Multiple Forms of the U2 Small Nuclear Ribonucleoprotein Auxiliary Factor U2AF Subunits Expressed in Higher Plants

Abstract

Requirements for intron recognition during pre-mRNA splicing in plants differ from those in vertebrates and yeast. Plant introns contain neither conserved branch points nor distinct 3 splice site-proximal polypyrimidine tracts characteristic of the yeast and vertebrate introns, respectively. However, they are strongly enriched in U residues throughout the intron, property essential for splicing. To understand the roles of different sequence elements in splicing, we are characterizing proteins involved in intron recognition in plants. Accurate and orderly splicing of nuclear pre-mRNAs requires that exon and intron sequences are effectively distinguished from each other and that matching 5Ј and 3Ј splice sites (5Јss and 3Јss) 1 are selected with precision and juxtaposed before the catalytic steps. Several short sequence elements in pre-mRNA contribute to intron recognition and splice site selection. The 5Јss and 3Јss represent the most universally conserved and functionally important elements. The 5Јss, containing the nearly invariant GU dinucleotide, is recognized through base pairing by the U1 small nuclear ribonucleoprotein (snRNP) early in spliceosome assembly and by other snRNPs and factors at later stages of the reaction. The precise function of the conserved AG at the 3Ј intron border is less well understood (reviewed in Refs. 1-4). Although the role of splice sites appears to be similar in different eukaryotes, the relative contribution of other signals differs significantly between different organisms. In vertebrates and insects, the characteristic and functionally important feature of introns is a polypyrimidine tract, which is located between the branch point and the 3Јss. The pyrimidine tract is recognized by the heterodimeric protein U2AF (U2 snRNP auxiliary factor) early in spliceosome assembly, and this interaction is essential for positioning the U2 snRNP at the branch site, the sequence of which is highly degenerate in metazoa (5-7). Another factor, mBBP/SF1, interacting directly with the branch point region, may also assist U2 snRNP to establish a base pairing interaction with the branch site (8 -10). The distinguishing feature of pre-mRNA introns in the yeast Saccharomyces cerevisiae is a highly conserved branch site sequence, UACUAAC (reviewed in Refs. 2 and 11). Early in spliceosome assembly, UACUAAC is specifically recognized by the branch point-bridging protein BBP, the yeast ortholog of mBBP/SF1 (8 -10). Most introns in S. cerevisiae lack 3Јss-proximal polypyrimidine tracts, and specific sequences positioned 3Ј of the branch site do not contribute to the first step of splicing despite the fact that a factor, Mud2p, sharing some structural and functional similarity with the large subunit of the metazoan U2AF, is present in yeast (8,12). Some yeast introns contain uridine stretches upstream of the 3Јss, but these sequences function during the second step of splicing (13,14).Requirements for intron recognition in higher plants differ from those in yeast and vertebrates. Plant introns contain n...

Molecular Characterization and Phylogeny of U2AF35 Homologs in Plants

“…Two U2AF 35 homologs are known in rice (Oryza sativa; Domon et al, 1998). We identified maize orthologs of the two rice genes by RT-PCR (Wang, 2005).…”

“…Plant introns have neither conserved branch-point sequences nor a Py tract. Two U2AF 65 homologs isolated from wild tobacco (Nicotiana plumbaginifolia) can complement the in vitro splicing of adenovirus pre-mRNA in HeLa cell extracts depleted of U2AF factor (Domon et al, 1998). Previous results revealed that Arabidopsis (Arabidopsis thaliana) has three copies of genes encoding the U2AF large subunit and one possible pseudogene (Wang and Brendel, 2004).…”

“…35 genes in Arabidopsis (Domon et al, 1998;Lorkovic and Barta, 2002;Wang and Brendel, 2004). These two genes are highly conserved with their metazoan counterparts on the sequence level, indicating that their functions may also be similar.…”

AtCyp59 is a multidomain cyclophilin from Arabidopsis thaliana that interacts with SR proteins and the C-terminal domain of the RNA polymerase II

“…In vitro transcription, UV cross-linking, and ribohomopolymer competition assays with Syn7 RNA (Goodall and Filipowicz 1989) and GST-AtCyp59 were performed as described by Domon et al (1998).…”

RBP45 and RBP47, two oligouridylate-specific hnRNP-like proteins interacting with poly(A)+ RNA in nuclei of plant cells

“…The apetala3-1 locus was PCR amplified from plasmid pD1103 (a kind gift from Thomas Jack, Dartmouth College, Hanover, New Hampshire) using oligonucleotides ATCATGTCGACAA AAAGATTAAACAAAGAG and CAGTAGGATCCTTCAAGAA GATGGAAGGTA+ The PCR product was cut with Sal I and BamHI and ligated into corresponding sites of pDEDH/Nco + Overexpression of proteins in E. coli RBP45 and RBP47 and their deletion mutants were expressed as N-terminal GST fusions in E. coli strain DH5a+ Purification was done as previously described for GST-U2AF 65 (Domon et al+, 1998)+ After elution from glutathione-Sepharose 4B, proteins were concentrated using Millipore Ultrafree-4 centrifugal filters (Millipore); in parallel the buffer was exchanged into 20 mM HEPES-KOH, pH 8+0, containing 100 mM KCl, 0+2 mM EDTA, and 1 mM DTT+…”