Inhaltszusammenfassung

Argonaute proteins bind small RNAs (sRNAs) and together regulate gene expression across all domains of life. Multiple sRNA pathways are involved in controlling selfish genetic elements, like transposable elements (TEs). The nematode Caenorhabditis elegans expresses multiple classes of sRNAs, including 21U-RNAs, which are considered the Piwi-interacting RNAs (piRNAs) of C. elegans, as well as 26G- and 22G-RNAs, which are primary and secondary endogenous small interfering RNAs (endo-siRNAs), re...Argonaute proteins bind small RNAs (sRNAs) and together regulate gene expression across all domains of life. Multiple sRNA pathways are involved in controlling selfish genetic elements, like transposable elements (TEs). The nematode Caenorhabditis elegans expresses multiple classes of sRNAs, including 21U-RNAs, which are considered the Piwi-interacting RNAs (piRNAs) of C. elegans, as well as 26G- and 22G-RNAs, which are primary and secondary endogenous small interfering RNAs (endo-siRNAs), respectively. GTSF1 proteins are evolutionarily conserved factors that are required for normal fertility and TE silencing in the animal germline, by physically interacting with Piwi Argonautes. Given the lack of conserved factors acting in the 21U-RNA/piRNA pathway, we wanted to dissect the role of the single GTSF-1 ortholog in C. elegans. To do this, we first created gtsf-1 mutants using CRISPR/Cas9 technology. We found that gtsf-1 mutants display fertility defects similar to its orthologs in mouse and flies. Surprisingly, we found that GTSF-1 is not required for TE silencing nor for 21U-RNA biogenesis or function. Instead, we have shown that GTSF-1 is required for the biogenesis of endo-siRNAs by interacting, via its CHHC zinc fingers, with the RNA-dependent RNA Polymerase RRF-3. Importantly, GTSF-1 is required for the assembly of the protein complex that assists RRF-3 in the biogenesis of endo-siRNAs. This work suggests that a common denominator of GTSF1 function may be to promote the assembly of multi-subunit effector complexes, in the context of sRNA pathways. Upon the genetic dissection of GTSF-1 function, we uncovered a remarkable maternal effect in the transmission of its mutant phenotypes. We used high-throughput sequencing and genetics to characterize the sRNA and target mRNA dynamics subjacent to this maternal effect. We found that primary endo-siRNAs are maternally deposited to initiate secondary endo-siRNA production, which, in turn, will exert target gene silencing throughout development. Furthermore, we explored additional aspects of gene regulation by endo-siRNAs. We have shown that two redundant paralog Argonaute proteins, termed ALG-3 and ALG-4, which interact with primary endo-siRNAs, fine-tune their own expression in a negative feedback loop. Moreover, we identified several determinants of the regulatory outcome of ALG-3/4 targets. The last facet of my PhD work concerns the characterization of GTSF-1 homologs in additional nematode species. We were driven to do so because of the apparent functional diversity of GTSF-1 proteins. To this end, we identified and mutated gtsf-1 homologs in C. briggsae and Pristionchus pacificus. We found that gtsf-1 is required for normal fertility in these two species, but is required for endo-siRNA biogenesis only in P. pacificus. Therefore, Cbr-GTSF-1 may not be required for the endo-siRNA pathway, in line with the robust evolutionary plasticity of GTSF-1 proteins.» weiterlesen» einklappen

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DDC Sachgruppe:
Biowissenschaften, Biologie