Project description:One of the most abundant RNA modifications is N6-methyladenosine (m6A). RNA from all forms of life, including viruses, contain m6A. This modification has been detected in many types of RNAs, such as mRNA, ribosomal RNA, long non-coding RNAs, small nuclear RNAs and microRNAs. Diverse set of proteins have been characterized to methylate, demethylate and specifically bind to this modification in different organisms. C. elegans is a unique model organism with abundant m6A modification, although its genome does not code for orthologs of the well characterized m6A methyltransferase METTL3/METTL14 complex or the demethylases FTO or ALKBH5. Furthermore, orthologs of the YTH family m6A reader proteins seem to be absent from the worm genome as well. To gain insights into how this modification is installed in this organism, we set out to identify enzymes that contribute to the abundant level of m6A in C. elegans. We designed a targeted RNAi screen by which the expression of 22 candidate putative RNA methyltransferase genes are knocked down. We measured global RNA methylation level by HPLC-MS/MS analysis after two generations of RNAi-mediated knock down. The knock down of two candidate methyltransferases resulted in a decrease in global m6A level in total RNA. The first methyltransferase, F33A8.4, is an ortholog of the human ZCCHC4 gene. The second methyltransferase, C38D4.9, is an ortholog of the human METTL5 gene. In order to determine if ZCCHC4 or METTL5 are involved in the deposition of m6A at the mRNA level, m6A-RIP-seq experiments were performed in mRNA derived from WT (N2), ZCCHC4 KO, METTL5 KO and ZCCHC4/METTL5 dKO C. elegans embryos.
Project description:The nematode Caenorhabditis elegans has evolutionarily conserved EV signaling pathways. In this study, we apply a recently published method for high specificity purification of EVs from C. elegans to carry out target-independent proteomic and RNA analysis of EVs from C. elegans. Our experiments uncovered diverse coding and non-coding RNA transcripts as well as protein cargo types commonly found in human EVs.
Project description:Methylation of carbon-5 of cytosines (m5C) is a conserved post-transcriptional nucleotide modification of RNA with widespread distribution across organisms. m5C has been shown to participate in mRNA transport and maintain mRNA stability through its recognition by the reader proteins ALYREF and YBX1, respectively. We recently showed that m5C is required for Caenorhabditis elegans development and fertility under heat stress. To contribute to the understanding of how m5C and its oxidative derivatives mediate their functions, we developed RNA baits bearing modified cytosines in diverse structural contexts to pulldown potential readers in C. elegans. Our mass spectrometry analyses reveal unique binding proteins for each of the modifications. We validate our dataset by demonstrating that the nematode ALYREF homologues ALY-1 and ALY-2 preferentially bind m5C in vitro. The dataset presented here serves as an important scientific resource that will support the discovery of new functions of m5C and its derivatives.
Project description:This SuperSeries is composed of the following subset Series: GSE28617: Function, targets and evolution of Caenorhabditis elegans piRNAs (small RNA-Seq) GSE37432: Function, targets and evolution of Caenorhabditis elegans piRNAs (mRNA) Refer to individual Series