Diversity of Vertebrate Splicing Factor U2AF35: IDENTIFICATION OF ALTERNATIVELY SPLICED U2AF1 mRNAs

Teresa R Pacheco,Anita Q Gomes,Nuno L Barbosa-Morais,Vladimir Benes,Wilhelm Ansorge,Matthew Wollerton,Christopher W Smith,Juan Valcárcel,Maria Carmo-Fonseca

doi:10.1074/jbc.m402136200

Abstract

U2 small nuclear ribonucleoprotein auxiliary factor small subunit (U2AF(35)) is encoded by a conserved gene designated U2AF1. Here we provide evidence for the existence of alternative vertebrate transcripts encoding different U2AF(35) isoforms. Three mRNA isoforms (termed U2AF(35)a-c) were produced by alternative splicing of the human U2AF1 gene. U2AF(35)c contains a premature stop codon that targets the resulting mRNA to nonsense-mediated mRNA decay. U2AF(35)b differs from the previously described U2AF(35)a isoform in 7 amino acids located at the atypical RNA Recognition Motif involved in dimerization with U2AF(65). Biochemical experiments indicate that isoform U2AF(35)b, which has been highly conserved from fish to man, maintains the ability to interact with U2AF(65), stimulates U2AF(65) binding to a pre-mRNA, and promotes U2AF splicing activity in vitro. Real time, quantitative PCR analysis indicates that U2AF(35)a is the most abundant isoform expressed in murine tissues, although the ratio between U2AF(35)a and U2AF(35)b varies from 10-fold in the brain to 20-fold in skeletal muscle. We propose that post-transcriptional regulation of U2AF1 gene expression may provide a mechanism by which the relative cellular concentration and availability of U2AF(35) protein isoforms are modulated, thus contributing to the finely tuned control of splicing events in different tissues.

Highlights

U2 small nuclear ribonucleoprotein auxiliary factor small subunit (U2AF35) is encoded by a conserved gene designated U2AF1
Biochemical experiments indicate that isoform U2AF35b, which has been highly conserved from fish to man, maintains the ability to interact with U2AF65, stimulates U2AF65 binding to a pre-mRNA, and promotes U2AF splicing activity in vitro
Our results further show that the relative abundance of mRNAs encoding each U2AF35 isoform differs between cell types, arguing that expression of the U2AF1 gene is controlled in a tissue-specific manner

Summary

EXPERIMENTAL PROCEDURES

Isolation and Characterization of Chicken U2AF1 cDNA— Degenerate oligonucleotides were designed to regions of U2AF35 cDNA conserved among Homo sapiens, Drosophila melanogaster, and Schizosaccharomyces pombe. The PCR product was gel-purified using QIAEX II Gel Extraction Kit (Qiagen) and subcloned into pBluescript II KS (ϩ/Ϫ) by “TA cloning” [24] This 392-bp chicken U2AF35 cDNA fragment (PCR35) was random prime-labeled with [␣-32P]dCTP using the Prime-it® II Random Primer Labeling kit (Stratagene®) and used to screen a chicken embryo cDNA library constructed in ␭ZAP II (Stratagene®). Cloning of Chicken U2AF1 Gene—The chicken U2AF35 cDNA fragment PCR35 was random prime labeled and used to screen a DT40 cell line genomic library constructed in LambdaGEM®-11 (Promega), using standard procedures [25]. Hybridizations were carried out with the following probes: (a) chicken U2AF35 cDNA fragment PCR35; (b) a 1175-bp XbaI/XbaI (2403–3578) fragment obtained by restriction digest of pBluescript-chU2AF35III (a class III clone); a probe complementary to chicken ␤-actin was used as an internal control.

Amplicon size nm bp

RESULTS

Size bp

DISCUSSION