Wu, Monica et al. (2015) International Worm Meeting "Characterization of the Argonaute protein C04F12.1/VSRA-1 in C. elegans development."

Weng, Zhiping, Wu, Monica, Claycomb, Julie, Tu, Shikui

[International Worm Meeting, 2015]

At the core of all small RNA pathways are the Argonaute (AGO) proteins, which interact with particular small RNA binding partners to regulate gene expression. C. elegans possesses 26 AGO family proteins and four types of endogenous small RNAs. Thus far, the characterization of a handful of AGOs in the worm has shown that they bind to non-overlapping subsets of small RNAs to perform distinct regulatory functions throughout development. However, a large number of the AGOs remain to be characterized, which could ultimately lead to novel insights into small RNA mediated gene regulatory functions. We have investigated the role that an uncharacterized, but well-conserved AGO, C04F12.1, plays in small RNA-mediated gene regulation throughout development. C04F12.1 is broadly expressed across most developmental stages and tissues, but is particularly enriched in the germline. Excitingly, C04F12.1 localizes to cytoplasmic germ granules (P granules) and to the nucleus, implicating C04F12.1 in both transcriptional and/or post-transcriptional gene regulation. To dissect the molecular functions of C04F12.1, we have cloned and Illumina sequenced small RNAs in both the C04F12.1(tm1637) mutant background and from C04F12.1 complexes. Remarkably, our analyses reveal that C04F12.1 associates with multiple classes of small RNAs, including a subset of miRNAs, piRNAs, and 22G-RNAs, whereas previously characterized worm AGOs generally associate with only one particular type of small RNA. Because of its unique capacity to associate with multiple small RNA classes, we have named C04F12.1 VSRA-1 (Versatile Small RNAs Argonaute-1). Consistent with functional roles utilizing multiple small RNA classes, vsra-1 displays synthetic genetic interactions with the AGOs generally associated with these categories of small RNAs (alg-1, prg-1, csr-1). In vivo reporter assays and mRNA-seq data indicate the functional importance of VSRA-1 and point to a role in silencing gene expression. These findings are exciting and point to a novel activity for the AGO VSRA-1 in multiple small RNA pathways. Further characterization of VSRA-1 will likely reveal new molecular mechanisms of AGO/small RNA-mediated gene regulation, including points of cross-talk and specificity between small RNA pathways. Furthermore, due to its unique property of being able to associate with multiple classes of small RNAs that generally possess different biochemical features, VSRA-1 can be exploited as a valuable tool in dissecting the requirements for small RNA loading onto particular AGO proteins in the worm.

Svendsen, J. et al. (2017) International Worm Meeting "A probable link between primary small RNA methylation and secondary siRNA amplification."

Svendsen, J., Montgomery, T., Brown, K., Montgomery, B., Tucci, R.

[International Worm Meeting, 2017]

In C. elegans, primary small RNAs initiate both endogenous and exogenous RNAi by directing the association of target mRNAs with a secondary small interfering RNA (siRNA) amplification complex. The siRNA amplification complex contains an RNA-dependent RNA polymerase as well as several MUT/RDE proteins with unknown function. There are two classes of primary small RNAs, piRNAs and ERGO-1 class 26G-RNAs, that trigger mRNA entry into the MUT/RDE-dependent siRNA amplification pathway. Both piRNAs and ERGO-1 class 26G-RNAs are methylated at their 3' ends by the 2'-O methyltransferase henn-1, which is thought to be required for protection from exonuclease-mediated degradation. However, other small RNAs, including miRNAs, CSR-1 class 22G-RNAs, and ALG-3/4 class 22G-RNAs are not methylated. It is possible that primary small RNA methylation is in some way linked to secondary siRNA amplification. If so, primary small RNAs produced through the exogenous RNAi pathway, which are biochemically distinct from endogenous small RNAs, should also be methylated. To test this, we sequenced small RNAs from animals undergoing RNAi using an approach that allows for enrichment of methylated small RNAs. Indeed, primary, but not secondary, exogenous siRNAs are also methylated. Exogenous primary siRNAs bind to the Argonaute RDE-1. RDE-1 also binds a subset of endogenous small RNAs, most notably miRNAs. Although miRNAs are not typically methylated, our results indicate that those that associate with RDE-1 are methylated. We are currently exploring the mechanistic link between primary small RNA methylation and secondary siRNA amplification and the role of Argonautes in dictating which small RNAs are methylated.

Tabach, Yuval et al. (2013) International Worm Meeting "Identification of small RNA pathway genes using patterns of phylogenetic conservation and divergence."

Borowsky, Mark, Newman, Martin, Ruvkun, Gary, Hayes, Gabriel, Billi, Allison, Zuk, Or, Gabel, Harrison, Garcia, Susana, Chapman, Brab, Kamath, Ravi, Kim, John, Tabach, Yuval

[International Worm Meeting, 2013]

Genetic and biochemical analyses of RNA interference (RNAi) and microRNA (miRNA) pathways have revealed proteins such as Argonaute and Dicer as essential cofactors that process and present small RNAs to their targets. Well-validated small RNA pathway cofactors such as these show distinctive patterns of conservation or divergence in particular animal, plant, fungal and protist species. We compared 86 divergent eukaryotic genome sequences to discern sets of proteins that show similar phylogenetic profiles with known small RNA cofactors. A large set of additional candidate small RNA cofactors have emerged from functional genomic screens for defects in miRNA- or short interfering RNA (siRNA)-mediated repression in Caenorhabditis elegans and Drosophila melanogaster, and from proteomic analyses of proteins co-purifying with validated small RNA pathway proteins. The phylogenetic profiles of many of these candidate small RNA pathway proteins are similar to those of known small RNA cofactor proteins. We used a Bayesian approach to integrate the phylogenetic profile analysis with predictions from diverse transcriptional coregulation and proteome interaction data sets to assign a probability for each protein for a role in a small RNA pathway. Testing high-confidence candidates from this analysis for defects in RNAi silencing, we found that about one-half of the predicted small RNA cofactors are required for RNAi silencing. Many of the newly identified small RNA pathway proteins are orthologues of proteins implicated in RNA splicing. In support of a deep connection between the mechanism of RNA splicing and small-RNA-mediated gene silencing, the presence of the Argonaute proteins and other small RNA components in the many species analysed strongly correlates with the number of introns in those species.

Cheng, H et al. (2019) International Worm Meeting "A novel Zc3h12a ribonuclease protein participates in small RNA mediated spermatogenesis pathway in C. elegans."

Tsai, H, Cheng, H

[International Worm Meeting, 2019]

The importance of small RNA in sperm is conserved from C. elegans to mammals. Variety of small RNAs are known not only important for spermatogenesis but also affects how sperm influence its offspring. Previous studies have revealed a set of sperm-specific small RNAs and their associated argonautes in C. elegans. Though missing this small RNA-argonaute complex results in temperature sensitive (ts) spermatogenesis defect has established, however, the detail mechanism of how sperm get damaged at elevated temperature and how these small RNAs are synthesized and processed are still largely unknown. Here, we identify a novel zc3h12a ribonuclease gene in C. elegans, c29f5.3. Zc3h12a ribonuclease protein is found in all three domains of life but only few are characterized. Zc3h12a in mammals are known targeting 3' utr of selective mRNA to regulate transcript turnover. Interestingly, our allele of c29f5.3 have shown ts sperm defect as well as sperm specific small RNA downregulation in C. elegans similar to the phenotype observed in losing sperm-specific small RNA-argonaute complex suggesting c29f5.3 plays a role in sperm specific small RNA biogenesis. Our current efforts focus on generating crispr kncokin line to identify its localization and RNA protein interactors.

Wu, Monica Z. et al. (2013) International Worm Meeting "Characterization of the AGO protein VSR-1 in small RNA-mediated gene silencing pathways in the worm."

Wu, Monica Z., Claycomb, Julie M.

[International Worm Meeting, 2013]

Argonautes (AGOs) are the key effector components of RNA interference (RNAi) and related endogenous small RNA pathways. C. elegans possesses 26 AGO family proteins and although deletion mutant strains for each of the C. elegans AGOs have been generated, the functions of only a handful of these proteins are understood. The AGOs that have been characterized thus far have been shown to perform distinct functions. Therefore, further investigation of the remaining AGOs can lead to novel insights into small RNA mediated gene-silencing functions. We have been investigating the role that a relatively uncharacterized, but well-conserved AGO protein, which we have named VSR-1 (Versatile Small RNAs), plays in small RNA-mediated gene regulation in C. elegans. mRNA and protein expression analysis demonstrates that VSR-1 is expressed throughout development, but is enriched in later stages when the germline develops, implicating VSR-1 in germline small RNA functions. Immunolocalization studies show that VSR-1 localizes to embryonic P granules, consistent with localization patterns of other Argonautes, including WAGO-1 and CSR-1. VSR-1 also localizes to oocyte and embryonic chromatin, which has been confirmed through biochemical fractionation experiments, suggesting a role for this AGO in transcriptional regulation or other nuclear functions. To identify with which small RNAs VSR-1 interacts, we have cloned and Illumina sequenced small RNAs in both the vsr-1 mutant background and from VSR-1 complexes. Consistent with VSR-1 playing a role in the biogenesis of particular small RNAs, the small RNAs enriched in VSR-1 complexes are depleted in the mutant. Our initial analyses indicate that VSR-1 associates with particular subsets of small RNAs spanning multiple classes. These small RNAs have been implicated in playing key roles in germline and embryonic development. These findings are exciting and point to a novel activity for VSR-1 in multiple small RNA pathways, as all other AGOs studied thus far have been shown to associate with one particular class of small RNAs in the worm. Our ongoing studies will reveal additional insights into the roles of VSR-1 in worm development.

Rechavi, O. (2019) International Worm Meeting "Neuronal small RNAs control behavior transgenerationally."

Rechavi, O.

[International Worm Meeting, 2019]

Neuronal circuits can coordinate responses and integrate environmental cues like no other tissue. We hypothesized that small RNAs could transmit information originating from parental neuronal activity and impact the next generations. We found that neurons-specific synthesis of RDE-4-dependent small RNAs regulate the pool of heritable small RNAs and germline gene expression for multiple generations. Further, the production of small RNAs in neurons controls the chemotaxis behavior of the progeny for at least three generations via the germline Argonaute HRDE-1. Among other genes targeted by small RNAs derived from neuronal RDE-4 activity, we detected a conserved gene that is transgenerationally downregulated, and silencing of this gene by neuronal small RNAs is required for proper chemotaxis under stressful conditions. We will present new insights supporting a non-canonical small RNA-based mechanism for communication of neuronal processes transgenerationally. We will describe new results regarding the path that the signal takes from neurons to the germline. Through this route, neuronal responses to external stimuli or internal physiological states could be translated into inheritable information and affect the behavior, and possibly the fitness, of the next generations.

Wu, Monica et al. (2015) International Worm Meeting "Comparative Functional Characterization of the CSR-1 22G-RNA Pathway in Caenorhabditis."

Weng, Zhiping, Wu, Monica, Wang, Jie, Tu, Shikui, Julie, Claycomb, Cutter, Asher

[International Worm Meeting, 2015]

As a champion of small RNA research for two decades, C. elegans has revealed the essential Argonaute CSR-1 to play key nuclear roles in modulating chromatin, chromosome segregation, and germline gene expression via 22G-small RNAs. Despite CSR-1 being preserved among diverse nematodes (Clade III and Clade V), the conservation and divergence in function of the targets of small RNA pathways remains poorly resolved. Here we apply comparative functional genomic analysis between C. elegans and C. briggsae to characterize the CSR-1 small RNA pathway, its targets, and their evolution. C. briggsae CSR-1-associated small RNAs that we identified by immunoprecipitation-small RNA sequencing overlap with 22G-RNAs depleted in cbr-csr-1 RNAi-treated worms. Notably, our analysis of CSR-1 associated small RNAs in C. briggsae revealed a previously unappreciated class of 22A-RNAs that are also present in C. elegans. By comparing 22G-RNAs and target genes between species, we defined a set of CSR-1 target genes mostly with conserved germline expression, enrichment in operons, and more slowly-evolving coding sequences than other germline genes, along with a small group of evolutionarily labile targets. We demonstrate that the association of CSR-1 with chromatin is preserved between species, and show that depletion of cbr-csr-1 leads to chromosome segregation defects and embryonic lethality. This first comparative characterization of a small RNA pathway in Caenorhabditis establishes a conserved nuclear role for CSR-1 and highlights its key role in germline gene regulation across multiple animal species.

Zagoskin, Maxim et al. (2021) International Worm Meeting "Programmed DNA Elimination in the parasitic nematode Ascaris: Are Argonautes and their associated small RNAs involved?"

Zagoskin, Maxim, Davis, Richard, Wang, Jianbin

[International Worm Meeting, 2021]

Programmed DNA elimination in the nematode Ascaris is a developmentally regulated process that reduces the somatic genome, while leaving the germline genome intact. It occurs in early embryogenesis at 4 to 16 cell stage in all somatic lineages. The eliminated DNA consist of repetitive sequence as well as ~1,000 germline-expressed genes. The biological role of this process appears in part to be a gene silencing mechanism. Ascaris DNA elimination involves specific chromosomal DNA breaks and changes in the holocentric chromosomes. The DNA breaks and loss of CENP-A in specific chromosomal regions defines the retained and eliminated regions of chromosomes. In ciliate programmed DNA elimination, small RNAs are known to mark retained or eliminated genome regions during DNA elimination. To examine if small RNAs contribute to Ascaris programmed DNA elimination, we identified Ascaris small RNAs, 10 Argonautes, and characterized Ascaris small RNAs associated with 7 Argonaut proteins including all WAGOs. Immunostaining of embryos during programmed DNA elimination indicated that WAGO-2 specifically stains retained DNA while WAGO-3 predominantly localizes to chromosome fragments that will be eliminated. ChIP-seq data also showed some enrichment of WAGO-3 Argonaute on eliminated DNA regions. We also carried out small RNA sequencing and analysis in embryos enriched for DNA elimination mitoses by sorting with H3S10p labeled embryos or chromatin IP with WAGO-2 and WAGO-3. Small RNA sequencing of embryos enriched for DNA elimination (sorted) did not identify any small RNAs associated with the sites of chromosome breaks or changes in CENP-A localization that contribute to DNA elimination. Chromatin IP identified specific sets of small RNAs corresponding to repeats for WAGO-2 and mRNAs for WAGO-3. Overall, however, these small RNAs do not appear to specifically target retained vs eliminated regions as anticipated. WAGO-3 small RNAs appears to be enriched for nascent RNAs associated the eliminated regions. The possible role of WAGO-2 and WAGO-3 and small RNAs in Ascaris programmed DNA elimination will be further discussed.

Manoharan, Arun Prasad et al. (2009) International Worm Meeting "small RNA profiling in the C. elegans germline and germ cells."

Hirst, Martin, Khivansara, Vishal, Manoharan, Arun Prasad, Chu, Diana, Kim, John, Fitzpatrick, Colin, Marra, Marco, Han, Ting

[International Worm Meeting, 2009]

To identify classes of small RNAs expressed in the germline and germ cells, we performed a genome-wide analysis of the small RNA transcriptome in C. elegans. Small RNAs play central roles in regulating germline development. Mutations affecting various small RNA pathways are frequently associated with the loss of fertility. To uncover small RNAs enriched in the germline and germ cells, we sequenced the small RNAs expressed in N2 worms, the glp-4(bn2) temperature-sensitive germline mutant, purified sperm from him-8 and purified oocytes from fer-1, and in N2 embryos. By high-throughput deep sequencing, using the Solexa (Illumina) and 454 (Roche) platforms, we generated over 14 million sequence reads that map perfectly to the C. elegans genome. Comparative analysis allowed us to determine enrichment of particular small RNAs to different cell types. For example, comparing the expression of small RNAs in N2 vs. glp-4 identified 25 miRNAs that were enriched 5-fold or higher in the germline and 19 miRNAs that were enriched in the soma. Comparisons between oocyte and sperm samples identified 49 miRNAs that were enriched 5-fold or higher in oocytes but only 3 miRNAs that were enriched in sperm. Computational analysis indicates that the putative miRNA targets are also enriched in these particular tissues or cell types. In addition to comparative analysis of known classes of small RNAs, we have identified 70 potentially novel microRNAs as well as additional germline-expressed 21U RNAs (1-4), many of which we validated by northern blot analysis and a PCR-amplified sequencing method. Finally, deep sequencing revealed a class of endogenous siRNAs, the 26G RNAs, which are enriched in sperm, oocytes, and embryos (discussed in a separate abstract.). We are currently investigating a select number of small RNAs for further analysis. Our deep sequencing of the small RNA transcriptome may provide a useful resource for future studies of gene regulation mediated by different classes of small RNAs in the germline. 1.Ruby, J.G. et al., Cell (2006) 127:1193-1207. 2.Batista, P.J. et al., Mol. Cell (2008) 31: 67-78. 3.Das, P.P. et al., Mol Cell (2008) 31: 79-90. 4.Wang, G. and Reinke, V., Curr. Biol. (2008) 18: 861-867.

Batista, Pedro J. et al. (2009) International Worm Meeting "Characterization of the small RNA populations of C. briggsae and natural isolates of C. elegans."

Hammell, Molly C., Mello, Craig C., Shirayama, Masaki, Batista, Pedro J., Youngman, Elaine M., Gu, Weifeng

[International Worm Meeting, 2009]

Small-RNA-mediated pathways are involved in a myriad of processes that regulate gene expression. In Caenorhabditis elegans several classes of endogenous small RNAs have been identified. At least two of these classes - the Piwi-associated small RNAs (21U-RNAs) and the worm argonaute (WAGO)-associated small RNAs (22G-RNAs) - are expressed in and essential for the proper development and function of the germline. However the biogenesis and functions of these small RNA classes remain almost entirely mysterious. We have taken a phylogenetic approach to explore small RNA biogenesis and function in Caenorhabditis. We have sequenced small RNA libraries generated from wild isolates of C. elegans (AB1, KR314, JU258, RW7000 and CB4856), as well as from the related species Caenorhabditis briggsae. As expected, the C. elegans wild strains exhibited globally similar levels for the various known classes of small RNAs. However, substantial variations from the N2 levels were observed at fewer than 10% of the 22G-RNA-generating loci, and at fewer than 15% of the 21U loci. In some cases one or more wild isolates exhibited a complete lack of specific 22G or 21U species that are abundant in N2. Our analysis so far suggests that these changes rarely reflect deletions that remove the corresponding sequence, although in many cases there are small insertions or deletions in nearby regions. For those cases where the corresponding regions remain structurally intact we are now searching by DNA sequencing in the wild isolates for alterations that might underlie the absence or acquisition of 22G or 21U-RNA species. Small RNAs with the hallmarks of both 22G- and 21U-RNAs are present in C. briggsae, as are the Argonautes and other factors involved in their biogenesis. As in C. elegans, 22G-RNAs are the most abundant small RNAs in C. briggsae. The C. briggsae 21U-RNAs are highly divergent but associated to the same upstream motif originally characterized in C. elegans, and like their C. elegans counterparts begin predominanty with a U and are modified at the 3'' end. Curiously, while the vast majority of 21U-RNAs in C. elegans arise from 2 clusters on chromosome IV, in C. briggsae these small RNAs are generated from at least 3 clusters: 2 on chromosome IV and 1 on chromosome I. Ultimately, we believe this examination of small RNA populations in both closely- and distantly-related nematode strains will provide important insight into the largely mysterious mechanisms that underlie the biogenesis and function of these small RNA pathways.