POLYADENYLATION OF AN ESTROGEN REGULATED MESSENGER RNA

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DESCRIPTION (Adapted from the applicant's abstract): In Xenopus liver, major serum protein-coding mRNAs are destabilized in response to estrogen treatment (most notably albumin). These mRNAs may be "marked" for targeting to this pathway by the presence of a short (~20 residue) poly(A) tail. Preliminary results from the principal investigator indicate that the short poly(A) tail on albumin results from regulation of processing of its pre-mRNA. In Specific Aim 1, he plans to use transient transfections to map the location within albumin pre-mRNA of the cis-acting sequence responsible for regulating polyadenylation. This sequence is termed the poly(A) -limiting element or PLE. Experiments in this aim will also define its minimal sequence by mutagenesis and examine distance effects from AAUAAA for its function. Experiments in Specific Aim 2 will use RNA gel retardation and UV crosslinking to determine whether the PLE interacts directly with one or more of the components of the core protein of 3' processing, Cleavage and Polyadenylation Specificity Factor (CPSF), or with a specific PLE-binding protein (PLE-BP). Crosslinking of PLE-BP to specific sequences elements will be demonstrated using chimeric RNAs radiolabeled at known or suspected binding sites. Antibodies to known nuclear RNA-binding proteins will be used to determine the identity of PLE-binding proteins observed in nuclear extract, and if a known protein is identified, it will be tested in Specific Aim 3 for its role in regulating polyadenylation. Results from Specific Aims 1 and 2 will guide in vitro polyadenylation experiments in Specific Aim 3 using HeLa nuclear extract or purified and/or recombinant proteins of the 3' processing complex. Regulated polyadenylation will be reproduced using either uncleaved or "precleaved" transcripts bearing wild type PLE or PLE inactivated by mutation (DPLE). Three non-exclusive hypotheses to be tested in these Specific Aim are; 1) does the PLE alter the stability of CPSF binding, 2) does the PLE act to recruit a specific isoform of poly(A) polymerase to the albumin 3' processing complex that is capable of only adding a short poly(A) tail, and 3) does the PLE alter the binding of nuclear ply(A)-binding protein II, the protein that serves to stimulate addition of long poly (A) onto the processed pre-mRNA.