Functions of Multiple Poly(A) Polymerases in Arabidopsis

Poly(A) polymerases are central players in the process by which the 3' ends of eukaryotic mRNAs are formed. These enzymes add polyadenylate tracts to the ends of processed RNAs in the nucleus in a process that is controlled by a sophisticated machinery. The Arabidopsis genome possesses four potential genes whose predicted products are similar to other eukaryotic poly(A) polymerases (PAPs). These genes are expressed, and their m RNAs are alternatively-spliced,. This alternative splicing has the potential to yield numerous truncated PAP isoforms. These observations give rise to a host of questions regarding the roles of multiple PAP isoforms in Arabidopsis. This proposal seeks to address some of these questions. This will be done by exploring the following specific aims: .Establishing the essentiality (or lack thereof) of each of the four Arabidopsis nPAP genes .Determining the functional interchangeability of the products of the four Arabidopsis nPAP genes .Establishing the need (or lack thereof) of alternative splicing for the functionality of the different Arabidopsis nPAP genes .Studying the in vivo functionality of the promoters of each of the four Arabidopsis nPAP genes .and Cataloguing the interactions between the suite of Arabidopsis nPAP isoforms and other polyadenylation factor subunits. For these studies, a combination of genetic (e.g., antisense RNA, yeast two-hybrid) and biochemical (in vitro interaction assays) approaches will be used. This project will contribute significantly to our understanding of nuclear RNA metabolism in higher plants by lending insight into the functions of the myriad of PAP isoforms that are indicated by earlier results. Moreover, it will include a functional characterization of Arabidopsis genes that encode PAPs and other polyadenylation factor subunits. As such, this association is relevant to several categories mentioned in the 2001 Plant Genetic Mechanisms Program Description: "(a) small-scale functional analysis of agriculturally important plant genes and gene products"; and "(b) regulatory mechanisms of expression of nuclear and organellar genes including all stages from transcription to post-translational modification".