Cells of the reproductive system (germline) have a very important function of transmitting genetic information from parents to offspring. However, recent research has clearly established that in addition to DNA, germ cells also transmit information using epigenome. Epigenetic information includes DNA modification, modification of scaffold proteins involved in DNA packaging and small RNA (sRNA) molecules. My research is focused on studying sRNAs using the roundworm, Caenorhabditis elegans as a model. Apart from transmitting the life experience from parent to offspring, sRNAs also stop the expression of deleterious genes and provide defence against foreign elements in the germline to maintain genome integrity. However, in C. elegans germline, thousands of sRNAs are produced against self-germline genes. These sRNAs are loaded by a specialized Argonaute called CSR-1 which is an essential protein.
Adapted from Quarato, Singh, et al. BioEssays 2022
Function and biogenesis of small RNAs targeting all germline expressed genes.
CSR-1 is also the only essential Worm-specific AGO (WAGOs) with catalytic activity. Two antagonistic functions have been proposed for CSR-1: 1) direct cleavage of the CSR-1 germline targets to fine-tune their expression ('slicer’ activity), and/or 2) protection of endogenous germline mRNAs from piRNA-mediated silencing (‘anti-silencing’ activity). However, the biggest mystery in the field was- how CSR-1 22G-RNAs, which are antisense to self-transcripts, are produced. Using a worm-sorting strategy combined with proteomic, biochemical, and high-throughput genomic approaches, I showed that the biogenesis of CSR-1-associated 22G-RNAs on the coding sequence of target mRNAs requires CSR-1 slicer activity. Canonically, piRNA-triggered 22G-RNAs bound by other WAGOs in C. elegans (which participate in the silencing of non-self-genomic elements), are produced in phase-separated condensates called germ granules. But in this study, by integrating small RNA-seq and Ribo-seq, I showed that contrary to piRNA-dependent 22G-RNAs, CSR-1-associated 22G-RNAs are rather produced in cytosol on mRNAs engaged in translation. The levels of 22G-RNAs produced from target mRNAs inversely correlate with translational efficiency and are further dictated by the codon usage of the mRNAs. This is the first report on the regulation of small RNA biogenesis by translation in animals. This work was featured in the Editor’s highlight collection of “From molecules and cells to organisms” in Nature Communications.
Crosstalk of germline endogenous small RNA pathways.
Loss of piRNA and PIWI (Argonaute loading piRNAs) in C. elegans leads to transgenerational sterility. In this collaborative project and published manuscript where I am a co-first author, I used biochemical and proteomics tools to demonstrate that loss of PIWI led to a shift in interactors of downstream AGO (WAGO-1) of piRNA pathway, with an increased association with CSR-1 that is involved in cleaving the stem-loop at the 3’end of histone. This mislocalization and interaction of WAGO with CSR-1 resulted in the production of 22G-RNAs antisense to histone genes, which are transgenerationally inherited leading to a transgenerational sterility phenotype. With this study, we demonstrated the mechanism underlying transgenerational sterility of C. elegans PIWI mutants. This work was featured as Cover in Nature Cell Biology.
SARS-CoV-2 derived miRNA hijack host RNAi machinery to evade the immune response
With my expertise in small RNA biology, I collaborated with virologists and quickly adapted the methods and tools I had set up for C. elegans to sequence small RNAs from SARS-CoV-2 infected cells to identify virus-derived miRNA and elucidate its function. I discovered that virus-derived miRNAs13 are produced via host DICER1 and immunoprecipitation of human AGOs showed viral miRNAs are loaded by host AGO. I further identified Interferon-stimulated genes as targets for viral miRNA repression, revealing a role of viral miRNA in host immune evasion. This is the first report of a bona fide miRNA produced from a positive-strand RNA virus showing its role in hijacking host RNAi machinery to evade host immune response. This was published in EMBO Rep.
Role of Heat shock protein 90 and its multi-chaperone complex in the life cycle of protozoan parasites
During my integrated PhD at the Indian Institute of Science (IISc), Bangalore, I investigated the function of Heat Shock Protein 90 (Hsp90) in the life cycle and virulence of parasitic protozoa. Using three early branching protozoan parasites – Entamoeba histolytica, Trichomonas vaginalis and Theileria annulata, I addressed specific questions pertaining to the evolution of the Hsp90 multichaperone complex, its unique features, cellular functions, and regulation by co-chaperones. Using biochemical, cell biology and proteomics approaches I showed that Hsp90 is essential for the proliferation of all the three parasites. I specifically showed that Hsp90 regulates stage transition and phagocytosis in Entamoeba and identified a new co-chaperone regulating its ATPase activity. In collaboration with structural biologists, I identified a novel peptide inhibitor of Hsp90 binding to parasite Hsp90 with more efficiency than to the host. Lastly, I identified a secreted Hsp90 isoform in Trichomonas. Overall, I showed that Hsp90s from early branching protozoa differ from higher eukaryotes in terms of their biochemical and cell biological properties and play an essential role in their survival.