Mechanisms of epigenetic regulation by C. elegans nuclear RNA interference pathways.

RNA interference (RNAi) is a highly conserved gene regulatory phenomenon whereby Argonaute/small RNA (AGO/sRNA) complexes target transcripts by antisense complementarity to modulate gene expression. While initially appreciated as a cytoplasmic process, RNAi can also occur in the nucleus where AGO/sRNA complexes are recruited to nascent transcripts. Nuclear AGO/sRNA complexes recruit co-factors that regulate transcription by inhibiting RNA Polymerase II, modifying histones, compacting chromatin and, in some organisms, methylating DNA. C. elegans has a longstanding history in unveiling the mechanisms of RNAi and has become an outstanding model to delineate the mechanisms underlying nuclear RNAi. In this review we highlight recent discoveries in the field of nuclear RNAi in C. elegans and the roles of nuclear RNAi in the regulation of gene expression, chromatin organization, genome stability, and transgenerational epigenetic inheritance.

Two isoforms of the essential C. elegans Argonaute CSR-1 differentially regulate sperm and oocyte fertility.

The Caenorhabditis elegans genome encodes nineteen functional Argonaute proteins that use 22G-RNAs, 26G-RNAs, miRNAs or piRNAs to regulate target transcripts. Only one Argonaute is essential under normal laboratory conditions: CSR-1. While CSR-1 has been studied widely, nearly all studies have overlooked the fact that the csr-1 locus encodes two isoforms. These isoforms differ by an additional 163 amino acids present in the N-terminus of CSR-1a. Using CRISPR-Cas9 genome editing to introduce GFP::3xFLAG into the long (CSR-1a) and short (CSR-1b) isoforms, we found that CSR-1a is expressed during spermatogenesis and in several somatic tissues, including the intestine. CSR-1b is expressed constitutively in the germline. small RNA sequencing of CSR-1 complexes shows that they interact with partly overlapping sets of 22G-RNAs. Phenotypic analyses reveal that the essential functions of csr-1 described in the literature coincide with CSR-1b, while CSR-1a plays tissue specific functions. During spermatogenesis, CSR-1a integrates into an sRNA regulatory network including ALG-3, ALG-4 and WAGO-10 that is necessary for fertility at 25°C. In the intestine, CSR-1a silences immunity and pathogen-responsive genes, and its loss results in improved survival from the pathogen Pseudomonas aeruginosa. Our findings functionally distinguish the CSR-1 isoforms and highlight the importance of studying each AGO isoform independently.

A comprehensive survey of C. elegans argonaute proteins reveals organism-wide gene regulatory networks and functions.

Argonaute (AGO) proteins associate with small RNAs to direct their effector function on complementary transcripts. The nematode Caenorhabditis elegans contains an expanded family of 19 functional AGO proteins, many of which have not been fully characterized. In this work, we systematically analyzed every C. elegans AGO using CRISPR-Cas9 genome editing to introduce GFP::3xFLAG tags. We have characterized the expression patterns of each AGO throughout development, identified small RNA binding complements, and determined the effects of ago loss on small RNA populations and developmental phenotypes. Our analysis indicates stratification of subsets of AGOs into distinct regulatory modules, and integration of our data led us to uncover novel stress-induced fertility and pathogen response phenotypes due to ago loss.

Identification of tumor-agnostic biomarkers for predicting prostate cancer progression and biochemical recurrence.

The diverse clinical outcomes of prostate cancer have led to the development of gene signature assays predicting disease progression. Improved prostate cancer progression biomarkers are needed as current RNA biomarker tests have varying success for intermediate prostate cancer. Interest grows in universal gene signatures for invasive carcinoma progression. Early breast and prostate cancers share characteristics, including hormone dependence and BRCA1/2 mutations. Given the similarities in the pathobiology of breast and prostate cancer, we utilized the NanoString BC360 panel, comprising the validated PAM50 classifier and pathway-specific signatures associated with general tumor progression as well as breast cancer-specific classifiers. This retrospective cohort of primary prostate cancers (n=53) was stratified according to biochemical recurrence (BCR) status and the CAPRA-S to identify genes related to high-risk disease. Two public cohort (TCGA-PRAD and GSE54460) were used to validate the results. Expression profiling of our cohort uncovered associations between PIP and INHBA with BCR and high CAPRA-S score, as well as associations between VCAN, SFRP2, and THBS4 and BCR. Despite low levels of the ESR1 gene compared to AR, we found strong expression of the ER signaling signature, suggesting that BCR may be driven by ER-mediated pathways. Kaplan-Meier and univariate Cox proportional hazards regression analysis indicated the expression of ESR1, PGR, VCAN, and SFRP2 could predict the occurrence of relapse events. This is in keeping with the pathways represented by these genes which contribute to angiogenesis and the epithelial-mesenchymal transition. It is likely that VCAN works by activating the stroma and remodeling the tumor microenvironment. Additionally, SFRP2 overexpression has been associated with increased tumor size and reduced survival rates in breast cancer and among prostate cancer patients who experienced BCR. ESR1 influences disease progression by activating stroma, stimulating stem/progenitor prostate cancer, and inducing TGF-ß. Estrogen signaling may therefore serve as a surrogate to AR signaling during progression and in hormone-refractory disease, particularly in prostate cancer patients with stromal-rich tumors. Collectively, the use of agnostic biomarkers developed for breast cancer stratification has facilitated a precise clinical classification of patients undergoing radical prostatectomy and highlighted the therapeutic potential of targeting estrogen signaling in prostate cancer.

Connecting the Dots: Linking Caenorhabditis elegans Small RNA Pathways and Germ Granules.

Germ granules are non-membrane bound, phase-separated organelles, composed of RNAs and proteins. Germ granules are present only within the germ cells of animals, including model systems such as Caenorhabditis elegans, Drosophila, mice, and zebrafish, where they play critical roles in specifying the germ lineage, the inheritance of epigenetic information, and post-transcriptional gene regulation. Across species, conserved germ granule proteins reflect these essential functions. A significant proportion of proteins that localize to germ granules are components of RNA metabolism and small RNA (sRNA) gene regulatory pathways. Here we synthesize our current knowledge of the roles that germ granules and their components play in sRNA pathway functions, transgenerational inheritance, and fertility in the C. elegans germline.