We describe the application of a microarray platform, which combines information from exon body and splice-junction probes, to perform a quantitative analysis of tissue-specific alternative splicing (AS) for thousands of exons in mammalian cells. Through this system, we have analyzed global features of AS in major mouse tissues. The results provide numerous inferences for the functions of tissue-specific AS, insights into how the evolutionary history of exons can impact on their inclusion levels, and also information on how global regulatory properties of AS define tissue type. Like global transcription profiles, global AS profiles reflect tissue identity. Interestingly, we find that transcription and AS act independently on different sets of genes in order to define tissue-specific expression profiles. These results demonstrate the utility of our quantitative microarray platform and data for revealing important global regulatory features of AS.

Sequence-based analyses have predicted that ∼35% of mammalian alternative splicing (AS) events produce premature termination codon (PTC)-containing splice variants that are targeted by the process of nonsense-mediated mRNA decay (NMD). This led to speculation that AS may often regulate gene expression by activating NMD. Using AS microarrays, we show that PTC-containing splice variants are generally produced at uniformly low levels across diverse mammalian cells and tissues, independently of the action of NMD. Our results suggest that most PTC-introducing AS events are not under positive selection pressure and therefore may not contribute important functional roles. Alternative splicing (AS), the process by which exons in transcripts are joined in different combinations to generate multiple mRNA variants, represents an important mechanism for the expression of structurally and functionally distinct proteins from a limited number of genes (Graveley 2001;Black 2003). Regulation of AS plays critical roles in cell growth, differentiation, and cell death, and aberrant AS has been implicated in many human diseases (Smith and Valcarcel 2000;Caceres and Kornblihtt 2002;Cartegni et al. 2002). It has been estimated that at least 74% of human genes contain one or more alternative exons (Johnson et al. 2003), yet it is not known to what extent the resulting splice variants specify functionally relevant transcripts and proteins.Previous studies have shown that the introduction of premature termination codons (PTCs) in spliced transcripts can activate transcript degradation via the process of nonsense-mediated mRNA decay (NMD) (Hillman et al. 2004;Maquat 2004;Alonso 2005). NMD is important for the removal of nonfunctional PTC-containing transcripts (Mendell et al. 2004;Mitrovich and Anderson 2005). It has also been shown to function in the autoregulation of transcript levels of several RNAbinding proteins, including splicing factors (Morrison et al. 1997;Sureau et al. 2001;Wollerton et al. 2004; for review, see Lareau et al. 2004). In addition to a PTC, NMD requires a set of upstream frameshift (UPF) protein factors that associate with the spliced transcripts via interactions with a post-splicing exon junction complex Recent bioinformatics analyses of expressed sequence tag (EST) and cDNA sequence data have predicted that ∼35% of AS events have the potential to introduce PTCs that could elicit NMD of transcripts Lewis et al. 2003). This finding led to speculation that NMD activated by AS may represent a widely used mechanism by which gene expression is down-regulated (Hillman et al. 2004;Neu-Yilik et al. 2004;Alonso 2005;Lejeune and Maquat 2005), and it has also been proposed that the introduction of PTCs by AS serves as a mechanism for the tissue-specific regulation of gene expression (Hillman et al. 2004;Holbrook et al. 2004;Raes and Van de Peer 2005). However, no study has yet examined experimentally the actual levels of PTC-containing mRNA splice variants in normal cells and tissues, and it is not known to what extent AS f...

Alternative splicing (AS) plays a major role in the generation of proteomic diversity and in gene regulation. However, the role of the basal splicing machinery in regulating AS remains poorly understood. Here we show that the core snRNP (small nuclear ribonucleoprotein) protein SmB/B9 self-regulates its expression by promoting the inclusion of a highly conserved alternative exon in its own pre-mRNA that targets the spliced transcript for nonsense-mediated mRNA decay (NMD). Depletion of SmB/B9 in human cells results in reduced levels of snRNPs and a striking reduction in the inclusion levels of hundreds of additional alternative exons, with comparatively few effects on constitutive exon splicing levels. The affected alternative exons are enriched in genes encoding RNA processing and other RNA-binding factors, and a subset of these exons also regulate gene expression by activating NMD. Our results thus demonstrate a role for the core spliceosomal machinery in controlling an exon network that appears to modulate the levels of many RNA processing factors.[Keywords: alternative splicing; Sm proteins; snRNP; autoregulation; NMD; exon network] Supplemental material is available for this article. The production of multiple mRNA variants through alternative splicing (AS) is estimated to take place in transcripts from >95% of human multiexon genes Wang et al. 2008). AS represents a mechanism for gene regulation and expansion of the proteome. The most widely studied trans-acting factors regulating AS are proteins of the SR (Ser/Arg-rich) and hnRNP (heterogeneous ribonucleoprotein) families, as well as numerous tissue-restricted AS factors (for review, see Chen and Manley 2009;Nilsen and Graveley 2010). These factors generally regulate AS by recognizing cis-acting sequences in exons or introns and by promoting or suppressing the assembly of the spliceosome at adjacent splice sites. In contrast to these AS regulatory factors, much less is known about how or the extent to which components of the basal or ''core'' splicing machinery modulate splice site decisions.The spliceosome is a large RNP complex that carries out the removal of introns from pre-mRNAs. It comprises the U1, U2, U4/6, and U5 small nuclear RNPs (snRNPs) and several hundred protein factors (for review, see Wahl et al. 2009). Studies in yeast and metazoan systems have indicated that the levels of some of these core splicing components can affect splice site choice. Microarray profiling revealed transcript-specific effects on splicing in yeast strains harboring mutations in or deletions of core splicing components Pleiss et al. 2007). Knockdown of several core splicing factors in Drosophila cells resulted in transcript-specific effects on AS reporters (Park et al. 2004). Deficiency of the snRNP assembly factor SMN (survival of motor neuron) in a mouse model of spinal muscular atrophy (SMA) resulted in tissue-specific perturbations in snRNP levels and splicing defects (Gabanella et al. 2007;Zhang et al. 2008). Tiling microarray profiling analysis of fission yeast...

Alternative splicing (AS) can regulate gene expression by introducing premature termination codons (PTCs) into spliced mRNA that subsequently elicit transcript degradation by the nonsense-mediated mRNA decay (NMD) pathway. However, the range of cellular functions controlled by this process and the factors required are poorly understood. By quantitative AS microarray profiling, we find that there are significant overlaps among the sets of PTC-introducing AS events affected by individual knockdown of the three core human NMD factors, Up-Frameshift 1 (UPF1), UPF2, and UPF3X/B. However, the levels of some PTC-containing splice variants are less or not detectably affected by the knockdown of UPF2 and/or UPF3X, compared with the knockdown of UPF1. The intron sequences flanking the affected alternative exons are often highly conserved, suggesting important regulatory roles for these AS events. The corresponding genes represent diverse cellular functions, and surprisingly, many encode core spliceosomal proteins and assembly factors. We further show that conserved, PTC-introducing AS events are enriched in genes that encode core spliceosomal proteins. Where tested, altering the expression levels of these core spliceosomal components affects the regulation of PTC-containing splice variants from the corresponding genes. Together, our results show that AS-coupled NMD can have different UPF factor requirements and is likely to regulate many general components of the spliceosome. The results further implicate general spliceosomal components in AS regulation.The production of multiple mRNA variants through alternative splicing (AS) represents a widespread mechanism for the expansion of proteomic diversity, and regulated AS plays important roles in many physiological processes (for reviews, see references 4, 5, and 34). However, sequence-based predictions have revealed that approximately one-third or more of AS events have the potential to introduce a premature termination codon (PTC) that could target the resulting spliced transcript for nonsense-mediated mRNA decay (NMD) (32). While many predicted PTC-introducing AS events do not appear to be conserved or subject to regulation by NMD, AS microarray profiling experiments have revealed that approximately 10 to 20% of these events display substantial changes when NMD is disrupted (42). AS microarray profiling (40, 42) thus provides a basis for identifying new genes and functional processes regulated by AS-coupled NMD and for elucidating the factor requirements for this mode of gene regulation on a large scale.In mammals, NMD is generally, though not always (6, 35), dependent on a splicing event sufficiently downstream of a termination codon. Termination codons are usually recognized as premature when they occur more than 50 to 55 nucleotides upstream of a final splice junction (39), which is "marked" by the deposition a postsplicing exon junction complex (EJC) (30). NMD is believed to require three core UPF factors which are conserved from yeast to humans, namely, UPF1 (also kno...

Alternative splicing in the central nervous system

A microarray analysis provides new evidence suggesting that specific cellular processes in the mammalian CNS are coordinated at the level of alternative splicing, and that a complex splicing code underlies CNS-specific alternative splicing regulation.