Douglas L. Black

Protein Diversity from Alternative Splicing: A Challenge for Bioinformatics and Post-Genome Biology

With a painting, although the close view may be indistinct, the view of the whole can resolve itself into a discernable picture. Similarly, the global view of the genome will provide a unique vantage for understanding splicing. Since only a few systems of alternative splicing are likely to be analyzed in biochemical detail at least in the near term, bioinformatics approaches will be important in predicting alternative splicing patterns from genomic sequence. Simple sequence comparisons are a first step. Microexons and intronic splicing regulatory regions are often much more highly conserved between species than other intron sequence (see for example Thackeray and Ganetzky 1995xThackeray, J.R and Ganetzky, B. Genetics. 1995; 141: 203–214PubMedSee all ReferencesThackeray and Ganetzky 1995). Comparison of the mouse and human or the D. melanogaster and D. virilis genomes will yield a great deal of information on exon location and on splicing regulatory sequences.Work is also underway to align the EST and cDNA databases with genome sequences (9xKent, W.J and Zahler, A.M. Nucleic Acids Res. 2000; 28: 91–93Crossref | PubMedSee all References, 19xWolfsberg, T.G and Landsman, D. Nucleic Acids Res. 1997; 25: 1626–1632Crossref | PubMed | Scopus (102)See all References). Although this alignment approach again has difficulties in identifying short exons and exon termini correctly (Florea et al. 1998xFlorea, L, Hartzell, G, Zhang, Z, Rubin, G.M, and Miller, W. Genome Res. 1998; 8: 967–974PubMedSee all ReferencesFlorea et al. 1998), it can in principle identify all the splicing occurring within the sequenced portion of existing cDNAs. At the moment, this is only a small portion of the splicing events in the genome, but projects to generate large databases of full-length cDNA sequences will greatly improve its coverage (17xStrausberg, R.L, Feingold, E.A, Klausner, R.D, and Collins, F.S. Science. 1999; 286: 455–457Crossref | PubMed | Scopus (212)See all References, 13xRubin, G.M, Hong, L, Brokstein, P, Evans-Holm, M, Frise, E, Stapleton, M, and Harvey, D.A. Science. 2000; 287: 2222–2224Crossref | PubMed | Scopus (277)See all References). These efforts are unlikely to identify many low abundance mRNAs and will miss potentially important products. However, they will provide an initial reference splicing pattern for many genes. Such a large-scale identification of constitutive and regulated exons should also give us a great deal of information on the features and sequences that distinguish actual exons from the cryptic splice sites not normally recognized by the splicing apparatus. In addition to improving algorithms for predicting spliced segments within genomic sequence, this information will give insight into the mechanisms of alternative splicing and help experimentalists make sense of their complex biochemical systems.Much more difficult than identifying exons correctly will be predicting splicing regulatory patterns from genomic sequence. This issue can be seen in the DSCAM gene sequence where the alternative exons were relatively easy to identify through protein homology (Schmucker et al. 2000xSchmucker, D, Clemens, J, Shu, J, Worby, C, Xiao, J, Muda, M, Dixon, J, and Zipursky, L. Cell. 2000; 101: 671–684Abstract | Full Text | Full Text PDF | PubMedSee all ReferencesSchmucker et al. 2000). However, the same sequence gives no clues about how these exons are regulated or which exons might be used in particular cells. Alternative exons have binding sites for multiple regulatory proteins that often show only subtle variation between tissues, and the sequence elements that control exons of very different tissue specificity can look similar (Smith and Valcarcel 2000xSmith, C.W and Valcarcel, J. Trends Biochem. Sci. 2000; 25: 349–404Abstract | Full Text | Full Text PDF | PubMed | Scopus (143)See all ReferencesSmith and Valcarcel 2000). Thus, even where we identify important regulatory sequences and proteins for an exon, it will be difficult to predict the precise tissues or conditions that lead to splicing activation or repression.The combinatorial nature of splicing regulation is similar to the control of transcription through promoter and enhancer elements and poses similar problems (Smith and Valcarcel 2000xSmith, C.W and Valcarcel, J. Trends Biochem. Sci. 2000; 25: 349–404Abstract | Full Text | Full Text PDF | PubMed | Scopus (143)See all ReferencesSmith and Valcarcel 2000). Many of the whole genome experimental approaches already being applied to transcriptional regulation can be informative for splicing as well (Young 2000xYoung, R. Cell. 2000; 102: 9–15Abstract | Full Text | Full Text PDF | PubMedSee all ReferencesYoung 2000). Microarray technologies that allow the simultaneous assessment of the splicing of many exons within an RNA sample should prove particularly helpful. Unlike the detection of RNAs to measure whole transcript levels, these alternative splicing detector arrays will monitor the inclusion of particular exons in different populations of mRNAs. In one strategy, a position on the array would contain an oligonucleotide complementary to either a differentially included exon or to the exon/exon junction generated when this exon is skipped. The relative hybridization of a sample to these two sequences will give a measure of the relative inclusion of the exon. One limitation of this approach is that it does not give information correlating the exons within a single mRNA. For example, in the DSCAM gene, one would identify the alternatives for exons 4 and 6 that were present in a total mRNA sample, but which particular exons 4 and 6 were used together in the same mRNA molecule would not be discernable. Most importantly, however, such a technology permits one to examine the coordinate regulation of large groups of exons depending on development, cell type, or extracellular stimulus. This system-wide data about exon use may lead to the identification of sequence features that determine particular patterns of expression.The presence or absence of particular exons in an mRNA can also be correlated with data on the expression patterns of potential splicing regulators, such as SR proteins or hnRNPs. Combined with conditional knockouts of these regulators, we can ask precise questions about what combinations of regulatory proteins are needed for particular exons. Although it is recognized as a significant problem in the understanding transcriptional regulation, the question of combinatorial control of splicing is only beginning to be addressed. Nevertheless, this understanding of how exons are regulated on a system-wide scale will ultimately be essential in interpreting genome sequence and predicting how and when certain proteins are produced from it.Only a global view of splicing regulation combined with a detailed understanding of its mechanisms will allow us to paint a picture of an organisms total complement of proteins and of how this complement changes with development and the environment. Working toward this goal should keep bioinformatics researchers and molecular biologists busy for some time into the post-genome era.*E-mail: dougb@microbio.lifesci.ucla.edu

Alternative RNA splicing in the nervous system.

Tissue-specific alternative splicing profoundly effects animal physiology, development and disease, and this is nowhere more evident than in the nervous system. Alternative splicing is a versatile form of genetic control whereby a common pre-mRNA is processed into multiple mRNA isoforms differing in their precise combination of exon sequences. In the nervous system, thousands of alternatively spliced mRNAs are translated into their protein counterparts where specific isoforms play roles in learning and memory, neuronal cell recognition, neurotransmission, ion channel function, and receptor specificity. The essential nature of this process is underscored by the finding that its misregulation is a common characteristic of human disease. This review highlights the current views of the biological phenomenon of alternative splicing, and describes evidence for its intricate underlying biochemical mechanisms. The roles of RNA binding proteins and their tissue-specific properties are discussed. Why does alternative splicing occur in cosmic proportions in the nervous system? How does it affect integrated cellular functions? How are region-specific, cell-specific and developmental differences in splicing directed? How are the control mechanisms that operate in the nervous system distinct from those of other tissues? Although there are many unanswered questions, substantial progress has been made in showing that alternative splicing is of major importance in generating proteomic diversity, and in modulating protein activities in a temporal and spatial manner. The relevance of alternative splicing to diseases of the nervous system is also discussed.

Genome-wide Analysis of PTB-RNA Interactions Reveals a Strategy Used by the General Splicing Repressor to Modulate Exon Inclusion or Skipping

Recent transcriptome analysis indicates that > 90% of human genes undergo alternative splicing, underscoring the contribution of differential RNA processing to diverse proteomes in higher eukaryotic cells. The polypyrimidine tract-binding protein PTB is a well-characterized splicing repressor, but PTB knockdown causes both exon inclusion and skipping. Genome-wide mapping of PTB-RNA interactions and construction of a functional RNA map now reveal that dominant PTB binding near a competing constitutive splice site generally induces exon inclusion, whereas prevalent binding close to an alternative site often causes exon skipping. This positional effect was further demonstrated by disrupting or creating a PTB-binding site on minigene constructs and testing their responses to PTB knockdown or overexpression. These findings suggest a mechanism for PTB to modulate splice site competition to produce opposite functional consequences, which may be generally applicable to RNA-binding splicing factors to positively or negatively regulate alternative splicing in mammalian cells.

Neuronal regulation of alternative pre-mRNA splicing

Alternative pre-mRNA splicing has an important role in the control of neuronal gene expression. Many neuronal proteins are structurally diversified through the differential inclusion and exclusion of sequences in the final spliced mRNA. Here, we discuss common mechanisms of splicing regulation and provide examples of how alternative splicing has important roles in neuronal development and mature neuron function. Finally, we describe regulatory proteins that control the splicing of some neuronally expressed transcripts.

Cooperative Assembly of an hnRNP Complex Induced by a Tissue-Specific Homolog of Polypyrimidine Tract Binding Protein

ABSTRACT Splicing of the c-src N1 exon in neuronal cells depends in part on an intronic cluster of RNA regulatory elements called the downstream control sequence (DCS). Using site-specific cross-linking, RNA gel shift, and DCS RNA affinity chromatography assays, we characterized the binding of several proteins to specific sites along the DCS RNA. Heterogeneous nuclear ribonucleoprotein (hnRNP) H, polypyrimidine tract binding protein (PTB), and KH-type splicing-regulatory protein (KSRP) each bind to distinct elements within this sequence. We also identified a new 60-kDa tissue-specific protein that binds to the CUCUCU splicing repressor element of the DCS RNA. This protein was purified, partially sequenced, and cloned. The new protein (neurally enriched homolog of PTB [nPTB]) is highly homologous to PTB. Unlike PTB, nPTB is enriched in the brain and in some neural cell lines. Although similar in sequence, nPTB and PTB show significant differences in their properties. nPTB binds more stably to the DCS RNA than PTB does but is a weaker repressor of splicing in vitro. nPTB also greatly enhances the binding of two other proteins, hnRNP H and KSRP, to the DCS RNA. These experiments identify specific cooperative interactions between the proteins that assemble onto an intricate splicing-regulatory sequence and show how this hnRNP assembly is altered in different cell types by incorporating different but highly related proteins.

Transcript dynamics of proinflammatory genes revealed by sequence analysis of subcellular RNA fractions.

Macrophages respond to inflammatory stimuli by modulating the expression of hundreds of genes in a defined temporal cascade, with diverse transcriptional and posttranscriptional mechanisms contributing to the regulatory network. We examined proinflammatory gene regulation in activated macrophages by performing RNA-seq with fractionated chromatin-associated, nucleoplasmic, and cytoplasmic transcripts. This methodological approach allowed us to separate the synthesis of nascent transcripts from transcript processing and the accumulation of mature mRNAs. In addition to documenting the subcellular locations of coding and noncoding transcripts, the results provide a high-resolution view of the relationship between defined promoter and chromatin properties and the temporal regulation of diverse classes of coexpressed genes. The data also reveal a striking accumulation of full-length yet incompletely spliced transcripts in the chromatin fraction, suggesting that splicing often occurs after transcription has been completed, with transcripts retained on the chromatin until fully spliced.

hnRNP H Is a Component of a Splicing Enhancer Complex That Activates a c-src Alternative Exon in Neuronal Cells

ABSTRACT The regulation of the c-src N1 exon is mediated by an intronic splicing enhancer downstream of the N1 5′ splice site. Previous experiments showed that a set of proteins assembles onto the most conserved core of this enhancer sequence specifically in neuronal WERI-1 cell extracts. The most prominent components of this enhancer complex are the proteins hnRNP F, KSRP, and an unidentified protein of 58 kDa (p58). This p58 protein was purified from the WERI-1 cell nuclear extract by ammonium sulfate precipitation, Mono Q chromatography, and immunoprecipitation with anti-Sm antibody Y12. Peptide sequence analysis of purified p58 protein identified it as hnRNP H. Immunoprecipitation of hnRNP H cross-linked to the N1 enhancer RNA, as well as gel mobility shift analysis of the enhancer complex in the presence of hnRNP H-specific antibodies, confirmed that hnRNP H is a protein component of the splicing enhancer complex. Immunoprecipitation of splicing intermediates from in vitro splicing reactions with anti-hnRNP H antibody indicated that hnRNP H remains bound to the src pre-mRNA after the assembly of spliceosome. Partial immunodepletion of hnRNP H from the nuclear extract partially inactivated the splicing of the N1 exon in vitro. This inhibition of splicing can be restored by the addition of recombinant hnRNP H, indicating that hnRNP H is an important factor for N1 splicing. Finally, in vitro binding assays demonstrate that hnRNP H can interact with the related protein hnRNP F, suggesting that hnRNPs H and F may exist as a heterodimer in a single enhancer complex. These two proteins presumably cooperate with each other and with other enhancer complex proteins to direct splicing to the N1 exon upstream.

A CaMK IV responsive RNA element mediates depolarization-induced alternative splicing of ion channels

Calcium regulation of gene expression is critical for the long-lasting activity-dependent changes in cellular electrical properties that underlie important physiological functions such as learning and memory. Cellular electrical properties are diversified through the extensive alternative splicing of ion channel pre-messenger RNAs; however, the regulation of splicing by cell signalling pathways has not been well explored. Here we show that depolarization of GH3 pituitary cells represses splicing of the STREX exon in BK potassium channel transcripts through the action of Ca2+/calmodulin-dependent protein kinases (CaMKs). Overexpressing constitutively active CaMK IV, but not CaMK I or II, specifically decreases STREX inclusion in the mRNA. This decrease is prevented by mutations in particular RNA repressor sequences. Transferring 54 nucleotides from the 3′ splice site upstream of STREX to a heterologous gene is sufficient to confer CaMK IV repression on an otherwise constitutive exon. These experiments define a CaMK IV-responsive RNA element (CaRRE), which mediates the alternative splicing of ion channel pre-mRNAs. The CaRRE presents a unique molecular target for inducing long-term adaptive changes in cellular electrical properties. It also provides a model system for dissecting the effect of signal transduction pathways on alternative splicing.

Homologues of the Caenorhabditis elegans Fox-1 Protein Are Neuronal Splicing Regulators in Mammals†

ABSTRACT A vertebrate homologue of the Fox-1 protein from C. elegans was recently shown to bind to the element GCAUG and to act as an inhibitor of alternative splicing patterns in muscle. The element UGCAUG is a splicing enhancer element found downstream of numerous neuron-specific exons. We show here that mouse Fox-1 (mFox-1) and another homologue, Fox-2, are both specifically expressed in neurons in addition to muscle and heart. The mammalian Fox genes are very complex transcription units that generate transcripts from multiple promoters and with multiple internal exons whose inclusion is regulated. These genes produce a large family of proteins with variable N and C termini and internal deletions. We show that the overexpression of both Fox-1 and Fox-2 isoforms specifically activates splicing of neuronally regulated exons. This splicing activation requires UGCAUG enhancer elements. Conversely, RNA interference-mediated knockdown of Fox protein expression inhibits splicing of UGCAUG-dependent exons. These experiments show that this large family of proteins regulates splicing in the nervous system. They do this through a splicing enhancer function, in addition to their apparent negative effects on splicing in vertebrate muscle and in worms.

The splicing regulator Rbfox1 (A2BP1) controls neuronal excitation in the mammalian brain.

The Rbfox family of RNA binding proteins regulates alternative splicing of many important neuronal transcripts, but its role in neuronal physiology is not clear. We show here that central nervous system–specific deletion of the gene encoding Rbfox1 results in heightened susceptibility to spontaneous and kainic acid–induced seizures. Electrophysiological recording revealed a corresponding increase in neuronal excitability in the dentate gyrus of the knockout mice. Whole-transcriptome analyses identified multiple splicing changes in the Rbfox1−/− brain with few changes in overall transcript abundance. These splicing changes alter proteins that mediate synaptic transmission and membrane excitation. Thus, Rbfox1 directs a genetic program required in the prevention of neuronal hyperexcitation and seizures. The Rbfox1 knockout mice provide a new model to study the post-transcriptional regulation of synaptic function.