ResearcH
Research Overview:
Small RNA pathways are evolutionarily conserved mechanisms that regulate the expression of genetic elements. These pathways are responsible for maintaining homeostasis and appropriate gene expression, and silence deleterious foreign DNA, such as transposable elements (TEs) in eukaryotes. Integral components of small RNA pathways are RNA-induced silencing complexes (RISCs), made up of an Argonaute protein and its associated small RNA. RISC-associated small RNAs are typically ~20-30 nucleotides (nt) in length and guide Argonautes to target mRNAs for cleavage or epigenetic marker-mediated transcriptional repression. There are three major classes of small RNAs – microRNAs (miRNAs), small-interfering RNAs (siRNAs), and Piwi-interacting RNAs (piRNAs) - each defined by their mode of biogenesis, Argonaute cofactor, and mechanism of action on target mRNAs. In germ cells, many small RNA pathway components localize to distinct membrane-less, perinuclear germ granules, that act as hubs for RISC-mediated processing of mRNAs. One important process that occurs within germ granules is the amplification of small RNAs to promote efficient heritable silencing of targets. For example, in Caenorhabditis elegans, the recruitment of a transcript targeted by piRNAs or ‘primary’ siRNAs to a perinuclear germ granule, termed the Mutator focus, initiates the amplification of 22-nt ‘secondary’ siRNAs (22G-RNAs) by the mutator complex. The 22G-RNAs are then loaded into an Argonaute to perform transcriptional or post-transcriptional silencing. The amplification of 22G-RNAs is essential for the maintenance of small RNA-mediated regulation. Because small RNA pathway-mediated gene regulation is involved in many physiological processes, our research will have broad biomedical impacts.
The Rogers Lab is interested in regulatory mechanisms that promote genome stability and homeostasis through small RNA pathways. We aim to understand how evolutionarily-conserved small RNA pathways are regulated, and how mis-regulation of these pathways impact gene expression and physiological processes such as fertility and development by investigating three core questions:
What are the regulatory mechanisms that control small RNA pathways, and how does this regulation impact physiological processes?
How do small RNA and chromatin modifying pathways work together to maintain genome stability?
What are the subcellular and processing fates of small RNA pathway-targeted transcripts, and how are these fates altered by genetic and environmental perturbations?
Projects in the lab:
Homeostatic regulation of small RNA pathways
To date, most studies have focused on the mechanisms of gene regulation by small RNA pathways, but the mechanisms that regulate these pathways remain elusive. We expect diverse regulatory mechanisms may be used to maintain homeostasis of small RNA pathways. We are bioinformatically identifying the mechanisms that regulate small RNA pathways (loss of targeting by small RNAs, disruption of splicing, changes in chromatin modifications, modulation by sensor genes, etc.) and validating the existence and function of the regulatory mechanisms using a combination of RNA interference (RNAi) function assays and genetics. Subsequently, we will study the phenotypic effects of ablating the regulatory mechanisms identified using fertility assays, assessing small RNA populations and mRNA expression in the germline, and assessing maintenance of heritable epigenetic signals. Currently, we are using the Sensor of siRNAs-1 (sosi-1) feedback mechanism (Rogers & Phillips, Cell Reports, 2020) as a paradigm to study the long-term physiological effects of disrupting autoregulation of ERGO-1-class siRNA levels.
This work will have broad impacts on our understanding of gene regulation and make progress toward the lab's long-term goal of understanding how small RNA pathways are regulated to promote fertility and proper development and ensure homeostasis.
The synergism of small RNA and chromatin modifying pathways
Small RNA and chromatin modifying pathways both function to maintain genome integrity and regulate gene expression; however, how they work synergistically to maintain appropriate gene expression during adverse environmental conditions is unclear.
Heat stress causes a global increase in chromatin accessibility in the germline nuclei of C. elegans, which is further exacerbated upon loss of 22G-RNAs in mut-16 mutants, which lack the ability to amplify 22G-RNAs and are thus RNAi defective. Ultimately, the disruption of small RNA pathways in addition to the perturbation of chromatin accessibility in heat-stressed RNAi mutants, results in aberrant gene expression and a loss of germ cell identity, rendering the animals sterile. Intriguingly, the expression of only a subset of genes is susceptible to the global changes in chromatin state in mut-16 mutants (Rogers & Phillips, Nucleic Acids Research, 2020) . To further understand how gene regulation is impacted by environmental conditions, we will explore the interconnections between small RNA and chromatin modifying pathways.
This project will provide important insights into how small RNA and chromatin modifying pathways interact, and how these gene regulatory pathways are impacted by environmental perturbations.
Processing and subcellular fates of RISC-targeted transcripts
The subcellular localization, and subsequent processing fates, of mRNAs once they are targeted by RISC is unknown. To be able to harness distinct small RNA pathways for synthetic gene regulation, it is fundamental that we know how targets are channeled for selection and processing. We are using the heterologous in vivo tethering assay, I previously used in D. melanogaster (Rogers et. al, Genes and Development, 2017), to determine the effects of distinct small RNA pathway factors on (i) transcript subcellular localization, (ii) initiation of small RNA production, (iii) mRNA expression, and (iiii) recruitment of effector complexes to establish epigenetic silencing marks. Subsequently, we will determine the necessity and sufficiency of each factor to trigger these processing and subcellular fates.
It is unknown how small RNA pathway-mediated transcript fates are impacted by environmental perturbations. We will assess how transcript fates are altered in temperature-sensitive sterile small RNA pathway mutants by performing the above described experiments in wild-type and mut-16 mutant animals in normal and heat stress conditions.
This project will provide key insights to the mechanisms governing the sorting and processing of mRNAs by the distinct small RNA pathways and provide a foundation for future studies on the molecular mechanisms underlying mis-regulation of genes by small RNA pathways.
Research funded by:
