ReviewPre-mRNA splicing in the new millennium
Introduction
The precise removal of pre-mRNA introns is a critical aspect of gene expression. Not only must the splicing machinery recognize and remove introns to make the correct message for protein production but also, for many genes, alternative splicing mechanisms must be in place to generate functionally diverse protein isoforms in a spatially and temporally regulated manner. The splicing reaction is carried out by the spliceosome, which consists of five small nuclear ribonucleoprotein complexes U1, U2, U4, U5 and U6 snRNPs and a large number of non-snRNP proteins. The spliceosome acts through a multitude of RNA–RNA, RNA–protein and protein–protein interactions to precisely excise each intron and join the exons in the correct order [1].
For efficient splicing, most introns require a conserved 5′ splice site (5′ss), and a branch point sequence (BPS) followed by a polypyrimidine tract (Py tract) and a 3′ splice site (3′ss). Assembly of the spliceosome onto pre-mRNA is an ordered process with several distinct intermediates. In metazoans, current models hold that commitment of pre-mRNA to the splicing pathway occurs upon ATP-independent formation of the E complex. Assembly of the E complex involves recognition of the 5′ss by U1 snRNA base pairing and association of non-snRNP splicing factors, such as serine/arginine-rich (SR) proteins and the U2 auxiliary factor (U2AF), which binds to the Py tract and 3′ss. Next, U2 snRNA base pairs with the BPS during ATP-dependent formation of the A complex. The subsequent association of the U4/U6•U5 tri-snRNP with pre-mRNA results in formation of the B complex, and finally, the C complex is formed by remodelling of RNA–RNA and RNA–protein interactions to create the catalytically competent spliceosome.
Spliceosome assembly is facilitated, in part, by SR proteins, which are a family of splicing factors that have one or two copies of an RNA-recognition motif (RRM) followed by an arginine/serine-rich (RS) domain [2]. The RRMs mediate RNA binding and determine substrate specificity for individual SR proteins, whereas the RS domain appears to be required for protein–protein interactions. SR proteins have diverse roles in constitutive and alternative splicing. One such role is the recognition of exonic splicing enhancers (ESEs), which mediate splicing stimulation [3]. SR proteins act in a substrate-specific manner by binding to cognate ESEs, which consist of degenerate sequence motifs. The degeneracy of the consensus recognition motifs for SR proteins probably allows overlap in binding, and specificity may result from combinatorial effects, different SR protein levels, binding affinities and specific interactions with other proteins.
The mechanisms by which the splicing machinery recognizes pre-mRNA have been the focus of several key studies in the past year, and these will be highlighted in this review. The discovery of novel spliceosomal intermediates and their implications for the mechanism of early splice-site recognition will be discussed. Several studies that provide insights into the function of SR proteins in constitutive and alternative splicing will also be reviewed. In addition, we will describe reports that illustrate the physiological importance of alternative splicing and consequences of aberrant splicing. Several topics not discussed here are addressed in recent reviews on alternative splicing 4., 5., nuclear localization of splicing [6] and catalytic activity of the spliceosome [1]. In addition, systematic analyses of sequences from the entire Drosophilamelanogaster [7] and Schizosaccharomycespombe [8] genomes allowed useful comparisons of the human, yeast and fly splicing machinery.
Section snippets
Rethinking spliceosome assembly
The current model of spliceosome assembly proposes that U2 snRNP first associates with the pre-mRNA in an ATP-dependent manner in the A complex. However, U2 snRNP has now been identified as a component of a purified, functional E complex [9••]. U2 snRNP association with the E complex occurs in the absence of ATP and does not require BPS interactions. The most straightforward interpretation of the data is that the U2 snRNP first binds loosely to the pre-mRNA in the E complex via the integral U2
Insights into SR protein function
Previous studies showed that the RS domain of the SR protein SF2/ASF is required for constitutive splicing invitro and for cell viability invivo, although it is dispensable for dose-dependent switching between alternative 5′ss [2]. However, a report this year demonstrated that the RS domain of SF2/ASF is not required for invitro splicing of all pre-mRNAs [17•]. Specifically, the RS domain is dispensable for processing of several constitutively spliced pre-mRNAs with strong splice sites. In
Splice-site recognition
Although many sequences within mammalian transcripts match the consensus splice sites, most of them are not used. A recent report provides evidence that these pseudo-splice sites have multiple defects and are inhibited by surrounding splicing silencer sequences [27]. The arrangement of positive and negative cis-acting sequence elements is probably one solution to the problem of finding authentic splice sites. Positive elements promote splicing at appropriate times and at correct splice sites,
Applying splicing models to human disease and genetic diversity
An important mechanism for regulation of gene expression is alternative splicing, which can expand the coding capacity of a single gene to allow production of different protein isoforms, which often have very different functions [4]. An analysis of the prevalence of alternative splicing, on the basis of alignment of available EST sequences to the genome, estimates that at least 35% of human genes are alternatively spliced (39., 40.; see also Update). The Drosophila Dscam gene, which codes for a
Conclusions
In summary, the year 2000 has brought insights into spliceosome assembly, indicating that the recognition of the 5′ss and 3′ss by several components of the spliceosome takes place earlier than previously thought. In addition, the role of SR proteins in mediating splice-site recognition is complex and involves RS-domain-dependent and -independent activities that may be determined by the strength of the splice sites, as well as the presence of competing factors that negatively affect splicing.
Update
The recent completion of a draft of the human genome and initial analysis of the sequence has led to important insights into the process of pre-mRNA splicing. Of particular interest is the conclusion that the human genome appears to have only 30 000–40 000 protein-coding genes, which is only two to three fold more than the number of genes in invertebrates like C. elegans and D. melanogaster 51., 52.. In view of this finding, it seems likely that post-transcriptional mechanisms, especially
Acknowledgements
We thank Jim Duffy for artwork, and Mikko Frilander, Joan Steitz and Christine Guthrie for communicating results prior to publication. We acknowledge support from a post-doctoral fellowship from the American Cancer Society, grants GM42699 from the National Institutes of Health and CA13106 from the National Cancer Institute.
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest
•• of outstanding interest
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