[
International Worm Meeting,
2021]
One of the most fundamental questions in RNA biology is how transcriptional termination is executed in eukaryotes, and how the location of the cleavage reaction influences mRNA stability and its expression levels. The mechanism of this process is important because determines the length of the 3' Untranslated Regions (3'UTRs), which are defined as the sequences located between the STOP codon and the polyA tail of mature mRNAs. 3'UTRs are targeted by a variety of regulatory factors, including miRNAs and RNA Binding Proteins (RBPs). Here, we have used a genomic approach to map and study 3'UTR data from 1,094 transcriptome datasets downloaded from the public SRA repository at the NCBI. These datasets correspond to the entire collection of C. elegans transcriptomes stored in this repository from 2015 to 2018, which allowed us to map 3'UTRs with an unprecedented ultra-deep coverage of several magnitudes (the average coverage at the mRNA cleavage site is close to 220X). Given the amount of data used in this study, to our knowledge this is the most comprehensive and high-resolution analysis of 3'UTRs in a living organism performed anywhere to date. We have assigned novel 3'UTR isoforms to ~1,000 protein coding genes, refined and updated 3'UTR boundaries for ~23,000 3'UTR isoforms, and performed a detailed comparative genomic analysis of the C. elegans cleavage and polyadenylation complex (CPC) performing in vivo studies to probe principles of mRNA cleavage and polyadenylation. We found that the CPC in C. elegans is conserved to its human counterpart, with most of the functional domains and critical amino acids preserved. While most of the 3'UTRs possess a known Polyadenylation signal element (PAS) localized around -19 nt from the cleavage site (AAUAAA), non-canonical PAS 3'UTRs possess a less stringent requirement but preserve the chemical nature of the element which is RRYRRR. The majority of C. elegans 3'UTRs terminate with a terminal Adenosine nucleotide, which we speculate is included by the RNA polymerase II during the transcription step, since. This Adenosine nucleotide is required for proper cleavage since its removal impacts the location of the cleavage site in vivo.
[
International Worm Meeting,
2021]
The region downstream of the STOP codon in mRNA, referred to as the 3'Untranslated Region (3'UTR), governs the length of mature mRNA. Specifically, the cleavage site located in this region determines where mRNA cleavage will occur and where polyadenylation reaction will begin, thus terminating mRNA transcription. The mRNA cleavage and polyadenylation machinery in C. elegans is highly conserved to its human counterpart, with most functional domains and critical amino acids preserved. Dysregulation of 3'UTR processing has been observed in many diseases, such as cancer, Alzheimer's disease, and muscular dystrophies, but unfortunately the molecular mechanisms underlying the mRNA transcription termination remain elusive. Although the exact cleavage site is not precise, our lab has identified an adenosine consistently located at the mRNA cleavage site. It is unclear if this adenosine is maintained in the mature mRNA transcripts proceeding cleavage and/or is used as a template for the polymerization of the poly(A) tail. In order to answer this question, we developed a novel terminal adenosine RNA methyltransferase (TAM) assay that will sense the inclusion or exclusion of this terminal adenosine at the cleavage site of C. eleganstranscripts by taking advantage of the human nuclear methyltransferase, METTL16. METTL16 methylates the underscored adenosine in its binding motif, "UACAGAGAA", in both mRNA and snRNA. We have cloned both the human METTL16 gene and its RNA recognition motif at the cleavage site of the C. elegans gene M03A1.3and co-expressed them both in the pharynx tissue. Understanding this process is crucial to identifying the main mechanisms behind mRNA cleavage site determination, further advancing knowledge in gene regulation which influences development, growth, and disease.
[
International C. elegans Meeting,
1991]
The CGC has been in operation for 12 years, supplying Caenorhabditis strains and information to researchers. It is responsible for coordination of C. elegans genetic nomenclature, and acts as the central clearing house for naming strains, genes and alleles. Information distributed includes the genetic map, genetic map data, strain information, a bibliography of 1300 research articles and book chapters, and the C. elegans research newsletter, the Worm Breeder's Gazette, which is distributed to 450 subscribers worldwide. A collection of 1550 strains has been established, representing 30 wildtype isolates of C. elegans, wild-type strains of C. briggsae, C. remanei and C. vulgariensis, as well as alleles of 773 mutant genes and 254 chromosome rearrangements. Stocks are stored permanently in liquid nitrogen. Approximately 1350 different strains have been distributed to research laboratories in 7800 separate mailings. Investigators in 210 laboratories in 21 countries have either received strains from the CGC or contributed strains to the CGC collection. The current version of the C. elegans genetic map includes 26 pages, and contains 920 genes and 320 rearrangements. It is produced using the computer drawing program Designer (Micrografx, Inc., Richardson TX), which runs under Microsoft Windows on IBM-compatible microcomputers. The map is published biannually in Genetic Maps (S.J. O'Brien, editor; Cold Spring Harbor Laboratory) and semi-annually in the Worm Breeder's Gazette. Caenorhabditis strains and information are available upon request. All strain, bibliographic and genetic information is maintained on a microcomputer database system, and the various files are available as printouts or data on diskette. Operation of the CGC will be transferred to other investigators at the end of the current CGC contract (September 29,1992). One possible plan emerged at the last C. elegans meeting, in which Bob Herman will assume responsibility for the strain collection, and Jonathan Hodgkin will assume responsibility for the genetic map. Plans for continuation of other CGC services remain to be made. New developments in the use of computers to integrate all information on the worm will enhance the usefulness of data collected by the CGC (see Thierry-Mieg, Durbin, Schatz and Ward, this meeting). The CGC is supported by the NIH National Center for Research Resources.