Monika Priyadarshini

All Publications

• Reprogramming the piRNA pathway for multiplexed and transgenerational gene silencing in C. elegans. Nature methods Priyadarshini, M., Ni, J. Z., Vargas-Velazquez, A. M., Gu, S. G., Frøkjær-Jensen, C. 2022; 19 (2): 187-194

  Abstract

  Single-guide RNAs can target exogenous CRISPR-Cas proteins to unique DNA locations, enabling genetic tools that are efficient, specific and scalable. Here we show that short synthetic guide Piwi-interacting RNAs (piRNAs) (21-nucleotide sg-piRNAs) expressed from extrachromosomal transgenes can, analogously, reprogram the endogenous piRNA pathway for gene-specific silencing in the hermaphrodite germline, sperm and embryos of Caenorhabditis elegans. piRNA-mediated interference ('piRNAi') is more efficient than RNAi and can be multiplexed, and auxin-mediated degradation of the piRNA-specific Argonaute PRG-1 allows conditional gene silencing. Target-specific silencing results in decreased messenger RNA levels, amplification of secondary small interfering RNAs and repressive chromatin modifications. Short (300 base pairs) piRNAi transgenes amplified from arrayed oligonucleotide pools also induce silencing, potentially making piRNAi highly scalable. We show that piRNAi can induce transgenerational epigenetic silencing of two endogenous genes (him-5 and him-8). Silencing is inherited for four to six generations after target-specific sg-piRNAs are lost, whereas depleting PRG-1 leads to essentially permanent epigenetic silencing.

  View details for DOI 10.1038/s41592-021-01369-z

  View details for PubMedID 35115715

  View details for PubMedCentralID PMC9798472

• Target-dependent suppression of siRNA production modulates the levels of endogenous siRNAs in the Caenorhabditis elegans germline. Development (Cambridge, England) Gajic, Z., Kaur, D., Ni, J., Zhu, Z., Zhebrun, A., Gajic, M., Kim, M., Hong, J., Priyadarshini, M., Frøkjær-Jensen, C., Gu, S. 2022; 149 (16)

  Abstract

  Despite the prominent role of endo-siRNAs in transposon silencing, their expression is not limited to these 'nonself' DNA elements. Transcripts of protein-coding genes ('self' DNA) in some cases also produce endo-siRNAs in yeast, plants and animals. How cells distinguish these two populations of siRNAs to prevent unwanted silencing of active genes in animals is not well understood. To address this question, we inserted various self-gene or gfp fragments into an LTR retrotransposon that produces abundant siRNAs and examined the propensity of these gene fragments to produce ectopic siRNAs in the Caenorhabditis elegans germline. We found that fragments of germline genes are generally protected from production of ectopic siRNAs. This phenomenon, which we termed 'target-directed suppression of siRNA production' (or siRNA suppression), is dependent on the germline expression of target mRNA and requires germline P-granule components. We found that siRNA suppression can also occur in naturally produced endo-siRNAs. We suggest that siRNA suppression plays an important role in regulating siRNA expression and preventing self-genes from aberrant epigenetic silencing. This article has an associated 'The people behind the papers' interview.

  View details for DOI 10.1242/dev.200692

  View details for PubMedID 35876680

  View details for PubMedCentralID PMC9481970

• Acute and inherited piRNA-mediated silencing in a rde-3 ribonucleotidyltransferase mutant. microPublication biology Priyadarshini, M., AlHarbi, S., Frokjar-Jensen, C. 2022; 2022

  Abstract

  We recently developed a piRNA-based silencing assay (piRNAi) to study small-RNA mediated epigenetic silencing: acute gene silencing is induced by synthetic piRNAs expressed from extra-chromosomal array and transgenerational inheritance can be quantified after array loss. The assay allows inheritance assays by injecting piRNAs directly into mutant animals and targeting endogenous genes ( e.g. , him-5 and him-8 ) with obvious phenotypes (increased male frequency). Here we demonstrate the piRNAi assay by quantifying acute and inherited silencing in the ribonucleotidyltransferase rde-3 (ne3370) mutant. In the absence of rde-3, acute silencing was reduced but still detectable, whereas inherited silencing was abolished.

  View details for DOI 10.17912/micropub.biology.000638

  View details for PubMedID 36188099

• Engineering rules that minimize germline silencing of transgenes in simple extrachromosomal arrays in C. elegans. Nature communications Aljohani, M. D., El Mouridi, S., Priyadarshini, M., Vargas-Velazquez, A. M., Frøkjær-Jensen, C. 2020; 11 (1): 6300

  Abstract

  Transgenes are prone to progressive silencing due to their structure, copy number, and genomic location. In C. elegans, repressive mechanisms are particularly strong in the germline with almost fully penetrant transgene silencing in simple extrachromosomal arrays and frequent silencing of single-copy transgene insertions. A class of non-coding DNA, Periodic An/Tn Clusters (PATCs) can prevent transgene-silencing in repressive chromatin or from small interfering RNAs (piRNAs). Here, we describe design rules (codon-optimization, intron and PATC inclusion, elevated temperature (25 °C), and vector backbone removal) for efficient germline expression from arrays in wildtype animals. We generate web-based tools to analyze PATCs and reagents for the convenient assembly of PATC-rich transgenes. An extensive collection of silencing resistant fluorescent proteins (e.g., gfp, mCherry, and tagBFP) can be used for dissecting germline regulatory elements and a set of enhanced enzymes (Mos1 transposase, Cas9, Cre, and Flp recombinases) enable efficient genetic engineering in C. elegans.

  View details for DOI 10.1038/s41467-020-19898-0

  View details for PubMedID 33298957

  View details for PubMedCentralID PMC7725773