Research

A major gap in our understanding of RNA silencing lies in determining why some RNAs are targeted for silencing while others are not. We believe that a viable entry into this question is through studying the cell biology of RNA silencing. Germ granules are key hubs of RNA silencing, yet we know little about how they are organized and assembled. There are multiple subdomains of the germ granule, yet how RNAs are trafficked into and through these granules remains a mystery. This information is key to our understanding of how detrimental RNAs are identified and ultimately silenced. Because the proteins involved in RNA silencing are clustered together in subdomains of the germ granule, the mechanisms by which proteins and RNAs assemble at and are trafficked through these sites will be an essential piece in determining why particular RNAs are selected for silencing and others are not. Therefore, we seek to identify the factors that promote assembly of germ granule subdomains and examine how RNAs localize to and are regulated by germ granules.

For more details, see:

Uebel CJ, Rajeev, S,Phillips CM. Caenorhabditis elegans germ granules are present in distinct configurations that differentially associate with RNAi-targeted RNAs. BioRxiv. 2023  link

Manage KI et al. A Tudor domain protein, SIMR-1, promotes siRNA production at piRNA-targeted mRNAs in C. elegans. eLife. 2020  PDF

Uebel CJ et al. Distinct regions of the intrinsically disordered protein MUT-16 mediate assembly of a small RNA amplification complex and promote phase separation of Mutator foci. PLoS Genet. 2018  PDF

With 19 functional C. elegans Argonaute proteins, many of which localize to germ granules, the mechanisms by which small RNAs are robustly sorted into the correct Argonaute proteins are still mysterious. This sorting is critical because many Argonaute proteins bind different small RNAs from one another, target distinct groups of mRNAs, and have very different regulatory effects on these target mRNAs. We hypothesize that specificity for a particular class of small RNAs can be driven by Argonaute preference for certain small RNA features, protein-protein interactions made by each Argonaute protein, particularly with components of the small RNA biogenesis machinery, and/or spatial separation of distinct small RNA pathways. We seek to understand how these factors contribute individually and together to promote small RNA specificity and whether each Argonaute protein has distinct requirements.

For more details, see:

Chen S, Phillips CM. Germ granule association drives small RNA specificity for a nuclear Argonaute protein. BioRxiv. 2023 link

Nguyen DAH, Phillips CM. Arginine methylation promotes siRNA-binding specificity for a spermatogenesis-specific isoform of the Argonaute protein CSR-1. Nat Commun. 2021 PDF

Phillips CM et al. piRNAs and piRNA-dependent siRNAs protect conserved and essential C. elegans genes from misrouting into the RNAi pathway. Dev Cell. 2015 PDF

Beyond small RNA loading, additional layers of regulation are necessary to ensure that Argonaute proteins and other RNA silencing factors are properly localized, interacting with the correct protein partners, and able to perform their function in a dynamic manner. Protein modifications play a fundamental role in ensuring protein activity or function and can often be removed by deconjugating enzymes, allowing for much faster and more efficient modulation of a protein’s activity compared to protein degradation and
de novo synthesis. Thus, we are focusing on how protein modifications such as phosphorylation and methylation can regulate RNA pathways.

Recently, we have found that for one particular Argonaute protein, CSR-1A, we have found that methylation is essential to promote correct small RNA binding. We are currently investigating the role of methylation in the regulation of other Argonaute proteins and identifying factors that interact with Argonautes in a methylation-specific manner.

For more details, see:

Nguyen DAH, Phillips CM. Arginine methylation promotes siRNA-binding specificity for a spermatogenesis-specific isoform of the Argonaute protein CSR-1. Nat Commun. 2021 PDF