[
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.