An intron enhancer recognized by splicing factors activates polyadenylation

Area of Science:

• Molecular Biology
• RNA Processing
• Gene Regulation

Background:

• Alternative processing of pre-messenger RNA (mRNA) for calcitonin/calcitonin gene-related peptide (CT/CGRP) involves the inclusion of a specific exon (exon 4).
• Exon 4 inclusion occurs broadly across tissues, indicating widespread regulatory factors.
• An enhancer sequence within the downstream intron is crucial for exon 4 inclusion.

Purpose of the Study:

• To investigate the function of an intron-located enhancer in CT/CGRP pre-mRNA processing.
• To determine the mechanism by which this enhancer influences polyadenylation.
• To identify the specific factors that interact with the enhancer sequence.

Main Methods:

• In vitro polyadenylation assays using precursor RNAs with CT/CGRP or heterologous poly(A) sites.
• Site-directed mutagenesis of the enhancer sequence, including the 5' splice site and pyrimidine tract.
• Analysis of U1 small nuclear ribonucleoproteins (snRNPs), alternative splicing factor/splicing factor 2 (ASF/SF2), and polypyrimidine tract binding protein (PTB) interactions using UV cross-linking and competition assays.

Main Results:

• The intron enhancer activates in vitro polyadenylation cleavage at both the CT/CGRP exon 4 poly(A) site and heterologous sites.
• A 5' splice site sequence within the enhancer, recognized by U1 snRNPs and ASF/SF2, is essential for enhanced polyadenylation.
• The enhancer's pyrimidine tract binds PTB, and PTB binding is required for maximal enhancer-mediated polyadenylation.

Conclusions:

• This is the first demonstration of an intron-located enhancer that facilitates polyadenylation.
• Splicing factors (U1 snRNPs, ASF/SF2, PTB) bind to the enhancer and activate polyadenylation cleavage.
• Regulation of CT/CGRP alternative processing may occur at the polyadenylation step, not solely at the splicing step.