Eukaryotic cells often need to modify their gene expression patterns to adapt to environmental changes or to respond to signals regulating cell function. One rapid means of regulating expression is to vary the length of mRNAs. Length at the 3' end is determined by polyadenylation, an essential processing step involving cleavage of mRNA precursor at the polyA (pA) site, followed by addition of adenosines to the new 3' end. Changing the pA site can alter the amount of coding sequence or remove important regulatory sequences in the 3' untranslated region (UTR) that govern localization, translation, and stability. The majority of eukaryotic genes contain two or more pA sites which give mRNA isoforms of different length. Changes in the proportion of these isoforms occurs for a surprisingly large number of genes during development, differentiation, and tumorigenesis and in response to the cell's environment. These findings indicate that alternative polyadenylation (APA) joins transcription initiation and alternative splicing as an important, but under-appreciated way to modulate the amount and types of mRNAs needed for specific cellular states. However, the mechanisms leading to APA are not well defined, and the consequences on protein output and contribution to cell function remain poorly understood. Our overall objective is to understand the mechanism and functional consequences of these widespread changes in pA site usage. Previous work in the field has largely focused on changes to the 3'UTR, and the consequent changes in post-transcriptional regulation due to gain or loss of regulatory sequences. In contrast, relatively little attention has been given to transcripts ending at pA sites within the coding sequence (CDS-pA), despite clear evidence of their prevalence and their systematic variation in response to changes in cell state. We will therefore focus our studies on the CDS-pA transcripts. We will test the hypothesis that variations in the balance of these isoforms are controlled by a coordinated combination of changes in mRNA stability, mRNA polyadenylation, and transcription termination and that regardless of the mechanism, these changes will affect protein production and therefore are an integral component of an appropriate response to environmental changes. Our approach is innovative because it will use whole genome expression analysis to guide focused molecular biological experiments that will probe mechanisms underlying an important step in regulation of gene expression. This proposal is significant because of the wide-spread use of APA and its potential to rapidly affect the amount and type of protein made by a cell. Accomplishment of these aims is expected to yield novel insights broadly applicable to other cellular states modulated by alternative polyadenylation.
The alternative polyadenylation patterns of many genes change extensively depending on the growth rate of the cell, its differentiated state, and the environmental signals it is receiving. Defects in making these changes in mRNA length have the potential to disrupt normal cell function and lead to disease. Thus, the proposed research is relevant to the part of NIH's mission that pertains to advancing fundamental knowledge of important cell processes that could lead to novel treatment strategies for human disease, especially related to health issues such cancer.
