The SR proteins are essential metazoan pre-mRNA splicing factors that can also influence the selection of alternative 5' splice sites in a concentration-dependent manner. Their activity in alternative splicing in vitro is antagonized by members of the hnRNP A/B family of proteins. The opposite effects of members of these two families of antagonistic splicing factors in vitro and upon overexpression in vivo suggest that changes in their relative levels may be a natural mechanism for the regulation of alternative splicing in vivo. One prediction of this model is that the ratios of these antagonists should vary in different cell types and in other situations in which cellular or viral transcripts are differentially spliced. We raised monoclonal antibodies specific for SF2/ASF and used them to measure the abundance of SF2/ASF protein and its isoforms, its phosphorylation state in vivo and during splicing in vitro, and its association with the spliceosome. SF2/ASF exists predominantly or exclusively in a highly phosphorylated state in vivo in all cell types examined, and unphosphorylated protein was not detectable. Unphosphorylated recombinant SF2/ASF becomes rapidly phosphorylated under splicing conditions in HeLa cell extracts and associates stably with one or more exons of beta-globin pre-mRNA. This interaction appears to persist through the splicing reaction and SF2/ASF remains bound to spliced mRNA. We compared the distribution of SF2/ASF to that of its antagonist, hnRNP A1, in different rat tissues and in immortal and transformed cell lines. We found that the protein levels of these antagonistic splicing factors vary naturally over a very wide range, supporting the notion that changes in the ratio of these proteins can affect alternative splicing of a variety of pre-mRNAs in vivo.

The general splicing factor SF2/ASF binds in a sequence-specific manner to a purine-rich exonic splicing enhancer (ESE) in the last exon of bovine growth hormone (bGH) pre-mRNA. More importantly, SF2/ASF stimulates in vitro splicing of bGH intron D through specific interaction with the ESE sequences. However, another general splicing factor, SC35, does not bind the ESE sequences and has no effect on bGH intron D splicing. Thus, one possible function of SF2/ASF in alternative and, perhaps, constitutive pre-mRNA splicing is to recognize ESE sequences. The stimulation of bGH intron D splicing by SF2/ASF is counteracted by the addition of hnRNP A1. The relative levels of SF2/ASF and hnRNP A1 influence the efficiency of bGH intron D splicing in vitro and may be the underlying mechanism of this alternative pre-mRNA processing event in vivo.

Alternative splicing is a vast source of biological regulation and diversity that is misregulated in cancer and other diseases. To investigate global control of alternative splicing in human cells, we analyzed splicing of mRNAs encoding Bcl2 family apoptosis factors in a genome-wide siRNA screen. The screen identified many regulators of Bcl-x and Mcl1 splicing, notably an extensive network of cell-cycle factors linked to aurora kinase A. Drugs or siRNAs that induce mitotic arrest promote proapoptotic splicing of Bcl-x, Mcl1, and caspase-9 and alter splicing of other apoptotic transcripts. This response precedes mitotic arrest, indicating coordinated upregulation of prodeath splice variants that promotes apoptosis in arrested cells. These shifts correspond to posttranslational turnover of splicing regulator ASF/SF2, which directly binds and regulates these target mRNAs and globally regulates apoptosis. Broadly, our results reveal an alternative splicing network linking cell-cycle control to apoptosis.

hnRNP A1 is a pre-mRNA binding protein that antagonizes the alternative splicing activity of splicing factors SF2/ASF or SC35, causing activation of distal 5' splice sites. The structural requirements for hnRNP A1 function were determined by mutagenesis of recombinant human hnRNP A1. Two conserved Phe residues in the RNP-1 submotif of each of two RNA recognition motifs appear to be involved in specific RNA-protein interactions and are essential for modulating alternative splicing. These residues are not required for general pre-mRNA binding or RNA annealing activity. The C-terminal Gly-rich domain is necessary for alternative splicing activity, for stable RNA binding and for optimal RNA annealing activity. hnRNP A1B, which is an alternatively spliced isoform of hnRNP A1 with a longer Gly-rich domain, binds more strongly to pre-mRNA but has only limited alternative splicing activity. In contrast, hnRNP A2 and B1, which have 68% amino acid identity with hnRNP A1, bind more weakly to pre-mRNA and have stronger splice site switching activities than hnRNP A1. We propose that specific combinations of antagonistic hnRNP A/B and SR proteins are involved in regulating alternative splicing of distinct subsets of cellular premRNAs.

The transition from juvenile to adult life is accompanied by programmed remodeling in many tissues and organs, which is key for organisms to adapt to the demand of the environment. Here we report a novel regulated alternative splicing program that is crucial for postnatnal heart remodeling in the mouse. We identify the essential splicing factor ASF/SF2 as a key component of the program, regulating a restricted set of tissue-specific alternative splicing events during heart remodeling. Cardiomyocytes deficient in ASF/SF2 display an unexpected hypercontraction phenotype due to a defect in postnatal splicing switch of the Ca(2+)/calmodulin-dependent kinase IIdelta (CaMKIIdelta) transcript. This failure results in mistargeting of the kinase to sarcolemmal membranes, causing severe excitation-contraction coupling defects. Our results validate ASF/SF2 as a fundamental splicing regulator in the reprogramming pathway and reveal the central contribution of ASF/SF2-regulated CaMKIIdelta alternative splicing to functional remodeling in developing heart.

SC35 belongs to the family of SR proteins that regulate alternative splicing in a concentration-dependent manner in vitro and in vivo. We previously reported that SC35 is expressed through alternatively spliced mRNAs with differing 3' untranslated sequences and stabilities. Here, we show that overexpression of SC35 in HeLa cells results in a significant decrease of endogenous SC35 mRNA levels along with changes in the relative abundance of SC35 alternatively spliced mRNAs. Remarkably, SC35 leads to both an exon inclusion and an intron excision in the 3' untranslated region of its mRNAs. In vitro splicing experiments performed with recombinant SR proteins demonstrate that SC35, but not ASF/SF2 or 9G8, specifically activates these alternative splicing events. Interestingly, the resulting mRNA is very unstable and we present evidence that mRNA surveillance is likely to be involved in this instability. SC35 therefore constitutes the first example of a splicing factor that controls its own expression through activation of splicing events leading to expression of unstable mRNA.

The splicing-factor oncoprotein SRSF1 (also known as SF2/ASF or ASF/SF2) is upregulated in breast cancers. We investigated the ability of SRSF1 to transform human and mouse mammary epithelial cells in vivo and in vitro. SRSF1-overexpressing COMMA-1D cells formed tumors, following orthotopic transplantation to reconstitute the mammary gland. In three-dimensional (3D) culture, SRSF1-overexpressing MCF-10A cells formed larger acini than control cells, reflecting increased proliferation and delayed apoptosis during acinar morphogenesis. These effects required the first RNA-recognition motif and nuclear functions of SRSF1. SRSF1 overexpression promoted alternative splicing of BIM (also known as BCL2L11) and BIN1 to produce isoforms that lack pro-apoptotic functions and contribute to the phenotype. Finally, SRSF1 cooperated specifically with MYC to transform mammary epithelial cells, in part by potentiating eIF4E activation, and these cooperating oncogenes are significantly coexpressed in human breast tumors. Thus, SRSF1 can promote breast cancer, and SRSF1 itself or its downstream effectors may be valuable targets for the development of therapeutics.

The RNA-binding protein Sam68 is involved in apoptosis, but its cellular mRNA targets and its mechanism of action remain unknown. We demonstrate that Sam68 binds the mRNA for Bcl-x and affects its alternative splicing. Depletion of Sam68 by RNA interference caused accumulation of antiapoptotic Bcl-x(L), whereas its up-regulation increased the levels of proapoptotic Bcl-x(s). Tyrosine phosphorylation of Sam68 by Fyn inverted this effect and favored the Bcl-x(L) splice site selection. A point mutation in the RNA-binding domain of Sam68 influenced its splicing activity and subnuclear localization. Moreover, coexpression of ASF/SF2 with Sam68, or fusion with an RS domain, counteracted Sam68 splicing activity toward Bcl-x. Finally, Sam68 interacted with heterogenous nuclear RNP (hnRNP) A1, and depletion of hnRNP A1 or mutations that impair this interaction attenuated Bcl-x(s) splicing. Our results indicate that Sam68 plays a role in the regulation of Bcl-x alternative splicing and that tyrosine phosphorylation of Sam68 by Src-like kinases can switch its role from proapoptotic to antiapoptotic in live cells.

Higher eukaryotic cells express a family of essential splicing factors with a characteristic RNA-binding domain and serine/arginine-rich (SR) motif. These SR proteins, which include SC35 and SF2/ASF, are conserved from Drosophila to man, are required for early steps of spliceosome assembly and can influence splice-site selections. To address their mechanisms of action, SR proteins were examined for their role in committing pre-messenger RNA to the splicing pathway. I report here that SC35 was sufficient on its own to form a committed complex with human beta-globin pre-mRNA. Examination of other SR proteins and pre-mRNA substrates revealed that single SR proteins committed different pre-mRNAs to splicing with pronounced substrate specificity. These results suggest that splicing of different pre-mRNAs may require distinct sets of SR proteins, and that the commitment by SR proteins may be a critical step at which alternative and tissue-specific splicing is regulated.

Vascular endothelial growth factor A (VEGFA; hereafter referred to as VEGF) is a key regulator of physiological and pathological angiogenesis. Two families of VEGF isoforms are generated by alternate splice-site selection in the terminal exon. Proximal splice-site selection (PSS) in exon 8 results in pro-angiogenic VEGFxxx isoforms (xxx is the number of amino acids), whereas distal splice-site selection (DSS) results in anti-angiogenic VEGFxxxb isoforms. To investigate control of PSS and DSS, we investigated the regulation of isoform expression by extracellular growth factor administration and intracellular splicing factors. In primary epithelial cells VEGFxxxb formed the majority of VEGF isoforms (74%). IGF1, and TNFalpha treatment favoured PSS (increasing VEGFxxx) whereas TGFbeta1 favoured DSS, increasing VEGFxxxb levels. TGFbeta1 induced DSS selection was prevented by inhibition of p38 MAPK and the Clk/sty (CDC-like kinase, CLK1) splicing factor kinase family, but not ERK1/2. Clk phosphorylates SR protein splicing factors ASF/SF2, SRp40 and SRp55. To determine whether SR splicing factors alter VEGF splicing, they were overexpressed in epithelial cells, and VEGF isoform production assessed. ASF/SF2, and SRp40 both favoured PSS, whereas SRp55 upregulated VEGFxxxb (DSS) isoforms relative to VEGFxxx. SRp55 knockdown reduced expression of VEGF165b. Moreover, SRp55 bound to a 35 nucleotide region of the 3'UTR immediately downstream of the stop codon in exon 8b. These results identify regulation of splicing by growth and splice factors as a key event in determining the relative pro-versus anti-angiogenic expression of VEGF isoforms, and suggest that p38 MAPK-Clk/sty kinases are responsible for the TGFbeta1-induced DSS selection, and identify SRp55 as a key regulatory splice factor.

The SR protein SF2/ASF has been initially characterized as a splicing factor but has also been shown to mediate postsplicing activities such as mRNA export and translation. Here we demonstrate that SF2/ASF promotes translation initiation of bound mRNAs and that this activity requires the presence of the cytoplasmic cap-binding protein eIF4E. SF2/ASF promotes translation initiation by suppressing the activity of 4E-BP, a competitive inhibitor of cap-dependent translation. This activity is mediated by interactions of SF2/ASF with both mTOR and the phosphatase PP2A, two key regulators of 4E-BP phosphorylation. These findings suggest the model whereby SF2/ASF functions as an adaptor protein to recruit the signaling molecules responsible for regulation of cap-dependent translation of specific mRNAs. Taken together, these data suggest a novel mechanism for the activation of translation initiation of a subset of mRNAs bound by the shuttling protein SF2/ASF.

Human pre-mRNA splicing factor SF2/ASF has an activity required for general splicing in vitro and promotes utilization of proximal alternative 5' splice sites in a concentration-dependent manner by opposing hnRNP A1. We introduced selected mutations in the N-terminal RNA recognition motif (RRM) and the C-terminal Arg/Ser (RS) domain of SF2/ASF, and assayed the resulting recombinant proteins for constitutive and alternative splicing in vitro and for binding to pre-mRNA and mRNA. Mutants inactive in constitutive splicing can affect alternative splice site selection, demonstrating that these activities involve distinct molecular interactions. Specific protein-RNA contact mediated by Phe56 and Phe58 in the RNP-1 submotif of the SF2/ASF RRM are essential for constitutive splicing, although they are not required for RRM-mediated binding to pre-mRNA. The RS domain is also required for constitutive splicing activity and both Arg and Ser residues are important. Analysis of domain deletion mutants demonstrated strong synergy between the RRM and a central degenerate RRM repeat in binding to RNA. These two domains are sufficient for alternative splicing activity in the absence of an RS domain.

Alternative mRNA splicing of the fibronectin EDI exon is controlled by a purine-rich exonic splicing enhancer (ESE), postulated as a binding site for SR proteins. By using a transient expression alternative splicing assay combined with promoter swapping, we have demonstrated that the promoter can also control EDI splicing, arguing for coupling between the transcription and splicing machineries. We now report that the SR proteins SF2/ASF and 9G8 stimulate EDI splicing in vivo and that their effect requires an intact EDI ESE. Most importantly, we show that sensitivity to these SR proteins critically depends on the promoter structure, suggesting that the transcription machinery modulates their recruitment to the ESE.

Several metazoan splicing factors are characterized by ribonucleoprotein (RNP) consensus sequences and arginine-serine repeats (RS domain) which are essential for their function in splicing. These include members of the SR-protein family (SC35, SF2/ASF), the U1 small nuclear (sn) RNP protein (U1-70K) and the U2 snRNP auxiliary factor (U2AF). SR proteins are phosphorylated in vivo and the phosphorylation state of U1-70K's RS domain influences its splicing activity. Here we report the purification of a protein kinase that is specific for SR proteins and show that it is DNA topoisomerase I. This enzyme lacks a canonical ATP-binding motif but binds ATP with a dissociation constant of 50 nM. Camptothecin and derivatives, known to be specific inhibitors of DNA topoisomerase I, strongly inhibit the kinase activity in the presence of DNA and affect the phosphorylation state of SR proteins. Thus, DNA topoisomerase I may well be one of the SR protein kinases operating in vivo.

We have used nuclear extracts prepared from tobacco leaf tissue to characterize a factor binding site, designated as-2 (activating sequence-2), at the -100 region of the cauliflower mosaic virus 35S promoter. The activity of this factor, called ASF-2 (activating sequence factor-2), is not detected in tobacco root extracts. as-2 includes two GT motifs with sequence homology to the SV40 enhancer core A element and the Box II element of pea rbcS. Nevertheless, oligomers of these sequence elements do not compete for ASF-2 binding in gel retardation assays, indicating that the GT motifs may not be involved. Methylation interference studies identify two guanines (G93 and G98) that are required for interaction with ASF-2. Sequences surrounding these two critical guanines display homologies to a GATA repeat conserved among several light-responsive promoters. One such sequence from a petunia Cab promoter is able to compete with as-2 for factor binding. In transgenic plants, a tetramer of as-2 is able to confer leaf expression when fused 5' to the -90 derivative of the 35S promoter. The expression is not dependent on light and, thus, the as-2 tetramer does not function as a light-responsive element in this context. Histochemical localization of the reporter gene product suggests that the as-2 tetramer directs expression in trichomes, vascular elements, and epidermal and mesophyll cells.

The "altered Schaedler flora" (ASF) was developed for colonizing germfree rodents with a standardized microbiota. The purpose of this study was to identify each of the eight ASF strains by 16S rRNA sequence analysis. Three strains were previously identified as Lactobacillus acidophilus (strain ASF 360), Lactobacillus salivarius (strain ASF 361), and Bacteroides distasonis (strain ASF 519) based on phenotypic criteria. 16S rRNA analysis indicated that each of the strains differed from its presumptive identity. The 16S rRNA sequence of strain ASF 361 is essentially identical to the 16S rRNA sequences of the type strains of Lactobacillus murinis and Lactobacillus animalis (both isolated from mice), and all of these strains probably belong to a single species. Strain ASF 360 is a novel lactobacillus that clusters with L. acidophilus and Lactobacillus lactis. Strain ASF 519 falls into an unnamed genus containing [Bacteroides] distasonis, [Bacteroides] merdae, [Bacteroides] forsythus, and CDC group DF-3. This unnamed genus is in the Cytophaga-Flavobacterium-Bacteroides phylum and is most closely related to the genus Porphyromonas. The spiral-shaped strain, strain ASF 457, is in the Flexistipes phylum and exhibits sequence identity with rodent isolates of Robertson. The remaining four ASF strains, which are extremely oxygen-sensitive fusiform bacteria, group phylogenetically with the low-G+C-content gram-positive bacteria (Firmicutes, Bacillus-Clostridium group). ASF 356, ASF 492, and ASF 502 fall into Clostridium cluster XIV of Collins et al. Morphologically, ASF 492 resembles members of this cluster, Roseburia cecicola, and Eubacterium plexicaudatum. The 16S rRNA sequence of ASF 492 is identical to that of E. plexicaudatum. Since the type strain and other viable original isolates of E. plexicaudatum have been lost, strain ASF 492 is a candidate for a neotype strain. Strain ASF 500 branches deeply in the low-G+C-content gram-positive phylogenetic tree but is not closely related to any organisms whose 16S rRNA sequences are currently in the GenBank database. The 16S rRNA sequence information determined in the present study should allow rapid identification of ASF strains and should permit detailed analysis of the interactions of ASF organisms during development of intestinal disease in mice that are coinfected with a variety of pathogenic microorganisms.

Reversible phosphorylation plays an important role in pre-mRNA splicing in mammalian cells. Two kinases, SR protein-specific kinase (SRPK1) and Clk/Sty, have been shown to phosphorylate the SR family of splicing factors. We report here the cloning and characterization of SRPK2, which is highly related to SRPK1 in sequence, kinase activity, and substrate specificity. Random peptide selection for preferred phosphorylation sites revealed a stringent preference of SRPK2 for SR dipeptides, and the consensus derived may be used to predict potential phosphorylation sites in candidate arginine and serine-rich (RS) domain-containing proteins. Phosphorylation of an SR protein (ASF/SF2) by either SRPK1 or 2 enhanced its interaction with another RS domain-containing protein (U1 70K), and overexpression of either kinase induced specific redistribution of splicing factors in the nucleus. These observations likely reflect the function of the SRPK family of kinases in spliceosome assembly and in mediating the trafficking of splicing factors in mammalian cells. The biochemical and functional similarities between SRPK1 and 2, however, are in contrast to their differences in expression. SRPK1 is highly expressed in pancreas, whereas SRPK2 is highly expressed in brain, although both are coexpressed in other human tissues and in many experimental cell lines. Interestingly, SRPK2 also contains a proline-rich sequence at its NH2 terminus, and a recent study showed that this NH2-terminal sequence has the capacity to interact with a WW domain protein in vitro. Together, our studies suggest that different SRPK family members may be uniquely regulated and targeted, thereby contributing to splicing regulation in different tissues, during development, or in response to signaling.

The SR proteins, a group of abundant arginine/serine (RS)-rich proteins, are essential pre-mRNA splicing factors that are localized in the nucleus. The RS domain of these proteins serves as a nuclear localization signal. We found that RS domain-bearing proteins do not utilize any of the known nuclear import receptors and identified a novel nuclear import receptor specific for SR proteins. The SR protein import receptor, termed transportin-SR (TRN-SR), binds specifically and directly to the RS domains of ASF/SF2 and SC35 as well as several other SR proteins. The nuclear transport regulator RanGTP abolishes this interaction. Recombinant TRN-SR mediates nuclear import of RS domain- bearing proteins in vitro. TRN-SR has amino acid sequence similarity to several members of the importin beta/transportin family. These findings strongly suggest that TRN-SR is a nuclear import receptor for the SR protein family.

In metazoans, multiple RNA-binding proteins, including the shuttling serine/arginine-rich (SR)-splicing factors, function as adapters for mRNA nuclear export by interacting with the export receptor TAP/nuclear export factor 1 (NXF1). Yet, it is unclear how interactions between adapters and TAP are regulated. Here, we demonstrate that the SR proteins 9G8 and ASF/SF2 exhibit higher affinity for TAP/NXF1 when hypophosphorylated. 9G8 is recruited to the pre-mRNA in a hyperphosphorylated form but becomes hypophosphorylated during splicing both in vivo and in vitro. TAP preferentially binds spliced mRNA-protein complexes compared with pre-mRNA-protein complexes. Thus, the phosphorylation state of the SR protein adapters may underlie the selectivity of TAP-mediated export of spliced mRNA.

We have used protection against ribonuclease H to investigate the mechanisms by which U1 small nuclear ribonucleoprotein particles (snRNPs) determine the use of two alternative 5' splice sites. The initial binding of U1 snRNPs to alternative consensus splice sites was indiscriminate, and on a high proportion of pre-mRNA molecules both sites were occupied simultaneously. When the sites were close, this inhibited splicing. We propose that double occupancy leads to the use of the downstream site for splicing and that this is the cause of the proximity effect seen with strong alternative splice sites. This model predicts that splicing to an upstream site of any strength requires a low affinity of U1 snRNPs for the downstream site. This prediction was tested both by cleaving the 5' end of U1 snRNA and by altering the sequence of the downstream site of an adenovirus E1A gene. The enhancement of downstream 5' splice site use by splicing factor SF2/ASF appears to be mediated by an increase in the strength of U1 snRNP binding to all sites indiscriminately.

The Drosophila doublesex female-specific splicing enhancer consists of two classes of regulatory elements, six 13-nucleotide repeat sequences, and a single purine-rich element (PRE). Here, we show that the Drosophila regulatory proteins Transformer (Tra) and Transformer 2 (Tra2) recruit different members of the SR family of splicing factors to the repeats and the PRE. The complexes formed on the repeats in HeLa cell extract consist of Tra, Tra2, and the SR protein 9G8. in Drosophila Kc cell extract, Tra and Tra2 recruit the SR protein RBP1 to the repeats. These proteins are arranged in a specific order on the repeats, with the SR protein at the 5' end of each repeat, and Tra2 at each 3' end. Although Tra did not cross-link strongly to the repeats, its presence was essential for the binding of Tra2 to the 3' end of the repeat. Individual SR proteins were also recruited to the PRE by Tra and Tra2, but in this case they were SF2/ASF and dSRp30 in HeLa and Drosophila cell extracts, respectively. The binding of Tra2, Tra, and the specific SR proteins to the repeats or the PRE was highly cooperative within each complex. Thus, Tra2, which contains a single RNA binding domain, can recognize distinct sequences in the repeats and the PRE in conjunction with specific SR proteins. These observations show that the protein composition of each complex is determined by the RNA recognition sequence and specific interactions between SR proteins and Tra and Tra2.

Expression of most RNA polymerase II transcripts requires the coordinated execution of transcription, splicing, and 3' processing. We have previously shown that upon transcriptional activation of a gene in vivo, pre-mRNA splicing factors are recruited from nuclear speckles, in which they are concentrated, to sites of transcription (Misteli, T., J.F. Cáceres, and D.L. Spector. 1997. Nature. 387:523-527). This recruitment process appears to spatially coordinate transcription and pre-mRNA splicing within the cell nucleus. Here we have investigated the molecular basis for recruitment by analyzing the recruitment properties of mutant splicing factors. We show that multiple protein domains are required for efficient recruitment of SR proteins from nuclear speckles to nascent RNA. The two types of modular domains found in the splicing factor SF2/ ASF exert distinct functions in this process. In living cells, the RS domain functions in the dissociation of the protein from speckles, and phosphorylation of serine residues in the RS domain is a prerequisite for this event. The RNA binding domains play a role in the association of splicing factors with the target RNA. These observations identify a novel in vivo role for the RS domain of SR proteins and suggest a model in which protein phosphorylation is instrumental for the recruitment of these proteins to active sites of transcription in vivo.

The activity of the SR protein family of splicing factors in constitutive or alternative splicing requires direct interactions with the pre-mRNA substrate. Thus it is important to define the high affinity targets of the various SR species and to evaluate their ability to discriminate between defined RNA targets. We have analyzed the binding specificity of the 30-kDa SR protein 9G8, which contains a zinc knuckle in addition to the RNA binding domain (RBD). Using a SELEX approach, we demonstrate that 9G8 selects RNA sequences formed by GAC triplets, whereas a mutated zinc knuckle variant selects different RNA sequences, centered around a (A/U)C(A/U)(A/U)C motif, indicating that the zinc knuckle is involved in the RNA recognition specificity of 9G8. In contrast, SC35 selects sequences composed of pyrimidine or purine-rich motifs. Analyses of RNA-protein interactions with purified recombinant 30-kDa SR proteins or in nuclear extracts, by means of UV crosslinking and immunoprecipitation, demonstrate that 9G8, SC35, and ASF/SF2 recognize their specific RNA targets with high specificity. Interestingly, the RNA sequences selected by the mutated zinc knuckle 9G8 variant are efficiently recognized by SRp20, in agreement with the fact that the RBD of 9G8 and SRp20 are similar. Finally, we demonstrate the ability of 9G8 and of its zinc knuckle variant, or SRp20, to act as efficient splicing transactivators through their specific RNA targets. Our results provide the first evidence for cooperation between an RBD and a zinc knuckle in defining the specificity of an RNA binding domain.

Certain DNA and RNA sequences can form G-quadruplexes, which can affect promoter activity, genetic instability, RNA splicing, translation, and neurite mRNA localization. Amyotrophic lateral sclerosis and frontotemporal dementia were recently shown to be caused by expansion of a (GGGGCC)n·(GGCCCC)n repeat in the C9orf72 gene. Mutant r(GGGGCC)n-containing transcripts aggregate in nuclear foci possibly sequestering repeat-binding proteins, suggesting a toxic RNA pathogenesis. We demonstrate that the r(GGGGCC)n RNA but not the C-rich r(GGCCCC)n RNA forms extremely stable uni- and multimolecular parallel G-quadruplex structures (up to 95 °C). Multimolecular G-quadruplex formation is influenced by repeat number and RNA concentration. MBNL1, a splicing factor that is sequestered in myotonic dystrophy patients by binding to expanded r(CUG)n repeat hairpins, does not bind the C9orf72 repeats, but the splicing factor ASF/SF2 can bind the r(GGGGCC)n repeat. Because multimolecular G-quadruplexes are enhanced by repeat length, RNA-RNA interactions facilitated by G-quadruplex formation at expanded repeats might influence transcript aggregation and foci formation in amyotrophic lateral sclerosis-frontotemporal dementia cells. Tract length-dependent G-quadruplex formation by the C9orf72 RNA should be considered when assessing the role of this repeat in C9orf72 gene activity, protein binding, transcript foci formation, and translation of the C9orf72 product, including the noncanonical repeat-associated non-ATG translation (RAN translation) into pathologic dipeptide repeats, as well as any oligonucleotide repeat-based therapy.