Abstract
Human pre-mRNA introns vary in size from under fifty to over a million nucleotides. We searched for essential factors involved in the splicing of human short introns by screening siRNAs against 154 human nuclear proteins. The splicing activity was assayed with a model HNRNPH1 pre-mRNA containing short 56-nucleotide intron. We identify a known alternative splicing regulator SPF45 (RBM17) as a constitutive splicing factor that is required to splice out this 56-nt intron. Whole-transcriptome sequencing of SPF45-deficient cells reveals that SPF45 is essential in the efficient splicing of many short introns. To initiate the spliceosome assembly on a short intron with the truncated poly-pyrimidine tract, the U2AF-homology motif (UHM) of SPF45 competes out that of U2AF65 (U2AF2) for binding to the UHM-ligand motif (ULM) of the U2 snRNP protein SF3b155 (SF3B1). We propose that splicing in a distinct subset of human short introns depends on SPF45 but not U2AF heterodimer.
Highlights
Human pre-mRNA introns vary in size from under fifty to over a million nucleotides
To find potential factors involved in splicing of short introns, we screened an siRNA library targeting 154 human nuclear proteins for splicing activity of the HNRNPH1 pre-mRNA including 56-nt intron 710,11 (Fig. 1a; Supplementary Table S1)
HeLa cells were transfected with each siRNA and recovered total RNAs were analyzed by RT-PCR to examine splicing activity of the endogenous HNRNPH1 pre-mRNA containing a 56-nt intron (Fig. 1a)
Summary
Human pre-mRNA introns vary in size from under fifty to over a million nucleotides. The canonical splicing mechanisms were studied and established using model pre-mRNAs with a single relatively short intron of a few hundred nucleotides, which are efficiently spliced in cells and in vitro[4,5]. According to such optimal systems, the essential splicing sequences in pre-mRNA, namely the 5′ splice site, the branch-site sequence, and the poly-pyrimidine tract (PPT) followed by the 3′ splice site, are initially recognized by the U1 snRNP, SF1, and the U2AF heterodimer (U2AF65/U2AF35, U2AF2/U2AF1 as HGNC approved symbol), respectively. This raises the question of how such ultrashort introns can be recognized and committed to splicing by an ‘oversized’
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