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An intron enhancer recognized by splicing factors activates polyadenylation.
Genes Dev. 1996 Jan 15; 10(2):208-19.GD

Abstract

Alternative processing of the pre-messenger RNA encoding calcitonin/calcitonin gene-related peptide (CT/CGRP) involves alternative inclusion of a 3'-terminal exon (exon 4) embedded within a six exon primary transcript. Expression of CT/CGRP in transgenic mice indicates that inclusion of exon 4 occurs in a wide variety of tissues, suggesting that the factors responsible for exon 4 inclusion are widely distributed. Inclusion of exon 4 requires an enhancer sequence located within the intron downstream of the poly(A) site of exon 4. Here we show that the intron enhancer activated in vitro polyadenylation cleavage of precursor RNAs containing the CT/CGRP exon 4 poly(A) site or heterologous poly(A) sites. To our knowledge this is the first example of an intron-located enhancer that facilitates polyadenylation. Within the enhancer sequence is a 5' splice site sequence immediately preceded by a pyrimidine tract. This 5' splice site sequence was required for enhanced polyadenylation and was recognized by both U1 small nuclear ribonucleoproteins (snRNPs) and alternative splicing factor/splicing factor 2 (ASF/SF2). Enhancement of polyadenylation required U1 RNA, suggesting that the 5' splice site sequence within the enhancer mediates enhancement via interaction with factors normally associated with functional 5' splice sites. Mutation of the polypyrimidine track of the enhancer also inhibited in vitro polyadenylation cleavage. Oligonucleotide competitions and UV cross-linking indicated that the enhancer pyrimidine track binds the polypyrimidine tract binding protein (PTB), but not U2 snRNP auxiliary factor (U2AF), and that binding of PTB was required for maximal enhancer-mediated polyadenylation. These results suggest that the enhancer binds known splicing factors, and that binding of these factors activates polyadenylation cleavage. Furthermore, these results suggest that regulation of alternative processing of CT/CGRP could occur at the level of polyadenylation, rather than splicing.

Authors+Show Affiliations

Department of Medical Specialities, University of Texas M.D. Anderson Cancer Center, Houston 77030, USA.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

8566754

Citation

Lou, H, et al. "An Intron Enhancer Recognized By Splicing Factors Activates Polyadenylation." Genes & Development, vol. 10, no. 2, 1996, pp. 208-19.
Lou H, Gagel RF, Berget SM. An intron enhancer recognized by splicing factors activates polyadenylation. Genes Dev. 1996;10(2):208-19.
Lou, H., Gagel, R. F., & Berget, S. M. (1996). An intron enhancer recognized by splicing factors activates polyadenylation. Genes & Development, 10(2), 208-19.
Lou H, Gagel RF, Berget SM. An Intron Enhancer Recognized By Splicing Factors Activates Polyadenylation. Genes Dev. 1996 Jan 15;10(2):208-19. PubMed PMID: 8566754.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - An intron enhancer recognized by splicing factors activates polyadenylation. AU - Lou,H, AU - Gagel,R F, AU - Berget,S M, PY - 1996/1/15/pubmed PY - 1996/1/15/medline PY - 1996/1/15/entrez SP - 208 EP - 19 JF - Genes & development JO - Genes Dev VL - 10 IS - 2 N2 - Alternative processing of the pre-messenger RNA encoding calcitonin/calcitonin gene-related peptide (CT/CGRP) involves alternative inclusion of a 3'-terminal exon (exon 4) embedded within a six exon primary transcript. Expression of CT/CGRP in transgenic mice indicates that inclusion of exon 4 occurs in a wide variety of tissues, suggesting that the factors responsible for exon 4 inclusion are widely distributed. Inclusion of exon 4 requires an enhancer sequence located within the intron downstream of the poly(A) site of exon 4. Here we show that the intron enhancer activated in vitro polyadenylation cleavage of precursor RNAs containing the CT/CGRP exon 4 poly(A) site or heterologous poly(A) sites. To our knowledge this is the first example of an intron-located enhancer that facilitates polyadenylation. Within the enhancer sequence is a 5' splice site sequence immediately preceded by a pyrimidine tract. This 5' splice site sequence was required for enhanced polyadenylation and was recognized by both U1 small nuclear ribonucleoproteins (snRNPs) and alternative splicing factor/splicing factor 2 (ASF/SF2). Enhancement of polyadenylation required U1 RNA, suggesting that the 5' splice site sequence within the enhancer mediates enhancement via interaction with factors normally associated with functional 5' splice sites. Mutation of the polypyrimidine track of the enhancer also inhibited in vitro polyadenylation cleavage. Oligonucleotide competitions and UV cross-linking indicated that the enhancer pyrimidine track binds the polypyrimidine tract binding protein (PTB), but not U2 snRNP auxiliary factor (U2AF), and that binding of PTB was required for maximal enhancer-mediated polyadenylation. These results suggest that the enhancer binds known splicing factors, and that binding of these factors activates polyadenylation cleavage. Furthermore, these results suggest that regulation of alternative processing of CT/CGRP could occur at the level of polyadenylation, rather than splicing. SN - 0890-9369 UR - https://www.unboundmedicine.com/medline/citation/8566754/An_intron_enhancer_recognized_by_splicing_factors_activates_polyadenylation_ L2 - http://www.genesdev.org/cgi/pmidlookup?view=long&pmid=8566754 DB - PRIME DP - Unbound Medicine ER -