Messenger RNAs don't usually correspond exactly to DNA — portions of the primary transcript, known as introns, are removed by splicing. A study reveals new ways in which splicing can be regulated.
Complex eukaryotes, such as animals, have extensive RNA splicing to remove sequences that don't encode proteins (introns) and to connect those that do (exons). Often, several messenger RNAs can be generated by a single gene, because different patterns of splicing place different exons into the final mRNAs. As an example, the Dscam gene of the fruitfly Drosophila contains 24 exons, and is thought to encode 38,016 different protein isoforms by alternative splicing (although we're not sure anyone has counted)1. In this particular case, 'docking' and 'selector' sequences within the primary transcript help regulate splicing. But in general, understanding exactly how a cell picks and chooses among the many possible combinations of splices has been a long-standing problem2. Reporting on page 997 of this issue, Moldón et al.3 investigate how a gene's promoter region — the sequence that regulates gene expression — can regulate splicing.
As ever, one approach is to work with a simpler eukaryote. The fission yeast Schizosaccharomyces pombe has a sophisticated splicing apparatus, and regulates splicing, although this regulation is somewhat different from that in more complex eukaryotes. In the latter, introns are humongous, and the splicing system may therefore focus on recognizing the relatively small exons. When splicing is altered, it may fail to recognize an exon, leaving that exon out of the final product, and giving alternative splicing. In yeast, introns are tiny, and the splicing system seems to focus on recognizing the introns. Thus, when splicing is altered, the system may fail to recognize an intron, retaining the intron in the final product. Whatever the reason, the usual nature of regulated splicing in animals is alternative splicing (an altered selection of exons), whereas the usual nature of regulated splicing in yeast is intron retention4. Nevertheless, the mechanisms underlying these two kinds of regulated splicing may be similar.
In S. pombe, regulated splicing occurs in meiosis5. Many meiosis-specific proteins are toxic to vegetative (that is, mitotic) cells, and their expression is kept turned off by multiple mechanisms6. For many meiosis-specific genes, transcription is repressed in vegetative cells, and furthermore, if and when small amounts of transcript are made, their splicing is repressed; that is, introns are retained, so that no active protein is made. When the cells enter meiosis, transcription is induced, and so is splicing.
Moldón et al.3 have studied the splicing of rem1, a meiosis-specific gene of S. pombe. Transcription of rem1 is repressed in vegetative growth, and any transcript that does get made does not get spliced. On entry into meiosis, the rem1 transcript is induced and spliced5,7. The major finding of Moldón et al.3 is both simple and remarkable: the information that specifies meiosis-specific splicing lies entirely inside the promoter, and not in the transcribed region. For example, when the rem1 transcript is expressed from some other promoter, it is spliced in both vegetative and meiotic cells. Conversely, when the authors used the rem1 promoter to drive transcription of a normal vegetative gene (cdc2, which has four introns, and which is usually spliced in both vegetative and meiotic cells), then splicing occurred only in meiosis, and in the same temporal pattern as for the wild-type rem1 gene.
In meiosis, the rem1 promoter is bound by Mei4, a meiosis-specific protein belonging to the forkhead family of transcription factors. S. pombe has three other forkhead transcription factors, and Moldón et al. suggest that, in vegetative cells, the Mei4-binding sites in the rem1 promoter are probably occupied by one of these other factors, Fkh2. When the authors deleted the fkh2 gene from S. pombe, vegetative transcription of rem1 was slightly increased, and some of this transcript was spliced. This suggests that Fkh2 represses both transcription and splicing in vegetative cells. The authors show that Mei4, which is made only in meiosis, binds to rem1 and turns on both transcription and splicing.
Why would one forkhead transcription factor induce splicing, but not the other? On the basis of co-immunoprecipitation experiments, Moldón et al. found that Mei4, but not Fkh2, forms complexes with the spliceosome. The authors therefore suggest that the Mei4 transcription factor actively recruits splicing factors to the rem1 gene and transcript, whereas Fkh2 does not. They suggest that it is this recruitment of the spliceosome by a meiosis-specific transcription factor that is responsible for meiosis-specific splicing. However, less direct explanations are also possible: transcription factors can affect the conformation of chromatin, the rate of mRNA elongation, and the 5′ capping and 3′ poly-adenylation processing of transcripts, and all these are interrelated with splicing8.
The model proposed for rem1 is exciting, but still leaves us with a major puzzle. How can Fkh2 at a promoter inhibit splicing of rem1 and even an unrelated gene such as cdc2 whose transcript has good splicing signals? Maybe control of RNA processing (5′ capping and 3′ polyadenylation as well as splicing) will be a more general feature of promoters. It now seems that most of the genome is transcribed to at least some degree, so turning gene expression off completely may depend on regulating steps of RNA processing in addition to controlling efficiency of transcription.
These remarkable findings3 leave some loose ends. First, Fkh2 regulates many vegetative transcripts, and many of these are spliced. Thus, Fkh2 is not repressing vegetative splicing at most of its targets. Second, the rem1 promoter does not impose its usual temporalpattern of splicing when fused to the crs1 gene5, in contrast to the results obtained here with cdc2 (ref. 3). Third, in the model proposed, the failure of vegetative splicing for the rem1-driven cdc2 transcript seems to suggest little or no ability to splice rem1-driven transcripts post-transcriptionally, in contrast to Saccharo-myces cerevisiae, in which most splicing may be post-transcriptional9. It should also be noted that the study of intron retention is complicated by two subtle and nasty artefacts: unlike mRNAs generated by alternative splicing, mRNAs generated by complete intron retention are perfectly co-linear with the DNA. Thus, the polymerase chain reaction following reverse transcription (RT-PCR), which is used to amplify an mRNA with retained introns, can amplify genomic DNA instead, with results that appear identical results (an artefact Moldón et al. addressed). Second, if a gene has an anti-sense transcript, this will never be spliced, but, in RT-PCR, will yield the same product as an unspliced sense transcript.
Still, these issues do not significantly undercut the core result for rem1: splicing depends on which transcription factor is bound to the promoter. Understanding exactly how Mei4 turns splicing on, and how Fkh2 keeps splicingturned off, will be interesting investigations for the future. In addition, it is a reminder that efforts to understand splicing by searching for signals inside the transcript have limitations, as some of the information is elsewhere.
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