protein Like 1 (FUBL-1) has drawn attention. It is not only predicted to be an RNA binding protein, but fubl-1 mutants also show phenotypes related to RNAi. Knockout mutants show increased RNAi in generations after initiation as compared to wild type, in a system known as RNAi inheritance. Further, knockout mutants have been shown to have increased expression of ERGO-1 target genes. Visualisation experiments of FUBL-1 have shown that it localises primarily to the nucleus in somatic tissues, but it has not been known whether FUBL-1 is also expressed in the germline. In this report, the aim was not only to uncover the potential germline localisation of FUBL-1, but also to unveil the global effects of a knockout mutant. Further, by characterising potential transcripts bound by FUBL-1, another aim was to discover the potential pathway by which the protein acts. Using fluorescent wide-field microscopy and fluorescent antibodies, I could in this report show that FUBL-1 localises to the nucleus in the germline. Importantly, I also developed a method to grow large amounts of developmentally synchronised worms without starvation, which allowed for immunoprecipitation of FUBL-1 to extract bound transcripts. These could later be identified using next generation sequencing and bioinformatic analyses. By doing this I could present 839 likely targets of FUBL-1. Out of these, the vast majority turned out to be messenger RNAs, but several non-coding RNAs were also identified. Further, I also present data that shows the transcriptomes of fubl-1 knockout mutant and wild type to overall be very similar as only six genes are significantly deregulated. Together, these findings point towards FUBL-1 being a rather general RNA binding protein that acts in redundancy for most genes, except for a few, or that it through splicing causes proteomic changes not detected in this study. Together with earlier data, this work will hopefully be sufficient for a robust hypothesis on the function of FUBL-1.