Genomic mRNA Profiling Reveals Compensatory Mechanisms for the Requirement of the Essential Splicing Factor U2AF

Abstract

The large subunit of the U2 auxiliary factor (U2AF) recognizes the polypyrimidine tract (Py-tract) located adjacent to the 3 splice site to facilitate U2 snRNP recruitment. While U2AF is considered essential for pre-mRNA splicing, its requirement for splicing on a genome-wide level has not been analyzed. Using Solexa sequencing, we performed mRNA profiling for splicing in the Schizosaccharomyces pombe U2AF 59 (prp2.1) temperature-sensitive mutant. Surprisingly, our analysis revealed that introns show a range of splicing defects in the mutant strain. While U2AF 59 inactivation (nonpermissive) conditions inhibit splicing of some introns, others are spliced apparently normally. Bioinformatics analysis indicated that U2AF 59 -insensitive introns have stronger 5 splice sites and higher A/U content. Most importantly, features that contribute to U2AF 59 insensitivity of an intron unexpectedly reside in its 5-most 30 nucleotides. These include the 5 splice site, a guanosine at position 7, and the 5 splice site-to-branch point sequence context. A differential requirement (similar to U2AF 59 ) for introns may also apply to other general splicing factors (e.g., prp10). Our combined results indicate that U2AF insensitivity is a common phenomenon and that varied intron features support the existence of unrecognized aspects of spliceosome assembly.Pre-mRNA splicing plays a major role in gene regulation (23). Furthermore, alternative splicing serves as an important mechanism to generate molecular diversity (5). In the initial stages of splicing, the U1 snRNP recognizes the 5Ј splice site (5Ј SS), and the U2 snRNP auxiliary factor (U2AF) recognizes the polypyrimidine tract (Py-tract)/3Ј splice site (3Ј SS), leading to U2 snRNP recruitment to the branch point sequence (BPS) (27).In humans, the essential splicing factor U2AF, which has been extensively studied, is a heterodimeric protein containing a large subunit (U2AF65) and a small subunit (U2AF35). These subunits bind to the Py-tract and the 3Ј SS, respectively (37,65,68,71). Both subunits of U2AF are essential for viability in many model organisms, such as zebrafish, the fruit fly, the nematode worm, and fission yeast Schizosaccharomyces pombe (U2AF59) (17,28,42,46,47,59,72). However, in the budding yeast Saccharomyces cerevisiae, the large subunit is dispensable and the small subunit is absent (1). In humans, U2AF65 interacts with several splicing factors (BBP/SF1, UAP56, SAP155 [or SF3b155], and p54) (1,14,19,43,45,70) and facilitates branch point recognition (2, 4). The mammalian U2AF65 is found to be dispensable for splicing of some introns in vitro in the presence of the SR protein SC35 (36). Microarray analyses revealed an unexpected role of the Drosophila melanogaster large subunit (dU2AF50) in the nuclear export of intronless mRNAs (8). Several splicing regulators, such as SXL, PTB, hnRNP A1, ASF/SF2, SC35, and TRA, can facilitate or antagonize U2AF activity for splicing regulation (5, 53).Biochemical and structural studies indicate that the essential splicing factor...

Evolution of a tissue-specific splicing network

“…Although it is curious that knockdown of a core splicing factor like dU2AF 50 resulted in splicing changes at specific junctions and not a global down-regulation in splicing, this was consistent with previous studies that had shown similar effects with S. pombe U2AF temperature-sensitive mutants and RNAi depletion of Drosophila core spliceosome proteins (Park et al 2004;Sridharan et al 2011).…”

“…dU2AF 50 is a core splicing factor, and as such may not be expected to specifically regulate distinct transcripts. Nevertheless, our data are consistent with previous studies in which core spliceosomal factors did have such specificity (Park et al 2004;Sridharan et al 2011). Although the collection of splice junctions sensitive to dU2AF 50 and LS2 depletion overlapped to a small extent, the majority of them were unique to either protein (Fig.…”

The Fission Yeast Pre-mRNA-processing Factor 18 (prp18+) Has Intron-specific Splicing Functions with Links to G1-S Cell Cycle Progression

“…Mutations in the nucleotides of the 5Јss consensus hexamer motif (G 1 -A and U2-A/G) in budding yeast cause lariat intermediate accumulation and second step arrest, whereas surprisingly, mutations in all nucleotides in the 5Јss hexamer sequence cause a block before any catalysis in fission yeast (51). Among the trans-acting factors, studies from our laboratory on SpSlu7, the ortholog of budding yeast second step factor, ScSlu7, also clearly show splicing arrest before catalysis (23). Fission yeast cells deleted for lariat debranching gene (dbr1⌬) have more severe growth defects than the corresponding budding yeast mutant.…”

“…Genome-wide splicing analyses of transcripts in a missense mutant of spslu7 ϩ revealed its requirement before the first step of the splicing reaction, its widespread but intron-specific functions, and its novel interaction with another precatalytic splicing factor (22). The dependence on fission yeast factors SpPrp2 (23) and SpPrp4 (24) correlates with specific intronic features in each case. Other studies with cwf10-⌬NTE mutant of the SpCwf10 splicing factor revealed that its role in splicing is general and not transcript-specific (25).…”

The Prp4 Kinase: Its Substrates, Function and Regulation in Pre-mRNA Splicing

“…There are introns which are spliced independently of the large subunit of the U2 auxiliary factor (U2AF 59 ). Noteworthy here, U2AF 59 is essential for growth [56][57][58]. Moreover, there is some evidence that the recruitment of U2AF 59 to auxiliary factor dependent introns might be mediated by the SR protein Srp2, which has been shown above to be an in vitro substrate of Prp4 kinase [26].…”