94 Program for Research in Science and Engineering Understanding the Role of AT-Rich Regions in Determining Alternative Polyadenylation Sites in Saccharomyces cerevisiae

Authors:

Holly Curtis, Zarmik Moqtaderi, Kevin Struhl

Date Created:

2025-01-01

Course Title:

Professor:

Not specified

About Paper:

Eukaryotic mRNAs undergo 3′ end processing through cleavage targeting the ORFs of CHD1, DST1, and ISY1 were used along followed by polyadenylation at multiple potential sites within theirith newly-designed CRISPR repair templates to replace the ORF 3′ untranslated regions (3′ UTRs). Understanding how these sites and proximal regions of 3’ UTRs of these genes with the AT-rich are selected is important, as shorter mRNA isoforms are more region of BYE1. Variants of this AT-rich region including only prevalent in some cancers. Deletion of the BYE1 open reading the first and last 22 nucleotides and their complements were also frame(ORF)placesitsAT-rich5’UTRadjacenttothe3’UTR.This introduced to test if specific subregions of the AT-rich sequence causes an upstream shift in polyadenylation sites suggesting AT- affect polyadenylation, and if the effect is strand-specific. Strains rich regions may play a role in determining these sites. However, will undergo whole genome sequencing to profile 3’ isoforms. We it remains unclear which parts of this region could lead to this expect the results to show that this AT-rich region is sufficient to shift and whether it acts similarly across different genes. This cause an upstream shift in poly(A) profiles across different genes. study uses CRISPR-Cas9 to investigate how a 40-nucleotide AT- Overall, this work provides insight into the cis-regulatory features rich region from the BYE1 5’UTR affects poly(A) site usage in controlling alternative polyadenylation, with broader implications Saccharomyces cerevisiae. Cas9-guide plasmids targeting the for understanding how shortened 3’ isoforms can contribute to ORF of PDB1 and the 3’ UTRs of PDB1 and BYE1 were designed cancer. and constructed. These new molecules and existing derivatives

Abstract:

Eukaryotic mRNAs undergo 3′ end processing through cleavage targeting the ORFs of CHD1, DST1, and ISY1 were used along followed by polyadenylation at multiple potential sites within theirith newly-designed CRISPR repair templates to replace the ORF 3′ untranslated regions (3′ UTRs). Understanding how these sites and proximal regions of 3’ UTRs of these genes with the AT-rich are selected is important, as shorter mRNA isoforms are more region of BYE1. Variants of this AT-rich region including only prevalent in some cancers. Deletion of the BYE1 open reading the first and last 22 nucleotides and their complements were also frame(ORF)placesitsAT-rich5’UTRadjacenttothe3’UTR.This introduced to test if specific subregions of the AT-rich sequence causes an upstream shift in polyadenylation sites suggesting AT- affect polyadenylation, and if the effect is strand-specific. Strains rich regions may play a role in determining these sites. However, will undergo whole genome sequencing to profile 3’ isoforms. We it remains unclear which parts of this region could lead to this expect the results to show that this AT-rich region is sufficient to shift and whether it acts similarly across different genes. This cause an upstream shift in poly(A) profiles across different genes. study uses CRISPR-Cas9 to investigate how a 40-nucleotide AT- Overall, this work provides insight into the cis-regulatory features rich region from the BYE1 5’UTR affects poly(A) site usage in controlling alternative polyadenylation, with broader implications Saccharomyces cerevisiae. Cas9-guide plasmids targeting the for understanding how shortened 3’ isoforms can contribute to ORF of PDB1 and the 3’ UTRs of PDB1 and BYE1 were designed cancer. and constructed. These new molecules and existing derivatives

Source:

Harvard / Addison Crider, Kibum Park, Eugene Shakhnovich / 2025

Topics:

region, site, polyadenylation, bye1, understanding, orf, utrs, gene, isoform, shift, role, determining

Co-authors:

@hollycurtis266 , @zarmikmoqtaderi267 , @kevinstruhl268