The exon͞intron architecture of genes determines whether components of the spliceosome recognize splice sites across the intron or across the exon. Using in vitro splicing assays, we demonstrate that splice-site recognition across introns ceases when intron size is between 200 and 250 nucleotides. Beyond this threshold, splice sites are recognized across the exon. Splice-site recognition across the intron is significantly more efficient than splice-site recognition across the exon, resulting in enhanced inclusion of exons with weak splice sites. Thus, intron size can profoundly influence the likelihood that an exon is constitutively or alternatively spliced. An EST-based alternative-splicing database was used to determine whether the exon͞intron architecture influences the probability of alternative splicing in the Drosophila and human genomes. Drosophila exons flanked by long introns display an up to 90-foldhigher probability of being alternatively spliced compared with exons flanked by two short introns, demonstrating that the exon͞ intron architecture in Drosophila is a major determinant in governing the frequency of alternative splicing. Exon skipping is also more likely to occur when exons are flanked by long introns in the human genome. Interestingly, experimental and computational analyses show that the length of the upstream intron is more influential in inducing alternative splicing than is the length of the downstream intron. We conclude that the size and location of the flanking introns control the mechanism of splice-site recognition and influence the frequency and the type of alternative splicing that a pre-mRNA transcript undergoes.alternative splicing ͉ bioinformatics ͉ EST database ͉ intron length P re-mRNA splicing is an essential process that accounts for many aspects of regulated gene expression. Of the Ϸ25,000 genes encoded by the human genome (1), Ͼ60% are believed to produce transcripts that are alternatively spliced. Thus, alternative splicing of pre-mRNAs can lead to the production of multiple protein isoforms from a single pre-mRNA, exponentially enriching the proteomic diversity of higher eukaryotic organisms (2, 3). Because regulation of this process can determine when and where a particular protein isoform is produced, changes in alternative-splicing patterns modulate many cellular activities.The spliceosome assembles onto the pre-mRNA in a coordinated manner by binding to sequences located at the 5Ј and 3Ј ends of introns. Spliceosome assembly is initiated by the stable associations of the U1 small nuclear ribonucleoprotein particle with the 5Ј splice site, branch-point-binding protein͞SF1 with the branch point, and U2 snRNP auxiliary factor with the pyrimidine tract (4). ATP hydrolysis then leads to the stable association of U2 snRNP at the branch-point and functional splice-site pairing (5).Intron size has been correlated with rates of evolution (6) and the regulation of genome size (7,8). The exon͞intron architecture has also been shown to influence splice-site recognition (9-11)....

The extensive alternative splicing in higher eukaryotes has initiated a debate whether alternative mRNA isoforms are generated by an inaccurate spliceosome or are the consequence of highly degenerate splice sites within the human genome. Here, we established a quantitative assay to evaluate the accuracy of splicesite pairing by determining the number of incorrect exon-skipping events made from constitutively spliced pre-mRNA transcripts. We demonstrate that the spliceosome pairs exons with an astonishingly high degree of accuracy that may be limited by the quality of pre-mRNAs generated by RNA pol II. The error rate of exon pairing is increased by the effects of the neurodegenerative disorder spinal muscular atrophy because of reduced levels of Survival of Motor Neuron, a master assembler of spliceosomal components. We conclude that all multi-intron-containing genes are alternatively spliced and that the reduction of SMN results in a general splicing defect that is mediated through alterations in the fidelity of splice-site pairing.pre-mRNA splicing ͉ spinal muscular atrophy ͉ splice-site pairing ͉ splicing fidelity ͉ survival of motor neuron A critical step in pre-mRNA splicing is the recognition and correct pairing of 5Ј and 3Ј splice sites. Given the complexity of higher eukaryotic genes and the relatively low level of splice-site conservation (1), the precision of the splicing machinery in choosing and pairing splice sites is impressive. Introns ranging in size from less than 100 up to 10 5 bases are removed efficiently. At the same time, a large number of alternative splicing events accompany the processing of pre-mRNAs (2, 3). In addition, minor perturbations, such as single base mutations, can frequently lead to aberrant splicing (4), predominantly exon skipping and alternative splice-site activation (2, 3). Although the correct sequence context is imperative for splice-site selection, a number of splicing factors have also been implicated in maintaining splicing accuracy, including core components of the spliceosome, as well as Isy1, Prp8p, Slu7p, and Sky1p (5-8).More recent studies have shown that Prp16 and Prp22p act as ATP-dependent proofreading factors for the first and second step of splicing, respectively (8, 9). However, even with proofreading steps at the catalytic core, many alternative mRNA isoforms are generated through alternative splice-site pairing. The sheer number of alternative mRNA isoforms has triggered an ongoing debate as to whether the majority of these transcripts are generated by mistake or with a biological purpose (10). As of today, biological functions for mRNA isoforms have been demonstrated in a large number of the cases studied (11,12). Yet, it is possible that a significant number of the mRNA isoforms that survive quality-control steps, such as nonsensemediated decay (NMD) (13), nonstop decay (NSD) (14), or no-go decay (NGD) (15), are ultimately translated without an obvious biological function.These considerations raise the question whether the apparent promiscuity of the ...

Alternative pre-mRNA splicing may be the most efficient and widespread mechanism to generate multiple protein isoforms from single genes. Here, we describe the genomic analysis of one of the most frequent types of alternative pre-mRNA splicing, alternative 59-and 39-splice-site selection. Using an EST-based alternative splicing database recording >47,000 alternative splicing events, we determined the frequency and location of alternative 59-and 39-splice sites within the human genome. The most common alternative splice sites used in the human genome are located within 6 nucleotides (nt) of the dominant splice site. We show that the EST database overrepresents alternative splicing events that maintain the reading frame, thus supporting the concept that RNA quality-control steps ensure that mRNAs that encode for potentially harmful protein products are destroyed and do not serve as templates for translation. The most frequent location for alternative 59-splice sites is 4 nt upstream or downstream from the dominant splice site. Sequence analysis suggests that this preference is a consequence of the U1 snRNP binding sequence at the 59-splice site, which frequently contains a GU dinucleotide 4 nt downstream from the dominant splice site. Surprisingly, ;50% of duplicated 39-YAG splice junctions are subject to alternative splicing. This high probability of alternative 39-splice-site activation in close proximity of the dominant 39-splice site suggests that the second step of the splicing may be prone to violate splicing fidelity.

RNA-seq technologies are now replacing microarrays for profiling gene expression. Here we describe a robust RNA-seq strategy for multiplex analysis of RNA samples based on deep sequencing. First, an oligo-dT linked to an adaptor sequence is used to prime cDNA synthesis. Upon solid phase selection, second strand synthesis is initiated using a random primer linked to another adaptor sequence. Finally, the library is released from the beads and amplified using a bar-coded primer together with a common primer. This method, referred to as Multiplex Analysis of PolyA-linked Sequences (MAPS), preserves strand information, permits rapid identification of potentially new polyadenylation sites, and profiles gene expression in a highly cost effective manner. We have applied this technology to determine the transcriptome response to knockdown of the RNA binding protein TLS, and compared the result to current microarray technology, demonstrating the ability of MAPS to robustly detect regulated gene expression.

Pre-mRNA splicing, once thought to be a strictly posttranscriptional event in gene expression, is subject to a multitiered network of regulation. Luco et al. now report in Science that this regulation seems to begin with chromatin modifications, suggesting that the histone code may be a prequel to the splicing code.