[
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.
[
International Worm Meeting,
2021]
Nematoda is a diverse phylum that includes many free-living as well as parasitic species. The model nematode C. elegans has a diverse set of small RNAs and an expanded group of Argonautes, particularly the worm-specific clade Argonautes (WAGOs) and their endogenous siRNAs that are largely amplified by RNA-dependent RNA Polymerases (RdRPs). Our understanding of small RNA types, Argonautes, and pathways in other nematodes is limited. We carried out comparative studies of the small RNA pathways in the Clade III parasitic nematode Ascaris. Ascaris has 10 Argonautes. However, a PIWI Argonaute and piRNAs are absent. Five of the Argonautes are from the WAGO clade. We generated antibodies against all five Ascaris WAGOs as well as AsALG-1 (miRNAs) and AsALG-4 (26G-RNAs) and used them to identify their associated small RNAs in the early embryo, ovary, and testis including discrete developmental stages during spermatogenesis. We found that in general, AsALG-4, AsCSR-1 and AsWAGO-3 small RNAs target mRNAs while AsWAGO-1, AsWAGO-2 and AsNRDE-3 small RNAs target repetitive sequences. Notably, AsNRDE-3 small RNAs change their targets from repetitive sequences to mRNAs during male meiosis at the pachytene stage. RNA-seq data identified a group of genes expressed at pachytene during spermatogenesis which are rapidly degraded at the end of pachytene. Degradation of these mRNAs is associated with AsALG-4 26G-RNAs specifically expressed only in late pachytene and diplotene. The timing and expression of AsCSR-1 and AsALG-4 Argonautes and their small RNAs during spermatogenesis suggests they likely function independently, with AsCSR-1 and its 22-24G-RNAs fine-tuning expression of a broad set of transcripts and AsALG-4 and its 26G-RNAs down-regulating male meiosis-specific mRNAs. Genomic regions with transposons and their derivates are in general enriched for H3K9me3 and are targeted by an expansive set of 22G-RNAs associated with AsWAGO-1, AsWAGO-2 and AsNRDE-3 throughout development. Overall, there is clear conservation in the miRNA, 22G-RNA, and 26G-RNA pathways between the distantly related Ascaris and C. elegans nematodes. However, our data demonstrate the complexity and plasticity of small RNA pathways in a Clade III nematode without PIWI and a piRNA pathway and provide an in depth of analysis of the dynamics of small RNA pathways throughout spermatogenesis.