Thomas A. Cooper

RNA and Disease

Cellular functions depend on numerous protein-coding and noncoding RNAs and the RNA-binding proteins associated with them, which form ribonucleoprotein complexes (RNPs). Mutations that disrupt either the RNA or protein components of RNPs or the factors required for their assembly can be deleterious. Alternative splicing provides cells with an exquisite capacity to fine-tune their transcriptome and proteome in response to cues. Splicing depends on a complex code, numerous RNA-binding proteins, and an enormously intricate network of interactions among them, increasing the opportunity for exposure to mutations and misregulation that cause disease. The discovery of disease-causing mutations in RNAs is yielding a wealth of new therapeutic targets, and the growing understanding of RNA biology and chemistry is providing new RNA-based tools for developing therapeutics.

Splicing in disease: disruption of the splicing code and the decoding machinery

Human genes contain a dense array of diverse cis-acting elements that make up a code required for the expression of correctly spliced mRNAs. Alternative splicing generates a highly dynamic human proteome through networks of coordinated splicing events. Cis- and trans-acting mutations that disrupt the splicing code or the machinery required for splicing and its regulation have roles in various diseases, and recent studies have provided new insights into the mechanisms by which these effects occur. An unexpectedly large fraction of exonic mutations exhibit a primary pathogenic effect on splicing. Furthermore, normal genetic variation significantly contributes to disease severity and susceptibility by affecting splicing efficiency.

Aberrant regulation of insulin receptor alternative splicing is associated with insulin resistance in myotonic dystrophy

Myotonic dystrophy type 1 (DM1) is caused by a CTG trinucleotide expansion in the 3′ untranslated region of the DM protein kinase gene. People with DM1 have an unusual form of insulin resistance caused by a defect in skeletal muscle. Here we demonstrate that alternative splicing of the insulin receptor (IR) pre-mRNA is aberrantly regulated in DM1 skeletal muscle tissue, resulting in predominant expression of the lower-signaling nonmuscle isoform (IR-A). IR-A also predominates in DM1 skeletal muscle cultures, which exhibit a decreased metabolic response to insulin relative to cultures from normal controls. Steady-state levels of CUG-BP, a regulator of pre-mRNA splicing proposed to mediate some aspects of DM1 pathogenesis, are increased in DM1 skeletal muscle; overexpression of CUG-BP in normal cells induces a switch to IR-A. The CUG-BP protein mediates this switch through an intronic element located upstream of the alternatively spliced exon 11, and specifically binds within this element in vitro. These results support a model in which increased expression of a splicing regulator contributes to insulin resistance in DM1 by affecting IR alternative splicing.

Muscleblind proteins regulate alternative splicing

Although the muscleblind (MBNL) protein family has been implicated in myotonic dystrophy (DM), a specific function for these proteins has not been reported. A key feature of the RNA‐mediated pathogenesis model for DM is the disrupted splicing of specific pre‐mRNA targets. Here we demonstrate that MBNL proteins regulate alternative splicing of two pre‐mRNAs that are misregulated in DM, cardiac troponin T (cTNT) and insulin receptor (IR). Alternative cTNT and IR exons are also regulated by CELF proteins, which were previously implicated in DM pathogenesis. MBNL proteins promote opposite splicing patterns for cTNT and IR alternative exons, both of which are antagonized by CELF proteins. CELF‐ and MBNL‐binding sites are distinct and regulation by MBNL does not require the CELF‐binding site. The results are consistent with a mechanism for DM pathogenesis in which expanded repeats cause a loss of MBNL and/or gain of CELF activities, leading to misregulation of alternative splicing of specific pre‐mRNA targets.

Functional consequences of developmentally regulated alternative splicing

Genome-wide analyses of metazoan transcriptomes have revealed an unexpected level of mRNA diversity that is generated by alternative splicing. Recently, regulatory networks have been identified through which splicing promotes dynamic remodelling of the transcriptome to promote physiological changes, which involve robust and coordinated alternative splicing transitions. The regulation of splicing in yeast, worms, flies and vertebrates affects a variety of biological processes. The functional classes of genes that are regulated by alternative splicing include both those with widespread homeostatic activities and those with cell-type-specific functions. Alternative splicing can drive determinative physiological change or can have a permissive role by providing mRNA variability that is used by other regulatory mechanisms.

The CELF Family of RNA Binding Proteins Is Implicated in Cell-Specific and Developmentally Regulated Alternative Splicing

ABSTRACT Alternative splicing of cardiac troponin T (cTNT) exon 5 undergoes a developmentally regulated switch such that exon inclusion predominates in embryonic, but not adult, striated muscle. We previously described four muscle-specific splicing enhancers (MSEs) within introns flanking exon 5 in chicken cTNT that are both necessary and sufficient for exon inclusion in embryonic muscle. We also demonstrated that CUG-binding protein (CUG-BP) binds a conserved CUG motif within a human cTNT MSE and positively regulates MSE-dependent exon inclusion. Here we report that CUG-BP is one of a novel family of developmentally regulated RNA binding proteins that includes embryonically lethal abnormal vision-type RNA binding protein 3 (ETR-3). This family, which we call CELF proteins for CUG-BP- and ETR-3-like factors, specifically bound MSE-containing RNAs in vitro and activated MSE-dependent exon inclusion of cTNT minigenes in vivo. The expression of two CELF proteins is highly restricted to brain. CUG-BP, ETR-3, and CELF4 are more broadly expressed, and expression is developmentally regulated in striated muscle and brain. Changes in the level of expression and isoforms of ETR-3 in two different developmental systems correlated with regulated changes in cTNT splicing. A switch from cTNT exon skipping to inclusion tightly correlated with induction of ETR-3 protein expression during differentiation of C2C12 myoblasts. During heart development, the switch in cTNT splicing correlated with a transition in ETR-3 protein isoforms. We propose that ETR-3 is a major regulator of cTNT alternative splicing and that the CELF family plays an important regulatory role in cell-specific alternative splicing during normal development and disease.

A postnatal switch of CELF and MBNL proteins reprograms alternative splicing in the developing heart.

From a large-scale screen using splicing microarrays and RT-PCR, we identified 63 alternative splicing (AS) events that are coordinated in 3 distinct temporal patterns during mouse heart development. More than half of these splicing transitions are evolutionarily conserved between mouse and chicken. Computational analysis of the introns flanking these splicing events identified enriched and conserved motifs including binding sites for CUGBP and ETR-3-like factors (CELF), muscleblind-like (MBNL) and Fox proteins. We show that CELF proteins are down-regulated >10-fold during heart development, and MBNL1 protein is concomitantly up-regulated nearly 4-fold. Using transgenic and knockout mice, we show that reproducing the embryonic expression patterns for CUGBP1 and MBNL1 in adult heart induces the embryonic splicing patterns for more than half of the developmentally regulated AS transitions. These findings indicate that CELF and MBNL proteins are determinative for a large subset of splicing transitions that occur during postnatal heart development.

The pINDUCER lentiviral toolkit for inducible RNA interference in vitro and in vivo.

The discovery of RNAi has revolutionized loss-of-function genetic studies in mammalian systems. However, significant challenges still remain to fully exploit RNAi for mammalian genetics. For instance, genetic screens and in vivo studies could be broadly improved by methods that allow inducible and uniform gene expression control. To achieve this, we built the lentiviral pINDUCER series of expression vehicles for inducible RNAi in vivo. Using a multicistronic design, pINDUCER vehicles enable tracking of viral transduction and shRNA or cDNA induction in a broad spectrum of mammalian cell types in vivo. They achieve this uniform temporal, dose-dependent, and reversible control of gene expression across heterogenous cell populations via fluorescence-based quantification of reverse tet-transactivator expression. This feature allows isolation of cell populations that exhibit a potent, inducible target knockdown in vitro and in vivo that can be used in human xenotransplantation models to examine cancer drug targets.

The Regulation of Splice-Site Selection, and Its Role in Human Disease

The pre-mRNA splicing machinery recognizes exons ternative splicing events (Takagaki et al. 1996), this mechanism is unlikely to account for all regulated splicand joins them together with remarkable precision to form mRNAs with intact translational reading frames. ing observed in vertebrates. There are many examples in which different regulatory programs run concurrently Splicing requires canonical sequences at the intron/exon border, and mutation of these sequences may cause abwithin the same cell, suggesting that different alternatively spliced pre-mRNAs are regulated by distinct pronormal splicing patterns that affect gene expression and cause disease. Recent studies indicate that distinct segrams that use different sets of cis elements and transacting factors. Strong evidence that cell-specific factors quence elements that are distant from the splice sites are also needed for normal splicing. These elements can are responsible for alternative splicing comes from studies on intronic elements that mediate cell-specific splicing affect splice-site recognition during constitutive splicing and also play important roles in directing alternative (Guo et al. 1991; Tacke and Goridis 1991; Black 1992; Gooding et al. 1994; Huh and Hynes 1994; Ryan and splicing, a common phenomenon in which multiple mRNAs, encoding functionally distinct proteins, are Cooper 1996). One model system in which such elements have been identified is the cardiac troponin T generated by use of different combinations of splice junctions, according to developmentally regulated or tis(cTNT) gene (Ryan and Cooper 1996). Figure 1A shows a diagram of cTNT exons 4–6, in which the alternative sue-specific programs. A number of auxiliary splicing elements required for cell-specific modulation of alternaexon 5 is included in embryonic striated muscle and is skipped in the adult. Exon inclusion in embryonic mustive splicing have been found within introns that flank alternative exons. A second set of splicing elements, excle requires intronic elements, referred to as ‘‘musclespecific splicing enhancers’’ (MSEs), located a short disonic splicing enhancers (ESEs), are found within both coding and noncoding exons. These enhancers direct the tance upstream and downstream of the exon (shown as small boxes in fig. 1A). Evidence from transient transfecspecific recognition of splice sites during constitutive and alternative splicing. The prevalence of alternative spliction into embryonic muscle and nonmuscle cell cultures suggests that these elements regulate splicing via regulaing as a mechanism for regulation of gene expression makes it a likely target for alterations leading to human tory factors specific to embryonic muscle that promote disease. Below we summarize what is known about variinclusion of the exon (Ryan and Cooper 1996). ous sequences that affect splice-site selection and illusFew potential regulators of vertebrate alternative trate how changes in alternative splicing may lead either splicing have been found, but recent studies have identito disease or, conversely, to an amelioration of the effied one candidate, called ‘‘SWAP,’’ a homologue of a fects of certain genetic lesions. Drosophila splicing regulator, suppressor of white apricot (Zachar et al. 1987; Denhez and Lafyatis 1994; Intronic Splicing Elements and Splicing Regulators Spikes et al. 1994). In both humans and Drosophila, the SWAP splicing factor negatively regulates its own Although modulation of the nuclear concentrations expression at the posttranscriptional level. SWAP inhibof constitutive RNA processing factors causes some alits splicing of its own pre-mRNA, which, in its unspliced form, encodes a nonfunctional truncated protein. Recent evidence from transient-transfection experiments demReceived May 9, 1997; accepted for publication June 6, 1997. onstrates that overexpressed SWAP protein in mammaAddress for correspondence and reprints: Dr. Thomas A. Cooper, lian cells regulates splicing of several alternatively Department of Pathology, Room S201, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. E-mail: tcooper@bcm.tmc.edu spliced pre-mRNAs (Sarkissian et al. 1996). SignifiThis article represents the opinion of the authors and has not been cantly, in transient-transfection experiments, overexpeer reviewed. pression of the SWAP protein affects splicing patterns 1997 by The American Society of Human Genetics. All rights reserved. 0002-9297/97/6102-0002

The pathobiology of splicing.

02.00 differently than does overexpression of a general splicing