[
International Worm Meeting,
2021]
The cleavage and polyadenylation of pre-mRNAs is a critical step needed for RNA transcription termination and maturation. This process is executed by a large multi-subunit complex known as the RNA cleavage and polyadenylation complex (CPC). The CPC binds to the polyadenylation signal (PAS), which is a conserved hexameric element located at the end of the transcript's 3' Untranslated Region (3'UTR). The CPC then performs the cleavage reaction at the polyadenylation (PS) site. Despite their importance, PS element locations in eukaryotic genomes are poorly characterized. Prior research from our lab revealed that the distance between the PAS and the PS elements is not constant and a buffer region between 12-14 nt from the PAS element is needed by the CPC in order to perform a successful cleavage reaction. Our lab recently identified an enrichment of adenosine nucleotides at the PS site and demonstrated that their removal alters the location of the cleavage site in vivo, suggesting an important novel role of the PS element in pre-mRNA cleavage and polyadenylation. In order to further study the involvement of this terminal adenosine located at the PS site, we developed a novel in vivo cleavage and polyadenylation assay which we will use to determine the optimal buffer region length and further study the role of the terminal adenosine at the cleavage site. For these experiments, we have used the M03A1.3 3'UTR since it uses only one canonical PAS element (and thus does not use alternative polyadenylation) and has a buffer region of 14 nt. We have prepared a GFP reporter construct containing a mutated M03A1.3 3'UTR with no adenosines between the PAS and PS elements. This GFP reporter construct was used to prepare seven new mutants containing a terminal adenosine inserted at specific locations between 17 and 29 nucleotides downstream of the PAS site. The results of this assay will provide a working framework which we will use to model PS elements in the 5546 genes in the worm transcriptome, which currently lack annotated 3'UTR data. Our work will greatly improve our understanding of pre-mRNA cleavage and polyadenylation in C. elegans and will allow us to provide a needed reference for PS elements in the worm transcriptome to the scientific community.
[
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.