Pre-mRNA splicing in the new millennium

Abstract

The past year has witnessed refinements in models of spliceosome assembly pathways and in the understanding of how splicing factors of the serine/arginine-rich (SR) protein family function. The role of splicing in human genetic diseases has also received a lot of attention recently as exonic splicing enhancers become better understood.

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.