To a or not to a: investigating the inclusion of the terminal adenosine in mRNA
All pre-mRNAs must undergo cleavage and polyadenylation of their 3’ untranslated regions (3’UTRs) during processing. Dysregulation of this essential process is linked to diseases like cancer, due to its influence on post transcriptional regulation. Since RNA cleavage and polyadenylation is highly conserved across metazoans, we can utilize the model system C. elegans and apply our findings to humans.
In C. elegans, pre-mRNAs are typically cleaved and polyadenylated at the consensus sequence “UA”. The fate of the adenosine within this sequence, or the terminal adenosine, is unknown. By investigating the inclusion of the terminal adenosine into the poly(A) tail, we can gain further insight into the mechanism of cleavage and polyadenylation and potential additional functions of this nucleotide.
We will use a chimeric protein consisting of the methyltransferase domain of the human methyltransferase METTL16 fused to the C. elegans mRNA scaffolding protein FIP-1 (MTD::FIP-1) to mark the adenosine located at the cleavage site. We plan to verify MTD::FIP-1’s methylation capabilities by expressing it in BL21(DE3) cells, purifying it, and incubating it with mRNA containing its consensus sequence that we will then screen for methylation.
Currently, we are optimizing the expression of MTD::FIP-1 protein in BL21(DE3) cells. Once we have verified that MTD::FIP-1 is functional, we will use it in a novel in vivo assay to investigate the fate of the terminal adenosine during cleavage.
We constructed two transgenic worm strains: MTD::fipp-1, which expresses MTD::FIP-1, and GFP::M03A1.3, which expresses an mRNA probe that contains METTL16's methylation consensus sequence at the cleavage site. Crossing these strains will allow us to extract and screen the processed probe mRNA for the inclusion of the methylated terminal adenosine within the poly(A) tail, which in turn will allow us to better understand the cleavage and polyadenylation process.
