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. In this work we show that Nicotiana plumbaginifolia, a dicotyledonous plant, contains two genes encoding different homologs of the large 50-65-kDa subunit of the polypyrimidine tract binding factor U2AF, characterized previously in animals and Schizosaccharomyces pombe. Both plant U2AF65 isoforms, referred to as NpU2AF65a and NpU2AF65b, support splicing of an adenovirus pre-mRNA in HeLa cell nuclear extracts depleted of the endogenous U2AF factor. Both proteins interact with RNA fragments containing plant introns and show affinity for poly(U) and, to a lesser extend, poly(C) and poly(G). The branch point or the 3' splice site regions do not contribute significantly to intron recognition by NpU2AF65. The existence of multiple isoforms of U2AF may be quite general in plants because two genes expressing U2AF65 have been identified in Arabidopsis, and different isoforms of the U2AF small subunit are expressed in rice.
Highlights
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
The pyrimidine tract is recognized by the heterodimeric protein U2AF (U2 small nuclear ribonucleoprotein (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 [5,6,7]
The 2,204-bp-long cDNA encoding the full-length protein, named NpU2AF65b, containing this RBD domain was isolated by screening the N. plumbaginifolia cDNA library followed by the 5Ј-rapid amplification of cDNA ends
Summary
Requirements for intron recognition in higher plants differ from those in yeast and vertebrates. Other studies have shown that UA-rich elements play an important role in the definition of intron borders; the 3Ј and 5Ј splice sites preferentially selected for splicing were those present at the transition regions from the UA-rich to GC-rich sequences Stimulation of splicing by UA-rich elements from upstream locations in the intron and their importance for selection of both 5Ј and 3Ј splice sites make it unlikely that these elements act as plant counterparts to the metazoan polypyrimidine tracts. The latter are always located between the branch point and the 3Јss and usually function only in the definition of the 3Ј intron border Identification of the U2AF-like factor in plants raises the interesting question as to how the plant splicing machinery distinguishes between the 3Ј-proximal and upstream U-rich elements during intron definition
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