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Nucleus-Independent Transgenerational Small RNA Inheritance in Caenorhabditis elegans

ABSTRACT: In C. elegans worms, epigenetic information transmits transgenerationally. Still, it is unknown whether the effects transfer to the next generation inside or outside of the nucleus. Here we use the tractability of gene-specific double-stranded RNA-induced silencing to demonstrate that RNAi can be inherited independently of any nuclear factors via mothers that are genetically engineered to transmit only their ooplasm but not the oocytes’ nuclei to the next generation. We characterize the mechanisms and, utilizing RNA sequencing, chimeric worms and sequence polymorphism between different isolates, identify endogenous small RNAs which, similarly to exogenous siRNAs, are inherited in a nucleus-independent manner. From an historical perspective, these results might be regarded as partial vindication of discredited cytoplasmic inheritance theories from the 19th century, such as Darwin’s “pangenesis” theory.

ORGANISM(S): Caenorhabditis elegans

PROVIDER: GSE243434 | GEO | 2023/09/22

REPOSITORIES: GEO

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Project description:Deep sequencing was used to detect Flock House Virus (FHV)-derived small RNAs (viRNAs) that were inherited over generations. Small RNA libraries were constructed from 4 different samples: (a) FR1gfp-transgenic worms that can mount an RNAi response and should contain viRNAs (positive control); (b) rde-4(-/-) mutants (negative control as no viRNAs are expected to be produced); (c) FR1gfp; rde-4(-/-) worms that are two generations away from their rde-4(+/-) grandparents, i.e. worms that can themselves not produce viRNAs, but may have inherited viRNA from their grandparents; and (d) the F3 progeny of wild-type animals that contained the FR1gfp transgene (and therefore produced viRNAs), but have lost this transgene through outcrossing with non-transgenic wild-type worms. This tests whether the silencing reagent can exist without its template. The Small RNA libraries were constructed using a protocol that enriches for Dicer products which harbor a single phosphate at the 5M-bM-^@M-^Y end of the RNA (Zamore et al., 2000), such as primary siRNAs, and unlike RdRP products that harbor tri-phosphate ends (Parameswaran et al., 2010). This was the protocol of choice because we were interested in identifying viRNAs that are guaranteed to be derived from the original RNAi-competent parents (Alcazar et al., 2008), and rde-4 animals can not produce primary siRNAs (Grishok et al., 2000; Parrish and Fire, 2001). rde-4 animals are, however, not defective in secondary siRNA production (Blanchard et al., 2011), and can therefore continue to amplify secondary siRNAs de novo; such autonomously-produced siRNAs will not be distinguishable from inherited ones. The detection of rare primary siRNAs is important, as even a single M-bM-^@M-^\triggerM-bM-^@M-^] siRNA can induce a full-blown RNAi-response that is not proportional to the primary trigger (Groenenboom et al., 2005).In agreement with the functional assays we detected different viRNAs complementary to several regions of the viral genome in the positive control (FR1gfp), no viRNAs in the negative control (rde-4(-/-)), and a number of viRNAs in the worms that could not generate their own viRNAs and thus inherited these viRNAs from their grandparents (F3 generation FR1gfp; rde-4(-/-))(Table 2; Figure 4). Moreover, we detect viRNAs in the worms in which the FR1gfp transgene had been crossed out, confirming that the viRNA transmit in a template-independent manner. The inherited viRNA matched the two most abundant types of viRNAs detected in the positive control (Figure 4) and these viRNAs were all of the reverse orientation (negative strand, which typically exists in much lower quantities than the positive strand (Felix et al., 2011)); both observations make it highly unlikely that the detected viRNAs merely represent unspecific break-down products of the viral RNA. In regard to the low number of inherited viRNA reads it needs to be considered that our protocol enriches specifically for rare primary viRNA species. Moreover, these viRNA species are derived from a response mounted in RNAi-competent grandparents, and are therefore possibly diluted over the course of several generations. Taken together, our results support the genetic experiments that argued for the existence of trans-acting factors that are transmitted in a non-Mendelian manner to ensuing generations. 4 samples examined. Small RNA libraries generated from: C. elegans animals.

Project description:<p>The living and dried specimens in botanical collections play an important role in society for scientific and recreational purposes, offering the opportunity to obtain both macroscopic and molecular information for individual plants, ecosystems, and environmental studies. Untargeted metabolomics is an analytical approach that permits the simultaneous study of multiple small molecules present in an organism, which allows us to statistically compare different conditions of interest. Metabolomic approaches have been used on living specimens in botanical collections, but, until now, not on historical dried material. Using the Nicotiana genus herbaceous plant (tobacco) as a case study, we propose an untargeted metabolomic study to evaluate the potential of dried historical specimens as a source of metabolomic information on the past. The metabolomic profile from polar and less-polar/apolar aqueous extracts of four modern handmade tobacco cigars (split into wrapper, binder, and filler leaves), and a set of eight late-19th to early-20th century tobacco specimens (seven tobacco leaves and one snuff powder) from the collection of the Royal Botanical Gardens at Kew (London, UK) were analysed by liquid chromatography coupled to high resolution mass spectrometry. Results showed a wide range of polar and less-polar/apolar molecules which are preserved in dried botanical material, providing information optimal for metabolomic studies. The metabolomic profiles of historical dried samples were distinct enough to classify as Nicotiana tabacum or Nicotiana rustica, and showed differences based on geographic provenance or product transformation/processing. Statistical models based on the molecular data from the historical material permitted us to validate the labelling of the historical collection, which identified one possible mislabelled specimen and offered some clues as to the species of one unknown Nicotiana sample. Finally, metabolomic differences in profiles between Nicotiana tabacum and modern cigars showed that both share a large proportion of their metabolomic profile, where molecular differences could be possibly associated with both location of growth and anthropogenic transformation suffered by the plant in the last two centuries. This study demonstrates that dry botanical collections are a feasible source of information, and, if applied to a large set of individuals, conclusions may be drawn about the possible evolution and anthropogenic modification over time in plant material. The results are significant for disciplines interested in the history of plants, such as botany, history and archaeology.</p>

Project description:RNA-seq transcriptome analysis of C. elegans germlines that inherited only paternal chromosomes (non-Mendelian inheritance, 'red-head worms') and the germlines of their offspring ('offspring of red-head worms') vs. germlines that inherited both maternal and paternal chromosomes (Mendelian inheritance, HBR1280 control). Given that epigenetic marking of sperm chromosomes is faithfully transmitted through embryo cell divisions, and that sperm epigenetic marking is important in offspring, we tested if sperm epigenetic marking alone is sufficient for proper development of the germline in offspring. We utilized a mutant that, during the first embryonic division, delivers the sperm genome to the daughter cell that generates the germline and the oocyte genome to the other daughter cell (Besseling & Bringmann, 2016). This mutant over-expresses GPR-1, a protein involved in regulation of kinetochore pulling forces. GPR-1 over-expression results in excessive pulling forces, causing the paternal and maternal pronuclei to inappropriately move to opposite poles of the 1-cell embryo instead of merging in the center of the embryo. In this mutant background, ~60% of offspring undergo atypical chromosome segregation, generating mosaic embryos whose germlines are derived entirely from sperm chromosomes (Besseling & Bringmann, 2016). To track the parental genomes, differentially tagged histone transgenes were used: a GFP-tagged histone H2B encoded in the sperm genome, and a TdTomato-tagged histone H2B encoded in the oocyte genome. The mosaic embryos whose germline inherited only sperm chromosomes (‘red-head' worms) develop into fertile adults with a normal brood size, similar to control worms, in which the germline inherited both sperm and oocyte chromosomes. RNA-seq analysis demonstrated that the germline transcriptome of ‘red-head’ worms and their offspring show few (<80 genes) and minor changes compared to control worms. These findings demonstrate that epigenetic information provided by sperm can guide proper germ cell development.

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Nucleus-Independent Transgenerational Small RNA Inheritance in Caenorhabditis elegans

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