Crystal structures of Nova-1 and Nova-2 K-homology RNA-binding domains.

BACKGROUND: Nova-1 and Nova-2 are related neuronal proteins that were initially cloned using antisera obtained from patients with the autoimmune neurological disease paraneoplastic opsoclonus-myoclonus ataxia (POMA). Both of these disease gene products contain three RNA-binding motifs known as K-homology or KH domains, and their RNA ligands have been identified via binding-site selection experiments. The KH motif structure has been determined previously using NMR spectroscopy, but not using X-ray crystallography. Many proteins contain more than one KH domain, yet there is no published structural information regarding the behavior of such multimers. RESULTS: We have obtained the first X-ray crystallographic structures of KH-domain-containing proteins. Structures of the third KH domains (KH3) of Nova-1 and Nova-2 were determined by multiple isomorphous replacement and molecular replacement at 2.6 A and 2.0 A, respectively. These highly similar RNA-binding motifs form a compact protease-resistant domain resembling an open-faced sandwich, consisting of a three-stranded antiparallel beta sheet topped by three alpha helices. In both Nova crystals, the lattice is composed of symmetric tetramers of KH3 domains that are created by two dimer interfaces. CONCLUSIONS: The crystal structures of both Nova KH3 domains are similar to the previously determined NMR structures. The most significant differences among the KH domains involve changes in the positioning of one or more of the alpha helices with respect to the betasheet, particularly in the NMR structure of the KH1 domain of the Fragile X disease protein FMR-1. Loop regions in the KH domains are clearly visible in the crystal structure, unlike the NMR structures, revealing the conformation of the invariant Gly-X-X-Gly segment that is thought to participate in RNA-binding and of the variable region. The tetrameric arrangements of the Nova KH3 domains provide insights into how KH domains may interact with each other in proteins containing multiple KH motifs.     

Nova-1 regulates neuron-specific alternative splicing and is essential for neuronal viability

We have combined genetic and biochemical approaches to analyze the function of the RNA-binding protein Nova-1, the paraneoplastic opsoclonus-myoclonus ataxia (POMA) antigen. Nova-1 null mice die postnatally from a motor deficit associated with apoptotic death of spinal and brainstem neurons. Nova-1 null mice show specific splicing defects in two inhibitory receptor pre-mRNAs, glycine alpha2 exon 3A (GlyRalpha2 E3A) and GABA(A) exon gamma2L. Nova protein in brain extracts specifically bound to a previously identified GlyRalpha2 intronic (UCAUY)3 Nova target sequence, and Nova-1 acted directly on this element to increase E3A splicing in cotransfection assays. We conclude that Nova-1 binds RNA in a sequence-specific manner to regulate neuronal pre-mRNA alternative splicing; the defect in splicing in Nova-1 null mice provides a model for understanding the motor dysfunction in POMA.     

Functional domains of the human splicing factor ASF/SF2.

The human splicing factor ASF/SF2 displays two predominant activities in in vitro splicing assays: (i) it is an essential factor apparently required for all splices and (ii) it is able to switch utilization of alternative 5' splice sites in a concentration-dependent manner. ASF/SF2 is the prototype of a family of proteins typified by the presence of one or two RNP-type RNA binding domains (RBDs) and a region highly enriched in repeating arginine-serine dipeptides (RS regions). Here we describe a functional analysis of ASF/SF2, which defines several regions essential for one, or both, of its two principal activities, and provides insights into how this type of protein functions in splicing. Two isoforms of the protein, which arise from alternative splicing, are by themselves inactive, but each can block the activity of ASF/SF2, thereby functioning as splicing repressors. Some, but not all, mutations in the RS region prevent ASF/SF2 from functioning as an essential splicing factor. However, the entire RS region can be deleted without reducing splice site switching activity, indicating that it is not absolutely required for interaction with other splicing factors. Experiments with deletion and substitution mutants reveal that the protein contains two related, but highly diverged, RBDs, and that both are essential for activity. Each RBD by itself retains the ability to bind RNA, although optimal binding requires both domains.     

Functional analysis of pre-mRNA splicing factor SF2/ASF structural domains.

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.     

Molecular basis of RNA recognition by the human alternative splicing factor Fox-1

The Fox-1 protein regulates alternative splicing of tissue-specific exons by binding to GCAUG elements. Here, we report the solution structure of the Fox-1 RNA binding domain (RBD) in complex with UGCAUGU. The last three nucleotides, UGU, are recognized in a canonical way by the four-stranded beta-sheet of the RBD. In contrast, the first four nucleotides, UGCA, are bound by two loops of the protein in an unprecedented manner. Nucleotides U1, G2, and C3 are wrapped around a single phenylalanine, while G2 and A4 form a base-pair. This novel RNA binding site is independent from the beta-sheet binding interface. Surface plasmon resonance analyses were used to quantify the energetic contributions of electrostatic and hydrogen bond interactions to complex formation and support our structural findings. These results demonstrate the unusual molecular mechanism of sequence-specific RNA recognition by Fox-1, which is exceptional in its high affinity for a defined but short sequence element.     

Differential recognition of the polypyrimidine-tract by the general splicing factor U2AF65 and the splicing repressor sex-lethal

The polypyrimidine-tract (Py-tract) adjacent to 3' splice sites is an essential splicing signal and is recognized by several proteins, including the general splicing factor U2AF65 and the highly specific splicing repressor Sex-lethal (SXL). They both contain ribonucleoprotein-consensus RNA-binding motifs. However, U2AF65 recognizes a wide variety of Py-tracts, whereas SXL recognizes specific Py-tracts such as the nonsex-specific Py-tract of the transformer pre-mRNA. It is not understood how these seemingly similar proteins differentially recognize the Py-tract. To define these interactions, we used chemical interference and protection assays, saturation mutagenesis, and RNAs containing modified nucleotides. We find that these proteins recognize distinct features of the RNA. First, although uracils within the Py-tract are protected from chemical modification by both of these proteins, modification of any one of seven uracils by hydrazine, or any of eight phosphates by ethylnitrosourea strongly interfered with the binding of SXL only. Second, the 2' hydroxyl groups or backbone conformation appeared important for the binding of SXL, but not U2AF65. Third, although any of the bases (cytosine > adenine > guanine) could substitute for uracils for U2AF65 binding, only guanine partially substituted for certain uracils for SXL binding. The different dependence on individual contacts and nucleotide preference may provide a basis for the different RNA-binding specificities and thus functions of U2AF65 and SXL in 3' splice site choice.     

Mammalian splicing factor SF1 interacts with SURP domains of U2 snRNP-associated proteins

Splicing factor 1 (SF1) recognizes the branch point sequence (BPS) at the 3′ splice site during the formation of early complex E, thereby pre-bulging the BPS adenosine, thought to facilitate subsequent base-pairing of the U2 snRNA with the BPS. The 65-kDa subunit of U2 snRNP auxiliary factor (U2AF65) interacts with SF1 and was shown to recruit the U2 snRNP to the spliceosome. Co-immunoprecipitation experiments of SF1-interacting proteins from HeLa cell extracts shown here are consistent with the presence of SF1 in early splicing complexes. Surprisingly almost all U2 snRNP proteins were found associated with SF1. Yeast two-hybrid screens identified two SURP domain-containing U2 snRNP proteins as partners of SF1. A short, evolutionarily conserved region of SF1 interacts with the SURP domains, stressing their role in protein–protein interactions. A reduction of A complex formation in SF1-depleted extracts could be rescued with recombinant SF1 containing the SURP-interaction domain, but only partial rescue was observed with SF1 lacking this sequence. Thus, SF1 can initially recruit the U2 snRNP to the spliceosome during E complex formation, whereas U2AF65 may stabilize the association of the U2 snRNP with the spliceosome at later times. In addition, these findings may have implications for alternative splicing decisions.     

Ordered multi-site phosphorylation of the splicing factor ASF/SF2 by SRPK1

The human alternative splicing factor ASF/SF2, an SR (serine-arginine-rich) protein involved in mRNA splicing control, is activated by the multisite phosphorylation of its C-terminal RS domain, a segment containing numerous arginine-serine dipeptide repeats. The protein kinase responsible for this modification, SR-specific protein kinase 1 (SRPK1), catalyzes the selective phosphorylation of approximately a dozen serines in only the N-terminal portion of the RS domain (RS1). To gain insights into the nature of selective phosphate incorporation in ASF/SF2, region-specific phosphorylation in the RS domain was monitored as a function of reaction progress. Arg-to-Lys mutations were made at several positions to produce unique protease cleavage sites that separate the RS domain into identifiable N- and C-terminal phosphopeptides upon treatment with lysyl endoproteinase. These studies reveal that SRPK1 docks near the C-terminus of the RS1 segment and then moves in an N-terminal direction along the RS domain. Multiple quadruple Ser-to-Ala and deletion mutations did not disrupt the phosphorylation of other sites regardless of position, suggesting that the active site of SRPK1 docks in a flexible manner at the center of the RS domain. Taken together, these data suggest that SRPK1 uses a unique ‘grab-and-pull’ mechanism to control the regiospecific phosphorylation of its protein substrate.     

Interaction of poly(rC)-binding protein 2 domains KH1 and KH3 with coxsackievirus RNA

Recombinant hnRNP K-homology (KH) domains 1 and 3 of the poly(rC)-binding protein (PCBP) 2 were purified and assayed for interaction with coxsackievirus B3 RNA in electrophoretic mobility shift assays using in vitro transcribed RNAs which represent signal structures of the 5′-nontranslated region. KH domains 1 and 3 interact with the extended cloverleaf RNA and domain IV RNA of the internal ribosome entry site (IRES). KH1 but not KH3 interacts with subdomain IV/C RNA, whereas KH3 interacts with subdomain IV/B. All in vitro results are consistent with yeast three-hybrid experiments performed in parallel. The data demonstrate interaction of isolated PCBP2 KH1 and KH3 domains to four distinct target sites within the 5′-nontranslated region of the CVB3 genomic RNA.     

The role of RNA splicing factor PTBP1 in neuronal development

Alternative pre-mRNA splicing, which produces various mRNA isoforms with distinct structures and functions from a single gene, is regulated by specific RNA-binding proteins and is an essential method for regulating gene expression in mammals. Recent studies have shown that abnormal change during neuronal development triggered by splicing mis-regulation is an important feature of various neurological diseases. Polypyrimidine tract binding protein 1 (PTBP1) is a kind of RNA-binding proteins with extensive biological functions. As a well-known splicing regulator, it affects the neuronal development process through its involvement in axon formation, synaptogenesis, and neuronal apoptosis, according to the most recent studies. Here, we summarized the mechanism of alternative splicing, structure and function of PTBP1, and the latest research progress on the role of alternative splicing events regulated by PTBP1 in axon formation, synaptogenesis and neuronal apoptosis, to reveal the mechanism of PTBP1-regulated changes in neuronal development process.     

Regulation of alternative pre-mRNA splicing by hnRNP A1 and splicing factor SF2.

When messenger RNA precursors (pre-mRNAs) containing alternative 5' splice sites are spliced in vitro, the relative concentrations of the heterogeneous ribonucleoprotein (hnRNP) A1 and the essential splicing factor SF2 precisely determine which 5' splice site is selected. In general, an excess of hnRNP A1 favors distal 5' splice sites, whereas an excess of SF2 results in utilization of proximal 5' splice sites. The regulation of these antagonistic activities may play an important role in the tissue-specific and developmental control of gene expression by alternative splicing.     

Function of the conserved S1 and KH domains in polynucleotide phosphorylase

We have examined the roles of the conserved S1 and KH RNA binding motifs in the widely dispersed prokaryotic exoribonuclease polynucleotide phosphorylase (PNPase). These domains can be released from the enzyme by mild proteolysis or by truncation of the gene. Using purified recombinant enzymes, we have assessed the effects of specific deletions on RNA binding, on activity against a synthetic substrate under multiple-turnover conditions, and on the ability of truncated forms of PNPase to form a minimal RNA degradosome with RNase E and RhlB. Deletion of the S1 domain reduces the apparent activity of the enzyme by almost 70-fold under low-ionic-strength conditions and limits the enzyme to digest a single substrate molecule. Activity and product release are substantially regained at higher ionic strengths. This deletion also reduces the affinity of the enzyme for RNA, without affecting the enzyme's ability to bind to RNase E. Deletion of the KH domain produces similar, but less severe, effects, while deletion of both the S1 and KH domains accentuates the loss of activity, product release, and RNA binding but has no effect on binding to RNase E. We propose that the S1 domain, possibly arrayed with the KH domain, forms an RNA binding surface that facilitates substrate recognition and thus indirectly potentiates product release. The present data as well as prior observations can be rationalized by a two-step model for substrate binding.     

X-ray crystallographic and NMR studies of protein-protein and protein-nucleic acid interactions involving the KH domains from human poly(C)-binding protein-2

Poly(C)-binding proteins (PCBPs) are KH (hnRNP K homology) domain-containing proteins that recognize poly(C) DNA and RNA sequences in mammalian cells. Binding poly(C) sequences via the KH domains is critical for PCBP functions. To reveal the mechanisms of KH domain-D/RNA recognition and its functional importance, we have determined the crystal structures of PCBP2 KH1 domain in complex with a 12-nucleotide DNA corresponding to two repeats of the human C-rich strand telomeric DNA and its RNA equivalent. The crystal structures reveal molecular details for not only KH1-DNA/RNA interaction but also protein-protein interaction between two KH1 domains. NMR studies on a protein construct containing two KH domains (KH1 + KH2) of PCBP2 indicate that KH1 interacts with KH2 in a way similar to the KH1-KH1 interaction. The crystal structures and NMR data suggest possible ways by which binding certain nucleic acid targets containing tandem poly(C) motifs may induce structural rearrangement of the KH domains in PCBPs; such structural rearrangement may be crucial for some PCBP functions.     

Functional domains of the yeast splicing factor Prp22p

The essential Saccharomyces cerevisiae PRP22 gene encodes a 1145-amino acid DEXH box RNA helicase. Prp22p plays two roles during pre-mRNA splicing as follows: it is required for the second transesterification step and for the release of mature mRNA from the spliceosome. Whereas the step 2 function of Prp22p does not require ATP hydrolysis, spliceosome disassembly is dependent on the ATPase and helicase activities. Here we delineate a minimal functional domain, Prp22(262-1145), that suffices for the activity of Prp22p in vivo when expressed under the natural PRP22 promoter and for pre-mRNA splicing activity in vitro. The biologically active domain lacks an S1 motif (residues 177-256) that had been proposed to play a role in RNA binding by Prp22p. The deletion mutant Prp22(351-1145) can function in vivo when provided at a high gene dosage. We suggest that the segment from residues 262 to 350 enhances Prp22p function in vivo, presumably by targeting Prp22p to the spliceosome. We characterize an even smaller catalytic domain, Prp22(466-1145) that suffices for ATP hydrolysis, RNA binding, and RNA unwinding in vitro and for nuclear localization in vivo but cannot by itself support cell growth. However, the ATPase/helicase domain can function in vivo if the N-terminal region Prp22(1-480) is co-expressed in trans.     

The molecular basis of ceramide-1-phosphate recognition by C2 domains

Group IVA cytosolic phospholipase A₂ (cPLA₂α), which harbors an N-terminal lipid binding C2 domain and a C-terminal lipase domain, produces arachidonic acid from the sn-2 position of zwitterionic lipids such as phosphatidylcholine. The C2 domain has been shown to bind zwitterionic lipids, but more recently, the anionic phosphomonoester sphingolipid metabolite ceramide-1-phosphate (C1P) has emerged as a potent bioactive lipid with high affinity for a cationic patch in the C2 domain β-groove. To systematically analyze the role that C1P plays in promoting the binding of cPLA₂α-C2 to biological membranes, we employed biophysical measurements and cellular translocation studies along with mutagenesis. Biophysical and cellular translocation studies demonstrate that C1P specificity is mediated by Arg⁵⁹, Arg⁶¹, and His⁶² (an RxRH sequence) in the C2 domain. Computational studies using molecular dynamics simulations confirm the origin of C1P specificity, which results in a spatial shift of the C2 domain upon membrane docking to coordinate the small C1P headgroup. Additionally, the hydroxyl group on the sphingosine backbone plays an important role in the interaction with the C2 domain, further demonstrating the selectivity of the C2 domain for C1P over phosphatidic acid. Taken together, this is the first study demonstrating the molecular origin of C1P recognition.     

Structural basis for polypyrimidine tract recognition by the essential pre-mRNA splicing factor U2AF65

The essential pre-mRNA splicing factor, U2AF(65), guides the early stages of splice site choice by recognizing a polypyrimidine (Py) tract consensus sequence near the 3' splice site. Since Py tracts are relatively poorly conserved in higher eukaryotes, U2AF(65) is faced with the problem of specifying uridine-rich sequences, yet tolerating a variety of nucleotide substitutions found in natural Py tracts. To better understand these apparently contradictory RNA binding characteristics, the X-ray structure of the U2AF(65) RNA binding domain bound to a Py tract composed of seven uridines has been determined at 2.5 A resolution. Specific hydrogen bonds between U2AF(65) and the uracil bases provide an explanation for polyuridine recognition. Flexible side chains and bound water molecules form the majority of the base contacts and potentially could rearrange when the U2AF(65) structure adapts to different Py tract sequences. The energetic importance of conserved residues for Py tract binding is established by analysis of site-directed mutant U2AF(65) proteins using surface plasmon resonance.     

Regulatory roles of heterogeneous nuclear ribonucleoprotein M and Nova-1 protein in alternative splicing of dopamine D2 receptor pre-mRNA

The dopamine D2 receptor (D2R) plays a crucial role in the regulation of diverse key physiological functions, including motor control, reward, learning, and memory. This receptor is present in vivo in two isoforms, D2L and D2S, generated from the same gene by alternative pre-mRNA splicing. Each isoform has a specific role in vivo, underlining the importance of a strict control of its synthesis, yet the molecular mechanism modulating alternative D2R pre-mRNA splicing has not been completely elucidated. Here, we identify heterogeneous nuclear ribonucleoprotein M (hnRNP M) as a key molecule controlling D2R splicing. We show that binding of hnRNP M to exon 6 inhibited the inclusion of this exon in the mRNA. Importantly, the splicing factor Nova-1 counteracted hnRNP M effects on D2R pre-mRNA splicing. Indeed, mutations of the putative Nova-1-binding site on exon 6 disrupted Nova-1 RNA assembly and diminished the inhibitory effect of Nova-1 on hnRNP M-dependent exon 6 exclusion. These results identify Nova-1 and hnRNP M as D2R pre-mRNA-binding proteins and show their antagonistic role in the alternative splicing of D2R pre-mRNA.