Unproductive Splicing of SR Genes Associated with Highly Conserved and Ultraconserved DNA Elements
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Vol 446 | 19 April 2007 | doi:10.1038/nature05676 LETTERS Unproductive splicing of SR genes associated with highly conserved and ultraconserved DNA elements Liana F. Lareau1*, Maki Inada2*, Richard E. Green1{, Jordan C. Wengrod1 & Steven E. Brenner1,2 The human and mouse genomes share a number of long, perfectly SRP75 (SFRS4) and 9G8 (SFRS7) contained poison cassette exons conserved nucleotide sequences, termed ultraconserved elements1. that introduced in-frame PTCs. SRp38 (FUSIP1) had two linked Whereas these regions can act as transcriptional enhancers when alternative splicing events: an alternative 59 splice site followed by a upstream of genes, those within genes are less well understood. In cassette exon, with a PTC in the extended upstream exon (Fig. 1, particular, the function of ultraconserved elements that overlap panel 2). For most of these genes, we also observed ESTs in which the alternatively spliced exons of genes encoding RNA-binding pro- introns flanking the poison cassette exons were retained and intro- teins is unknown1,2. Here we report that in every member of the duced PTCs. SRp30c (SFRS9) had similar intron-retention ESTs, but human SR family of splicing regulators, highly or ultraconserved no ESTs supporting the cassette exon (discussed below). The remain- elements are alternatively spliced, either as alternative ‘poison ing genes, ASF/SF2 (also called SFRS1), SC35 (SFRS2) and SRP46 cassette exons’ containing early in-frame stop codons, or as alter- (SFRS2B), had alternative splicing in their 39 untranslated regions native introns in the 39 untranslated region. These alternative (UTRs) (Fig. 1, panel 2). Although splicing in the 39 UTR has no splicing events target the resulting messenger RNAs for degrada- effect on the protein-coding sequence, the introduction of a new tion by means of an RNA surveillance pathway called nonsense- exon–exon junction more than 50 nucleotides downstream of the mediated mRNA decay. Mouse orthologues of the human SR original stop codon marks that stop codon as premature and targets proteins exhibit the same unproductive splicing patterns. Three the transcript for NMD. SR proteins have been previously shown to direct splicing of their The existence of PTC1 isoforms for all of the SR genes led us to ask own transcripts, and one of these is known to autoregulate its ex- whether this alternative splicing is truly unproductive, leading to pression by coupling alternative splicing with decay3–5; our results downregulation of the spliced mRNA. We therefore measured the suggest that unproductive splicing is important for regulation of effect of NMD inhibition on mRNA isoform levels in HeLa cells using the entire SR family. We find that unproductive splicing assoc- quantitative polymerase chain reaction with reverse transcription iated with conserved regions has arisen independently in different (quantitative RT–PCR). We disrupted NMD using short interfering SR genes, suggesting that splicing factors may readily acquire this (si)RNAs against UPF1 (ref. 8), a key effector of the NMD pathway. If form of regulation. the PTCs trigger NMD as expected, the PTC1 isoforms would be The 11 human SR proteins comprise a homologous family of relatively stabilized upon UPF1 depletion. In all nine cases where the splicing factors (Supplementary Fig. 1) with diverse roles in RNA reference isoform could be detected, UPF1 depletion resulted in a 4- processing including control of export, translation, stability, and to 40-fold increase in the relative abundance of PTC-containing constitutive and alternative splicing6. To determine the extent of mRNAs (Fig. 2). We estimate that PTC1 isoforms comprised about alternative splicing of SR genes themselves, we mined expressed 2–14% of the spliced mRNA population from each gene in control sequence tag (EST) libraries for SR transcripts, aligned these ESTs conditions and 40–70% when NMD was inhibited (Supplementary to the human genome to infer splice junctions, and identified alterna- Table 5), suggesting that a substantial fraction of SR transcripts is tive junctions by comparison with a full-length reference mRNA of spliced into a form that is degraded by NMD. Time course experi- each gene. We found that all SR splicing regulators are alternatively ments using cycloheximide rather than RNA interference to inhibit spliced (Fig. 1, panel 2). NMD showed similar effects on PTC1 isoforms, supporting the Notably, we found that all human SR genes had alternative splice expectation that the stabilization is a direct effect of NMD (Sup- forms that are expected to be degraded by nonsense-mediated mRNA plementary Fig. 4). Overall, our results indicate that alternative splice decay (NMD) (Fig. 1, panel 2). The alternative splice forms contain forms of the human SR protein family are produced at appreciable stop codons that are thought to be recognized as premature by the levels and are subsequently downregulated by NMD. NMD machinery because they are located more than 50 nucleotides The 11 human SR genes correspond to 10 mouse SR genes (Sup- upstream of the final exon–exon junction7. In some cases, ESTs with plementary Fig. 1; human SRP46 is an expressed retropseudogene of premature termination codons (PTCs) comprised a sizeable fraction SC35). We used ESTs to identify alternative splicing of the mouse SR of the total ESTs for that gene (Supplementary Table 3 and Sup- genes. All of the genes except SRp30c had ESTs supporting the same plementary Fig. 2). Seven of the SR genes exhibited a distinctive unproductive splicing patterns in mouse as in human (Fig. 1, panel splicing pattern: an alternative, or cassette, exon was skipped in 3); the PTCs were in identical positions except in SRP75 (called Sfrs4 the reference isoform and included in an alternative isoform. SRp20 in mouse). Mouse SRp30c (also called Sfrs9) had a poison cassette (also called SFRS3), SRP40 (SFRS5), p54 (SFRS11), SRP55 (SFRS6), exon, but there was no EST evidence of an equivalent cassette exon in 1Departments of Molecular and Cell Biology and 2Plant and Microbial Biology, University of California, Berkeley, California 94720, USA. {Present address: Max Planck Institute for Evolutionary Anthropology, Leipzig D-04103, Germany. *These authors contributed equally to this work. 926 © 2007 Nature Publishing Group NATURE | Vol 446 | 19 April 2007 LETTERS the human orthologue (Fig. 1, panels 2 and 3). We identified a introns. The remaining gene, SRp30c, had shorter conserved stre- sequence within the human intron that was homologous to the tches of 42 and 44 nucleotides at the ends of its poison cassette mouse cassette exon and experimentally detected the predicted exon. The conserved sequences found in 9G8 (also called Sfrs7 in PTC1 splice form in HeLa cells (Fig. 2). Because alternative splice mouse), ASF/SF2 (Sfrs1), SRp20 (Sfrs3), p54 (Sfrs11) and SRP55 forms are not generally shared between human and mouse, those that (Sfrs6) were previously identified as ultraconserved genomic ele- are conserved may be functionally important9. ments, defined as regions of at least 200 nucleotides of perfect identity We next aligned the full genomic locus of each human SR protein between the human, mouse and rat genomes1; the conserved with its mouse counterpart. Remarkably, all but one SR gene with sequences in the other SR genes were shorter than that threshold, conserved unproductive splice patterns had long regions of 100% although still striking. nucleotide conservation between the human and mouse ortho- We compared the highly conserved regions among the human logues, ranging from 118 to 618 nucleotides in length (Fig. 1, panel paralogues in order to investigate whether these regions arose from 1). The conserved regions corresponded closely to the alternatively a common source. The most parsimonious scenario would be that spliced poison cassette exons or to sequences flanking the 39 UTR the highly conserved sequences and their unproductive splicing SRp20 SRp30c 618 nt 42 44 nt 1 100 1 100 50 50 % ID % ID 0 0 0 1,000 2,000 3,000 4,000 5,000 6,000 7,000 8,000 9,000 10,000 11,000 0 1,000 2,000 3,000 4,000 5,000 6,000 7,000 8,000 9,000 10,000 870 177 2 Human 2 Human 64 3 Mouse 3 Mouse SRp38 SRP40 144 nt 150 nt 1 100 1 100 50 50 % ID %ID 0 0 0 1,000 2,000 3,000 4,000 5,000 6,000 7,000 8,000 9,000 10,000 11,000 12,000 13,000 14,000 15,000 0 1,000 2,000 3,000 4,000 5,000 6,000 207 (AT/AC) 234 2 Human 2 Human 49 (GT/AG) 12 3 Mouse 3 Mouse p54 355 234 nt 1 100 50 % ID 0 0 5,000 10,000 15,000 20,000 25,000 30,000 35,000 40,000 45,000 50,000 125 2 Human 7 3 Mouse 9G8 228 nt SRP55 322 nt 1 100 1 100 50 50 % ID % ID 00 1,000 2,000 3,000 4,000 5,000 6,000 7,000 8,000 9,000 0 0 1,000 2,000 3,000 4,000 5,000 6,000 7,000 297 109 2 Human 2 Human 44 13 3 Mouse 3 Mouse SRP75 114 77 119 nt 1 100 50 % ID 0 0 5,000 10,000 15,000 20,000 25,000 30,000 35,000 122 129 2 Human 4 2 3 Mouse ASF/SF2 SC35 217 nt 188 nt 118 nt 132 nt 1 100 1 100 50 50 % ID % ID 0 0 0 1,000 2,000 3,000 4,000 5,000 0 1,000 2,000 3,000 4,000 5,000 117 2 Human 2 Human 7 21 16 3 Mouse 3 Mouse Figure 1 | SR gene alternative splice forms with PTCs are conserved alternative splicing (drawn below) and intron retention (dotted horizontal between human and mouse and are associated with highly conserved and lines).