Human splicing factor ASF/SF2 encodes for a repressor domain required for its inhibitory activity on pre-mRNA splicing.

The essential splicing factor ASF/SF2 activates or represses splicing depending on where on the pre-mRNA it binds. We have shown previously that ASF/SF2 inhibits adenovirus IIIa pre-mRNA splicing by binding to an intronic repressor element. Here we used MS2-ASF/SF2 fusion proteins to show that the second RNA binding domain (RBD2) is both necessary and sufficient for the splicing repressor function of ASF/SF2. Furthermore, we show that the completely conserved SWQDLKD motif in ASF/SF2-RBD2 is essential for splicing repression. Importantly, this heptapeptide motif is unlikely to be directly involved in RNA binding given its position within the predicted structure of RBD2. The activity of the ASF/SF2-RBD2 domain in splicing was position-dependent. Thus, tethering RBD2 to the IIIa intron resulted in splicing repression, whereas RBD2 binding at the second exon had no effect on IIIa splicing. The splicing repressor activity of RBD2 was not unique to the IIIa pre-mRNA, as binding of RBD2 at an intronic position in the rabbit beta-globin pre-mRNA also resulted in splicing inhibition. Taken together, our results suggest that ASF/SF2 encode distinct domains responsible for its function as a splicing enhancer or splicing repressor protein.     

Mutations in RRM4 uncouple the splicing repression and RNA-binding activities of polypyrimidine tract binding protein

The polypyrimidine tract binding protein (PTB, or hnRNP I) contains four RNA-binding domains of the ribonucleoprotein fold type (RRMs 1, 2, 3, and 4), and mediates the negative regulation of alternative splicing through sequence-specific binding to intronic splicing repressor elements. To assess the roles of individual RRM domains in splicing repression, a neural-specific splicing extract was used to screen for loss-of-function mutations that fail to switch splicing from the neural to nonneural pathway. These results show that three RRMs are sufficient for wild-type RNA binding and splicing repression activity, provided that RRM4 is intact. Surprisingly, the deletion of RRM4, or as few as 12 RRM4 residues, effectively uncouples these functions. Such an uncoupling phenotype is unique to RRM4, and suggests a possible regulatory role for this domain either in mediating specific RNA contacts, and/or contacts with putative splicing corepressors. Evidence of a role for RRM4 in anchoring PTB binding adjacent to the branch site is shown by mobility shift and RNA footprinting assays.     

An intronic signal for alternative splicing in the human genome

An important level at which the expression of programmed cell death (PCD) genes is regulated is alternative splicing. Our previous work identified an intronic splicing regulatory element in caspase-2 (casp-2) gene. This 100-nucleotide intronic element, In100, consists of an upstream region containing a decoy 3' splice site and a downstream region containing binding sites for splicing repressor PTB. Based on the signal of In100 element in casp-2, we have detected the In100-like sequences as a family of sequence elements associated with alternative splicing in the human genome by using computational and experimental approaches. A survey of human genome reveals the presence of more than four thousand In100-like elements in 2757 genes. These In100-like elements tend to locate more frequent in intronic regions than exonic regions. EST analyses indicate that the presence of In100-like elements correlates with the skipping of their immediate upstream exons, with 526 genes showing exon skipping in such a manner. In addition, In100-like elements are found in several human caspase genes near exons encoding the caspase active domain. RT-PCR experiments show that these caspase genes indeed undergo alternative splicing in a pattern predicted to affect their functional activity. Together, these results suggest that the In100-like elements represent a family of intronic signals for alternative splicing in the human genome.     

hnRNP A1 controls HIV-1 mRNA splicing through cooperative binding to intron and exon splicing silencers in the context of a conserved secondary structure

The removal of the second intron in the HIV-1 rev/tat pre-mRNAs, which involves the joining of splice site SD4 to SA7, is inhibited by hnRNP A1 by a mechanism that requires the intronic splicing silencer (ISS) and the exon splicing silencer (ESS3). In this study, we have determined the RNA secondary structure and the hnRNP A1 binding sites within the 3' splice site region by phylogenetic comparison and chemical/enzymatic probing. A biochemical characterization of the RNA/protein complexes demonstrates that hnRNP A1 binds specifically to primarily three sites, the ISS, a novel UAG motif in the exon splicing enhancer (ESE) and the ESS3 element, which are all situated in experimentally supported stem loop structures. A mutational analysis of the ISS region revealed that the core hnRNP A1 binding site directly overlaps with a major branchpoint used in splicing to SA7, thereby providing a direct explanation for the inhibition of U2 snRNP association with the pre-mRNA by hnRNP A1. Binding of hnRNP A1 to the ISS core site is inhibited by RNA structure but strongly stimulated by the exonic silencer, ESS3. Moreover, the ISS also stimulate binding of hnRNP A1 to the exonic splicing regulators ESS3 and the ESE. Our results suggest a model where a network is formed between hnRNP A1 molecules situated at discrete sites in the intron and exon and that these interactions preclude the recognition of essential splicing signals including the branch point.     

SR protein-mediated inhibition of CFTR exon 9 inclusion: molecular characterization of the intronic splicing silencer

The intronic splicing silencer (ISS) of CFTR exon 9 promotes exclusion of this exon from the mature mRNA. This negative influence has important consequences with regards to human pathologic events, as lack of exon 9 correlates well with the occurrence of monosymptomatic and full forms of CF disease. We have previously shown that the ISS element interacts with members of the SR protein family. In this work, we now provide the identification of SF2/ASF and SRp40 as the specific SR proteins binding to this element and map their precise binding sites in IVS9. We have also performed a functional analysis of the ISS element using a variety of unrelated SR-binding sequences and different splicing systems. Our results suggest that SR proteins mediate CFTR exon 9 exclusion by providing a ‘decoy’ sequence in the vicinity of its suboptimal donor site. The results of this study give an insight on intron ‘exonization’ mechanisms and provide useful indications for the development of novel therapeutic strategies aimed at the recovery of exon inclusion.     

Intronic CA-repeat and CA-rich elements: a new class of regulators of mammalian alternative splicing

We have recently identified an intronic polymorphic CA-repeat region in the human endothelial nitric oxide synthase (eNOS) gene as an important determinant of the splicing efficiency, requiring specific binding of hnRNP L. Here, we analyzed the position requirements of this CA-repeat element, which revealed its potential role in alternative splicing. In addition, we defined the RNA binding specificity of hnRNP L by SELEX: not only regular CA repeats are recognized with high affinity but also certain CA-rich clusters. Therefore, we have systematically searched the human genome databases for CA-repeat and CA-rich elements associated with alternative 5' splice sites (5'ss), followed by minigene transfection assays. Surprisingly, in several specific human genes that we tested, intronic CA RNA elements could function either as splicing enhancers or silencers, depending on their proximity to the alternative 5'ss. HnRNP L was detected specifically bound to these diverse CA elements. These data demonstrated that intronic CA sequences constitute novel and widespread regulatory elements of alternative splicing.     

Nova autoregulation reveals dual functions in neuronal splicing

The Nova family of neuron-specific RNA-binding proteins were originally identified as targets in an autoimmune neurologic disease characterized by failure of motor inhibition. Nova-1 regulates alternative splicing of pre-mRNAs encoding the inhibitory neurotransmitter receptor subunits GABA(A)Rgamma2 and GlyRalpha2 by directly binding intronic elements, resulting in enhancement of exon inclusion. Here we identify exon E4 in the Nova-1 pre-mRNA itself, encoding a phosphorylated protein domain, as an additional target of Nova-dependent splicing regulation in the mouse spinal cord. Nova binding to E4 is necessary and sufficient for Nova-dependent exon exclusion. E4 harbors five repeats of the known Nova-binding tetranucleotide YCAY and mutation of these elements destroys Nova-dependent regulation. Furthermore, swapping of the sites from Nova-1 and GABA(A)Rgamma2 indicates that the ability of Nova to enhance or repress alternative exon inclusion is dependent on the position of the Nova-binding element within the pre-mRNA. These studies demonstrate that in addition to its previously described role as a splicing activator, Nova autoregulates its own expression by acting as a splicing repressor.     

Solution Structure of the HIV-1 Intron Splicing Silencer and Its Interactions with the UP1 Domain of Heterogeneous Nuclear Ribonucleoprotein (hnRNP) A1

Splicing patterns in human immunodeficiency virus type 1 (HIV-1) are maintained through cis regulatory elements that recruit antagonistic host RNA-binding proteins. The activity of the 3′ acceptor site A7 is tightly regulated through a complex network of an intronic splicing silencer (ISS), a bipartite exonic splicing silencer (ESS3a/b), and an exonic splicing enhancer (ESE3). Because HIV-1 splicing depends on protein-RNA interactions, it is important to know the tertiary structures surrounding the splice sites. Herein, we present the NMR solution structure of the phylogenetically conserved ISS stem loop. ISS adopts a stable structure consisting of conserved UG wobble pairs, a folded 2X2 (GU/UA) internal loop, a UU bulge, and a flexible AGUGA apical loop. Calorimetric and biochemical titrations indicate that the UP1 domain of heterogeneous nuclear ribonucleoprotein A1 binds the ISS apical loop site-specifically and with nanomolar affinity. Collectively, this work provides additional insights into how HIV-1 uses a conserved RNA structure to commandeer a host RNA-binding protein.     

tau Exon 10 expression involves a bipartite intron 10 regulatory sequence and weak 5' and 3' splice sites

tau mutations that deregulate alternative exon 10 (E10) splicing cause frontotemporal dementia with parkinsonism chromosome 17-type by several mechanisms. Previously we showed that E10 splicing involved exon splicing enhancer sequences at the 5' and 3' ends of E10, an exon splicing silencer, a weak 5' splice site, and an intron splicing silencer (ISS) within intron 10 (I10). Here, we identify additional regulatory sequences in I10 using both non-neuronal and neuronal cells. The ISS sequence extends from I10 nucleotides 11-18, which is sufficient to inhibit use of a weakened 5' splice site of a heterologous exon. Furthermore, ISS function is location-independent but requires proximity to a weak 5' splice site. Thus, the ISS functions as a linear sequence. A new cis-acting element, the intron splicing modulator (ISM), was identified immediately downstream of the ISS at I10 positions 19-26. The ISM and ISS form a bipartite regulatory element, within which the ISM functions when the ISS is present, mitigating E10 repression by the ISS. Additionally, the 3' splice site of E10 is weak and requires exon splicing enhancer elements for efficient E10 inclusion. Thus far, tau FTDP-17 splicing mutations affect six predicted cis-regulatory sequences.     

A stem structure in fibroblast growth factor receptor 2 transcripts mediates cell-type-specific splicing by approximating intronic control elements

Alternative splicing of fibroblast growth factor receptor 2 (FGFR2) occurs in a cell-type-specific manner with the mutually exclusive use of exon IIIb or exon IIIc. Specific inclusion of exon IIIb is observed in epithelial cells, whereas exon IIIc inclusion is seen in mesenchymal cells. Epithelium-specific activation of exon IIIb and repression of exon IIIc are coordinately regulated by intronic activating sequence 2 (IAS2) and intronic splicing activator and repressor (ISAR) elements in FGFR2 pre-mRNA. Previously, it has been suggested that IAS2 and a 20-nucleotide core sequence of ISAR form a stem structure that allows for the proper regulation of FGFR2 alternative splicing. Replacement of IAS2 and the ISAR core with random sequences capable of stem formation resulted in the proper activation of exon IIIb and repression of exon IIIc in epithelial cells. Given the high degree of phylogenetic conservation of the IAS2-ISAR core structure and the fact that unrelated stem-forming sequences could functionally substitute for IAS2 and ISAR elements, we postulated that the stem structure facilitated the approximation of intronic control elements. Indeed, deletion of the entire stem-loop region and juxtaposition of sequences immediately upstream of IAS2 with sequences immediately downstream of the ISAR core maintained proper cell-type-specific inclusion of exon IIIb. These data demonstrate that IAS2 and the ISAR core are dispensable for the cell-type-specific activation of exon IIIb; thus, the major, if not the sole, role of the IAS2-ISAR stem in exon IIIb activation is to approximate sequences upstream of IAS2 with sequences downstream of the ISAR core. The downstream sequence is very likely a highly conserved GCAUG element, which we show was required for efficient exon IIIb activation.     

Antisense masking of an hnRNP A1/A2 intronic splicing silencer corrects SMN2 splicing in transgenic mice

Survival of motor neuron 2, centromeric (SMN2) is a gene that modifies the severity of spinal muscular atrophy (SMA), a motor-neuron disease that is the leading genetic cause of infant mortality. Increasing inclusion of SMN2 exon 7, which is predominantly skipped, holds promise to treat or possibly cure SMA; one practical strategy is the disruption of splicing silencers that impair exon 7 recognition. By using an antisense oligonucleotide (ASO)-tiling method, we systematically screened the proximal intronic regions flanking exon 7 and identified two intronic splicing silencers (ISSs): one in intron 6 and a recently described one in intron 7. We analyzed the intron 7 ISS by mutagenesis, coupled with splicing assays, RNA-affinity chromatography, and protein overexpression, and found two tandem hnRNP A1/A2 motifs within the ISS that are responsible for its inhibitory character. Mutations in these two motifs, or ASOs that block them, promote very efficient exon 7 inclusion. We screened 31 ASOs in this region and selected two optimal ones to test in human SMN2 transgenic mice. Both ASOs strongly increased hSMN2 exon 7 inclusion in the liver and kidney of the transgenic animals. Our results show that the high-resolution ASO-tiling approach can identify cis-elements that modulate splicing positively or negatively. Most importantly, our results highlight the therapeutic potential of some of these ASOs in the context of SMA.     

5'- and 3'-terminal nucleotides in the FGFR2 ISAR splicing element core have overlapping roles in exon IIIb activation and exon IIIc repression

The cell type-specific, mutually-exclusive alternative splicing of the fibroblast growth factor receptor 2 (FGFR2) pre-mRNA is tightly regulated. A sequence termed ISAR (intronic splicing activator and repressor) has been implicated as an important cis regulatory element in both activation of exon IIIb and repression of exon IIIc splicing in epithelial cells. In order to better understand how this single sequence could have dual roles, we transfected minigenes containing a series of 2-bp mutations in the 18 3′-most nucleotides of ISAR that we refer to as the ISAR core. Transfection of cells with dual-exon (IIIb and IIIc) minigenes revealed that mutation of terminal sequences of the core led to decreased exon IIIb inclusion and increased exon IIIc inclusion. Transfection of cells with single-exon IIIb minigenes and single-exon IIIc minigenes revealed that mutation of terminal sequences of the ISAR core led to decreased exon IIIb inclusion and increased exon IIIc inclusion, respectively. Nucleotides of the ISAR core responsible for exon IIIb activation appear to overlap very closely with those required for exon IIIc repression. We describe a model in which ISAR and a 5′ intronic sequence known as IAS2 form a stem structure required for simultaneous exon IIIb activation and exon IIIc repression.     

An Intronic Sequence Element Mediates Both Activation and Repression of Rat Fibroblast Growth Factor Receptor 2 Pre-mRNA Splicing

Alternative splicing of fibroblast growth factor receptor 2 (FGF-R2) is an example of highly regulated alternative splicing in which exons IIIb and IIIc are utilized in a mutually exclusive manner in different cell types. The importance of this splicing choice is highlighted by studies which indicate that deregulation of the FGF-R2 splicing is associated with progression of prostate cancer. Loss of expression of a IIIb exon-containing isoform of FGF-R2 [FGF-R2 (IIIb)] accompanies the transition of a well-differentiated, androgen-dependent rat prostate cancer cell line, DT3, to the more aggressive, androgen-independent AT3 cell line. We have used transfection of rat FGF-R2 minigenes into DT3 and AT3 cancer cell lines to study the mechanisms that control alternative splicing of rat FGF-R2. Our results support a model in which an important cis-acting element located in the intron between these alternative exons mediates activation of splicing using the upstream IIIb exon and repression of the downstream IIIc exon in DT3 cells. This element consists of 57 nucleotides (nt) beginning 917 nt downstream of the IIIb exon. Analysis of mutants further demonstrates that an 18-nt “core sequence” within this element is most crucial for its function. Based on our observations, we have termed this sequence element ISAR (for intronic splicing activator and repressor), and we suggest that factors which bind this sequence are required for maintenance of expression of the FGF-R2 (IIIb) isoform.