TRENDS in Genetics Vol.18 No.4 April 2002 186 Review Review http://tig.trends.com 0168-9525/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S0168-9525(01)02626-9 Javier F. Cáceres MRC Human Genetics Unit, Western General Hospital, Edinburgh, UK EH4 2XU. e-mail: Javier.Caceres@ hgu.mrc.ac.uk Alberto R. Kornblihtt Laboratorio de Fisiología y Biología Molecular, Departamento de Fisiologia, Biologia Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 2, (1428) Buenos Aires, Argentina. e-mail: ark@bg.fcen.uba.ar An important conclusion of recent genomic analysis is that a large proteomic complexity is achieved with a limited number of genes. This underscores the importance of post-transcriptional mechanisms of gene regulation, which contribute to the generation of an increased protein diversity, significantly through alternative splicing of mRNA precursors. Alternative splicing is a major mechanism for modulating the expression of cellular and viral genes and enables a single gene to increase its coding capacity, allowing the synthesis of several structurally and functionally distinct protein isoforms. An extreme example is the Drosophila Dscam gene, which codes for a cell surface protein involved in neuronal connectivity. Its pre-mRNA is alternatively spliced and can potentially generate 38 016 different protein isoforms, more than twice the number of genes in the entire Drosophila genome [1,2]. Pre-mRNA splicing takes place within the spliceosome, a large molecular complex composed of four small nuclear ribonucleoproteins (U1, U2, U4/U6 and U5 snRNPs) and approximately 50–100 non- snRNP splicing factors [3]. The heterogeneous nuclear ribonucleoproteins (hnRNPs) that associate with nascent transcripts to form hnRNPparticles have been implicated in splicing repression [4,5]. A recent review discusses biochemical aspects of pre-mRNA splicing [5]. Vertebrate genes have small exons separated by large introns, and interactions between the upstream 3′ splice site and the downstream 5′ splice sit e acr oss the exon, facilitate exon recognition [6]. The mechanisms of splice-site selection in alternative and constitutive splicing appear to be closely connected because components of the splicing machinery essential for the constitutive splicing reaction, also have a role in the regulation of alternative splicing [7]. Alternative exons often have suboptimal splice sites and/or a suboptimal length when compared with constitutive exons. Splicing of regulated exons is modulated by trans -acting factors that recognize an arrangement of positive (splicing enhancers) and/or negative (splicing silencers) ci s -acting sequence elements, which can be either exonic or intronic. Differences in the activities or amounts of general splicing factors and/or gene-specific splicing regulators during development or in different tissues can cause differential patterns of splicing. First, we will focus on some of the trans-acting factors known to have a role in alternative splicing regulation; then, we will discuss the integration of their activities with the transcription process itself; and finally, we will highlight recent examples of how alterations of RNA processing can lead to human disease. The SR family of proteins The SR proteins, a group of highly conserved proteins in metazoans, are required for constitutive splicing and also influence alternative splicing regulation [8,9]. They have a modular structure consisting of one or two copies of an RNA-recognition motif (RRM) and a C-terminal domain rich in alternating serine and arginine residues (the RS domain). The RRMs determine RNA-binding specificity, whereas the RS domain mediates protein–protein interactions that are thought to be essential for the recruitment of the splicing apparatus and for splice site pairing [10,11]. Another class of RS domain-containing proteins involved in splicing are the SR-related proteins (SRrps). These proteins, which might contain RRMs, include the U1-70K protein, both subunits of U2AF, SRm 160/300 (two SR-related nuclear matrix proteins of 160 and 300 kDa), as well as alternative splicing regulators such as Tra and Tra2 [8]. SR family and SR-related proteins function in the recognition of exonic splicing enhancers (ESEs) leading to the activation of suboptimal adjacent 3′ splice sites [12]. Actions of SR proteins and hnRNP A/ B proteins in splice site selection The first SR proteins to be identified had similar effects on 5′ splice-site selection: increased concentrations of the proteins resulted in the selection of intron-proximal 5′ splice sites in pre-mRNAs that contain two or more alternative 5′ splice sites. Strikingly, an excess of Alternative splicing is an important mechanism for controlling gene expression. It allow s large proteomic complexity from a limited number of genes. An interplay of cis-acting sequences and trans-acting factors modulates the splicing of regulated exons. Here, we discuss the roles of the SR and hnRNP families of proteins in this process. We also focus on the role of the transcriptional machinery in the regulation of alternative splicing, and on those alterations of alternative splicing that lead to human disease. Alternative splicing: multiple control mechanisms and involvement in human disease Javier F. Cáceres and Alberto R. Kornblihtt