טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentTerno Rinata
SubjectIdentifying the factors responsible for RRF-3 recruitment
DepartmentDepartment of Biology
Supervisor Professor Ayelet Lamm
Full Thesis textFull thesis text - English Version


Abstract

Gene silencing induced by small interfering RNAs (siRNAs), through the RNA interference (RNAi) machinery, is a widespread and diverse feature of eukaryotes. Small RNAs have been shown to participate in important processes such as meiotic and mitotic silencing, heterochromatin formation and epigenetic modifications. Internally triggered RNAi, much like exogenous RNAi, operates in a two-step manner in C. elegans: an initiating (primary) response and a secondary response, each involving different sets of proteins and siRNAs. Previous studies have characterized one of the endogenously triggered RNAi, the RRF-3 pathway. The primary phase of the RRF-3 pathway involves the dicer DCR-1, the dsRNA-binding protein RDE-4, and the RNA-directed RNA polymerase (RdRP) RRF-3. RdRP enzymes, found in plants, fungi and C. elegans, synthesize RNA from an RNA template. This primary phase leads to the production of a small pool primary siRNAs. Secondary siRNAs are generated by the RdRP, RRF-1, and are essential for efficient gene silencing. 23 genes were found to be targeted for silencing by RRF-3, however, it is not known why or how they are selected by this pathway. In this work, we attempted to find what makes RRF-3 select certain genes for silencing and what the role of the pathway in C. elegans is. We combined two approaches: (1) A bioinformatic approach in which we searched for common motifs in the target genes, which may be responsible for RRF-3 recruitment. For this purpose we extended the number of target genes to 112. We looked for common sequence and structure motifs using standard softwares, but we were not able to find a common feature. However, when looking at each gene separately, we were able to identify a secondary structure that involves the start codon in 83% of the target genes. In an additional analysis, we searched for modifications that change nucleotides, on the RNA level, which might be shared by the target genes. We could not find a common modification in the target genes. (2) An experimental approach in which we aimed at 2


finding the site necessary for RRF-3 recruitment in-vivo. We selected target genes, fused them to GFP and cloned them into vectors. Additional clones were prepared, in which one piece of the target gene was missing. We used the miniMos system, which utilizes transposons in order to insert cloned DNA fragments into the C. elegans genome. This method allowed us to insert the different clones and observe the change in GFP fluorescence, indicating the expression level. We expected to get increased fluorescence in clones lacking the region necessary for RRF-3 recruitment, since these clones would not be silenced by this pathway. We created 4 strains with different integrated clones and were able to rule out the 3’UTR as the region responsible for recruitment, strengthening our hypothesis that the 5’ structure is responsible for RRF-3 recruitment. Crossing a strain containing full length target gene, which showed no fluorescence, with RRF-3 mutants, resulted in fluorescent expression, proving for the first time in-vivo, the effect of RRF-3 on an endogenous gene.