Makeyeva, Yekaterina et al. (2021) International Worm Meeting "mRNA Splicing Promotes Polyadenylation and Counteracts Novel Default Argonaute Silencing in the Germline of Caenorhabditis elegans"

Shirayama, Masaki, Makeyeva, Yekaterina, Mello, Craig

[International Worm Meeting, 2021]

Mechanisms that safeguard the germline genome are essential for species preservation. For example, in metazoans the PIWI Argonaute pathway promotes genome integrity by silencing transposons and newly acquired foreign genes. PIWI Argonautes engage genomically-encoded piRNAs to detect their targets and to trigger gene regulation at both the mRNA and transcriptional level. However, the question of how foreign or aberrant gene expression is detected in the germline is still poorly understood. In yeast, genes with suboptimal mRNA splicing signals can trigger Argonaute silencing, and a similar connection has been made in the silencing of C. elegans early embryo transcripts with weak and abnormal splicing by the ERGO-1 ERI small RNA pathway. Here we show that the removal of introns from a gene and replacement with a cDNA-derived open reading frame results in complete silencing of the gene in the C. elegans germline. For example, a transgenic copy of gfp-tagged cdk-1 gene (cdk-1::gfp) yields robust germline and somatic expression. However, an intronless cdk-1::gfp transgene fails to express in the germline and can also transitively silence other intron-containing gfp transgenes. Transitive silencing depends on the WAGO Argonaute pathway and is correlated with robust production of RNA-dependent RNA Polymerase (RdRP)-derived antisense small RNAs from the unspliced RNA. Silencing and small RNA production were also observed when introns were removed from the endogenous oma-1 gene. Surprisingly, although transitive silencing requires the WAGO Argonaute pathway, the intronless genes (themselves) remain silenced in WAGO-pathway mutants, suggesting that an additional, cis-mode, of silencing occurs on intronless genes. The known primary Argonautes including prg-1, ergo-1, alg-3/4, and rde-1 are not required for the initiation of "intronless" silencing. Northern blot analysis revealed that intronless genes yield only a shorter mRNA isoform that appears to lack polyadenylation. These findings suggest mRNA splicing serves as a licensing step in germline gene expression promoting both polyadenylation and protein translation in cis, while preventing mRNAs from becoming default templates for RdRP-dependent transitive Argonaute silencing.

Ishidate, Takao et al. (2021) International Worm Meeting "Proteolysis dependent gene silencing in C.elegans germline"

Makeyeva, Yekaterina, Ishidate, Takao, Shirayama, Masaki, Mello, Craig

[International Worm Meeting, 2021]

Animal germ cells employ small RNAs and Argonaute proteins to surveil transcripts and silence transposons and foreign genetic elements. Here we report that, in C.elegans germline, expression of a transgene encoding codon optimized Cas9 gene fused to GFP is silenced via small RNA dependent and small RNA independent mechanisms. Interestingly, small RNA-dependent silencing was initiated and small RNAs targeting Cas9::GFP sequence were generated even in the absence of prg-1 activity. Furthermore, disarming the small RNA pathway led to Cas9::GFP expression only in the most distal region of the gonad, suggesting that a second small RNA-independent silencing mechanism maintains the silencing throughout the pachytene region and in oocytes. A forward genetic screen revealed that inactivation of the ubiquitin-proteasome pathway causes full de-silencing of the CAS9::GFP throughout the germline, suggesting that the second silencing mechanism is mediated by protein degradation. Remarkably, insertion of virus derived T2A sequence that causes ribosome skipping, between Cas9 and GFP, lead to the expression of the downstream GFP moiety throughout the gonad. This observation suggests that the upstream Cas9 ORF is triggering protein degradation and that the mRNA is not degraded and can still encode downstream GFP. Thus ribosome subunits are likely not dissociated as in the RQC response to ribosome stalling. We propose that some modification within the upstream Cas9 mRNA signals for co-translational ubiquitylation and degradation of the peptide. Interestingly, a Cas9 transgene that contains multiple extended AT rich introns has been shown to escape this mode of silencing, pointing toward mRNA splicing as the triggering event and linking the phenomenon to the recently described intronless gene silencing. We are currently investigating the nature of the triggering signal and whether our findings extend to the regulation of endogenous targets.