RNA structure replaces the need for U2AF2 in splicing

RNA secondary structure plays an integral role in catalytic, ribosomal, small nuclear, micro, and transfer RNAs. Discovering a prevalent role for secondary structure in pre-mRNAs has proven more elusive. By utilizing a variety of computational and biochemical approaches, we present evidence for a class of nuclear introns that relies upon secondary structure for correct splicing. These introns are defined by simple repeat expansions of complementary AC and GT dimers that co-occur at opposite boundaries of an intron to form a bridging structure that enforces correct splice site pairing. Remarkably, this class of introns does not require U2AF2, a core component of the spliceosome, for its processing. Phylogenetic analysis suggests that this mechanism was present in the ancestral vertebrate lineage prior to the divergence of tetrapods from teleosts. While largely lost from land dwelling vertebrates, this class of introns is found in 10% of all zebrafish genes.RNA splicing is a process that removes an internal segment of RNA (i.e., the intron) and rejoins together the two flanking segments (exons). Distinct but evolutionarily related versions of this processing reaction are found in prokaryotes and eukaryotes in a variety of different contexts. In eukaryotes, the splicing of nuclear introns is catalyzed by a large riboprotein complex called the spliceosome (Matlin and Moore 2007). RNA encoded by genes in organelles and some bacterial genomes contain self-splicing group I and II introns which catalyze their own removal (Cech et al. 1981). A basic problem for all introns is the correct identification and pairing of the splice sites. In group I and II introns, this pairing function is performed by RNA secondary structure alone, whereas in spliceosomal introns, small nuclear ribonucleoproteins (snRNPs) recognize and pair together the correct 5′ splice site (5′ ss) and branchpoint site (BP). However, there are some examples where the pairing of sites is assisted by intramolecular secondary structure in the intron (Goguel and Rosbash 1993; Libri et al. 1995; Charpentier and Rosbash 1996; Howe and Ares 1997; Spingola et al. 1999). In addition, there are some fascinating examples of how secondary structures can regulate mutually exclusive alternative splicing (Warf and Berglund 2007; McManus and Graveley 2011): Several regions of the Dscam1 pre-mRNA undergo extensive alternative splicing. In one of these regions, an upstream “selector” sequence near exon 5 can select from an array of 48 complementary downstream “docking” sequences. Each “docking” sequence can potentially base-pair with the “selector” sequence, thereby bringing an alternate version of exon 6 to splice to exon 5 (Celotto and Graveley 2001; Graveley et al. 2004; Graveley 2005; Kreahling and Graveley 2005; May et al. 2011). As only a single hairpin can form, only a single 3′ splice site (3′ ss) can pair. Recent work suggests analogous mechanisms may explain regulated splicing at several other loci (Yang et al. 2011).Secondary structure in RNA can be identified experimentally or computationally. There are currently around a thousand publicly available structures—53% determined by X-ray crystallography and 47% by solution NMR (Bernstein et al. 1977). There have been a great many advances in computational approaches to predicting secondary structures (Mathews 2006; Mathews et al. 2007; Seetin and Mathews 2012). A variety of algorithms are currently in use, the most common being free energy minimization, which are increasingly used in combination with comparative sequence analysis and protection/enzymatic mapping approaches (Mathews 2006; Bellamy-Royds and Turcotte 2007; Low and Weeks 2010). A functional role for a predicted secondary structure has typically been explored by a two-step process of introducing mutations to disrupt predicted structure, followed by compensatory mutations at a second site designed to restore structure (Chen and Stephan 2003).Here, we report a functional role for expansions of simple repeats that is mediated by RNA secondary structure. These simple repeats were discovered using a computational method for detecting rapidly evolving noncoding splicing elements (Lim et al. 2011). A combination of chemical mapping of RNA structure, compensatory mutation analysis, and in silico RNA folding was utilized to define a novel class of structured introns.     

The U2AF35-related protein Urp contacts the 3' splice site to promote U12-type intron splicing and the second step of U2-type intron splicing

The U2AF35-related protein Urp has been implicated previously in splicing of the major class of U2-type introns. Here we show that Urp is also required for splicing of the minor class of U12-type introns. Urp is recruited in an ATP-dependent fashion to the U12-type intron 3' splice site, where it promotes formation of spliceosomal complexes. Remarkably, Urp also contacts the 3' splice site of a U2-type intron, but in this case is specifically required for the second step of splicing. Thus, through recognition of a common splicing element, Urp facilitates distinct steps of U2- and U12-type intron splicing.     

Suppressors of a U4 snRNA mutation define a novel U6 snRNP protein with RNA-binding motifs

U4 and U6 small nuclear RNAs are associated by an extensive base-pairing interaction that must be disrupted and reformed with each round of splicing. U4 mutations within the U4/U6 interaction domain destabilize the complex in vitro and cause a cold-sensitive phenotype in vivo. Restabilization of the U4/U6 helix by dominant (gain-of-function), compensatory mutations in U6 results in wild-type growth. Cold-insensitive growth can also be restored by two classes of recessive (loss-of-function) suppressors: (1) mutations in PRP24, which we show to be a U6-specific binding protein of the RNP-consensus family; and (2) mutations in U6, which lie outside the interaction domain and identify putative PRP24-binding sites. Destabilization of the U4/U6 helix causes the accumulation of a PRP24/U4/U6 complex, which is undetectable in wild-type cells. The loss-of-function suppressor mutations inhibit the binding of PRP24 to U6, and thus presumably promote the release of PRP24 from the PRP24/U4/U6 complex and the reformation of the base-paired U4/U6 snRNP. We propose that the PRP24/U4/U6 complex is normally a highly transient intermediate in the spliceosome cycle and that PRP24 promotes the reannealing of U6 with U4.     

Remote protein homology detection and fold recognition using two-layer support vector machine classifiers

Remote protein homology detection and fold recognition refer to detection of structural homology in proteins where there are small or no similarities in the sequence. To detect protein structural classes from protein primary sequence information, homology-based methods have been developed, which can be divided to three types: discriminative classifiers, generative models for protein families and pairwise sequence comparisons. Support Vector Machines (SVM) and Neural Networks (NN) are two popular discriminative methods. Recent studies have shown that SVM has fast speed during training, more accurate and efficient compared to NN. We present a comprehensive method based on two-layer classifiers. The 1st layer is used to detect up to superfamily and family in SCOP hierarchy using optimized binary SVM classification rules. It used the kernel function known as the Bio-kernel, which incorporates the biological information in the classification process. The 2nd layer uses discriminative SVM algorithm with string kernel that will detect up to protein fold level in SCOP hierarchy. The results obtained were evaluated using mean ROC and mean MRFP and the significance of the result produced with pairwise t-test was tested. Experimental results show that our approaches significantly improve the performance of remote protein homology detection and fold recognition for all three different version SCOP datasets (1.53, 1.67 and 1.73). We achieved 4.19% improvements in term of mean ROC in SCOP 1.53, 4.75% in SCOP 1.67 and 4.03% in SCOP 1.73 datasets when compared to the result produced by well-known methods. The combination of first layer and second layer of BioSVM-2L performs well in remote homology detection and fold recognition even in three different versions of datasets.     

Confirmation of the genetic association between the U2AF homology motif (UHM) kinase 1 (UHMK1) gene and schizophrenia on chromosome 1q23.3

UHMK1 has previously been implicated as a susceptibility gene for schizophrenia in the 1q23.3 region by significant evidence of allelic and haplotypic association between schizophrenia and several genetic markers at UHMK1 in a London-based case-control sample. Further fine mapping of the UHMK1 gene locus in the University College London schizophrenia case-control sample was carried out with tagging SNPs. Two additional SNPs were found to be associated with schizophrenia (rs6604863 P = 0.02, rs10753578 P = 0.017). Tests of allelic and haplotypic association were then carried out in a second independent sample from Aberdeen consisting of 858 individuals with schizophrenia and 591 controls. Two of these SNPs also showed association in the Aberdeen sample (rs7513662 P = 0.0087, rs10753578 P = 0.022) and several haplotypes were associated (global permutation P = 0.0004). When the UCL and Aberdeen samples were combined three SNPs (rs7513662 P = 0.0007, rs6427680 P = 0.0252, rs6694863 P = 0.015) and several haplotypes showed association (eg HAP-A, HAP-B, HAP-C permutation P = 0.00005). The finding of allelic association with markers in the UHMK1 gene might help explain why it has not been possible, despite great effort, to satisfactorily confirm previously reported associations between schizophrenia and the genes RGS4 and NOS1AP/CAPON. These genes flank UHMK1 and all three loci are within a 700 kb region showing linkage to schizophrenia. The confirmation of association between UHMK1 and schizophrenia, rather than RGS4 and NOS1AP in the London sample, points to the possibility that previous efforts to accurately fine map a gene in the 1q23.3 region have lacked accuracy or may have suffered from methodological flaws.     

U2AF2 variant in a patient with developmental delay,dysmorphic features,and epilepsy

Variants in the RNA binding protein (RBP) U2AF2 are hypothesized to cause a novel neurodevelopmental disorder. Here, we report a patient with a de novo missense variant in U2AF2, the second case report of the same variant, and third case report overall. The patient in this report has a history of global developmental delay, dysmorphic features, and epilepsy. This presentation is consistent with the previous case report with the same U2AF2 variant and with a recent case report of another U2AF2 variant, strengthening the evidence that variants in U2AF2 are the cause of a novel neurodevelopmental disorder.     

CDART: protein homology by domain architecture

The Conserved Domain Architecture Retrieval Tool (CDART) performs similarity searches of the NCBI Entrez Protein Database based on domain architecture, defined as the sequential order of conserved domains in proteins. The algorithm finds protein similarities across significant evolutionary distances using sensitive protein domain profiles rather than by direct sequence similarity. Proteins similar to a query protein are grouped and scored by architecture. Relying on domain profiles allows CDART to be fast, and, because it relies on annotated functional domains, informative. Domain profiles are derived from several collections of domain definitions that include functional annotation. Searches can be further refined by taxonomy and by selecting domains of interest. CDART is available at http://www.ncbi.nlm.nih.gov/Structure/lexington/lexington.cgi.     

Confusing dinosaurs with mammals: tetrapod phylogenetics and anatomical terminology in the world of homology

At present, three different systems of anatomical nomenclature are available to researchers describing new tetrapod taxa: a nonstandardized traditional system erected in part by Sir Richard Owen and subsequently elaborated by Alfred Romer; a standardized system created for avians, the Nomina Anatomica Avium (NAA); and a standardized system for extant (crown-group) mammals, the Nomina Anatomica Veterinaria (NAV). Conserved homologous structures widely distributed within the Tetrapoda are often granted different names in each system. The recent shift toward a phylogenetic system based on homology requires a concomitant shift toward a single nomenclatural system also based on both evolutionary and functional morphological homology. Standardized terms employed in the NAA and NAV should be perpetuated as far as possible basally in their respective phylogenies. Thus, NAA terms apply to nonavian archosaurs (or even all diapsids) and NAV terms apply to noncrown-group mammals and more basal synapsids. Taxa equally distant from both avians and crown-group mammals may maintain the traditional nonstandardized terminology until a universal anatomical nomenclature for all tetrapods is constructed.     

A novel protein with RNA-binding motifs interacts with ataxin-2

Spinocerebellar ataxia type 2 (SCA2) is caused by expansion of a polyglutamine tract in ataxin-2, a protein of unknown function. Using the yeast two-hybrid system, we identified a novel protein, A2BP1 (ataxin-2 binding protein 1) which binds to the C-terminus of ataxin-2. Northern blot analysis showed that A2BP1 was predominantly expressed in muscle and brain. By immunocfluorescent staining, A2BP1 and ataxin-2 were both localized to the trans -Golgi network. Immunocytochemistry showed that A2BP1 was expressed in the cytoplasm of Purkinje cells and dentate neurons in a pattern similar to that seen for ataxin-2 labeling. Western blot analysis of subcellular fractions indicated enrichment of A2BP1 in the same fractions as ataxin-2. Sequence analysis of the A2BP1 cDNA revealed an RNP motif that is highly conserved among RNA-binding proteins. A2BP1 had striking homology with a human cDNA clone, P83A20, of unknown function and at least two copies of A2BP1 homologs are found in the Caenorhabditis elegans genome database. A2BP1 and related proteins appear to form a novel gene family sharing RNA-binding motifs.     

Clinical features and biological implications of different U2AF1 mutation types in myelodysplastic syndromes

U2AF1 mutations (U2AF1MT) occur commonly in myelodysplastic syndromes (MDS) without ring sideroblasts. The aim of this study was to investigate the clinical and biological implications of different U2AF1 mutation types in MDS. We performed targeted gene sequencing in a cohort of 511 MDS patients. Eighty‐six patients (17%) were found to have U2AF1MT, which occurred more common in younger patients (P = .001) and represented ancestral lesions in a substantial proportion (71%) of cases. ASXL1MT and isolated +8 were significantly enriched in U2AF1MT‐positive cases, whereas TP53MT, SF3B1MT, and complex karyotypes were inversely associated with U2AF1MT. U2AF^S34 subjects were enriched for isolated +8 and were inversely associated with complex karyotypes. U2AF1MT was significantly associated with anemia, thrombocytopenia, and poor survival in both lower‐risk and higher‐risk MDS. U2AF1^S34 subjects had more frequently platelet levels of <50 × 10⁹/L (P = .043) and U2AF1^Q157/U2AF1^R156 subjects had more frequently hemoglobin concentrations at <80 g/L (P = .008) and more often overt fibrosis (P = .049). In conclusion, our study indicates that U2AF1MT is one of the earliest genetic events in MDS patients and that different types of U2AF1MT have distinct clinical and biological characteristics.     

T cell recognition motifs of an immunodominant peptide of myelin basic protein in patients with multiple sclerosis: structural requirements and clinical implications

Myelin basic protein (MBP)-reactive T cells may play an important role in the pathogenesis of multiple sclerosis (MS). The T cell response to the 83 – 99 region of MBP represents a dominant autoreactive response to MBP in MS patients of DR2 haplotype. In this study, a large panel of DR2- and DR4-restricted T cell clones specific for the MBP83 – 99 peptide were examined for the recognition motifs and structural requirements for antigen recognition using alanine-substituted peptides. Our study revealed that although the recognition motifs of the T cell clones were diverse, the TCR contact residues within the 83 – 99 region of MBP were highly conserved. Two central residues (Phe90 and Lys91) served as the critical TCR contact points for both DR2- and DR4-restricted T cell clones. Single alanine substitution at residue 90 or residue 91 abolished the responses of 81 – 95 % of the T cell clones while a double alanine substitution rendered all T cell clones unresponsive. It was also demonstrated in this study that the substituted peptides altered the cytokine profile of some, but not all, T cell clones. Some MBP83 – 99-specific T cell clones were able to sustain alanine substitutions and were susceptible to activation by microbial antigens. The study has an important implication in designing a peptide-based therapy for MS.     

Predominant expression of the src homology 2-containing tyrosine phosphatase protein SHP2 in vascular smooth muscle cells

src homology 2 (SH2)-containing protein-tyrosine phosphatase SHP2 is known to transduce positive signals from activated receptor protein-tyrosine kinases such as platelet-derived growth factor receptor (PDGFR) and insulin receptor. Here, we demonstrate the physiological expression of SHP2 in rats. In northern and western blot analyses, SHP2 expressions were recognized in all tissues, but their expression levels varied significantly among tissues; it is lowest in the liver and kidney. Immunohistochemical staining and in situ hybridization showed SHP2 was expressed ubiquitously but predominantly in vascular smooth muscle cells (SMC). During the development of granulations, SHP2 was expressed predominantly in vascular SMC and also highly expressed in capillary cells. The functional associations of SHP2 with PDGFR, which transduces major growth signals in vascular SMC, identify a crucial function of SHP2 in blood vessels in consert with PDGFR.     

Characterization of a Trypanosoma brucei SR domain-containing protein bearing homology to cis-spliceosomal U1 70 kDa proteins

The protozoan parasite Trypanosoma brucei relies on trans-splicing of a common spliced leader (SL) RNA to maturate mRNAs. Using the yeast two-hybrid system a protein (TSR1IP) was identified that interacts with the T. brucei serine-arginine (SR) protein termed TSR1. TSR1IP shows homology to U1 70 kDa proteins, and contains an SR rich domain as well as an acidic/arginine domain homologous to the U1 70 kDa poly(A) polymerase inhibiting domain. This protein is localized in the nucleoplasm and excluded from the nucleolus in trypanosomal bloodstream and procyclic forms. Based on structural modelling predictions and on the identification of a RNA recognition motif (RRM), it was possible to demonstrate by the yeast three-hybrid system that TSR1IP interacts with the 5' splice region of the SL RNA. All the above characteristics suggest that TSR1IP could be involved in trans-splicing.     

Perturbation of U2AF65/NXF1-mediated RNA nuclear export enhances RNA toxicity in polyQ diseases

Expanded CAG RNA has recently been reported to contribute to neurotoxicity in polyglutamine (polyQ) degeneration. In this study, we showed that RNA carrying an expanded CAG repeat progressively accumulated in the cell nucleus of transgenic Drosophila that displayed degeneration. Our gene knockdown and mutant analyses demonstrated that reduction of U2AF50 function, a gene involved in RNA nuclear export, intensified nuclear accumulation of expanded CAG RNA and resulted in a concomitant exacerbation of expanded CAG RNA-mediated toxicity in vivo. We found that the human U2AF50 ortholog, U2AF65, interacted directly and specifically with expanded CAG RNA via its RRM3 domain. We further identified an RNA/protein complex that consisted of expanded CAG RNA, U2AF65 and the NXF1 nuclear export receptor. The U2AF65 protein served as an adaptor to link expanded CAG RNA to NXF1 for RNA export. Finally, we confirmed the nuclear accumulation of expanded CAG RNA in symptomatic polyQ transgenic mice and also observed a neurodevelopmental downregulation of U2AF65 protein levels in mice. Altogether, our findings demonstrate that the cell nucleus is a site where expanded CAG RNA exerts its toxicity. We also provide a novel mechanistic explanation to how perturbation of RNA nuclear export would contribute to polyQ degeneration.     

Deletion of MUD2, the yeast homolog of U2AF65, can bypass the requirement for sub2, an essential spliceosomal ATPase

Mammalian U2AF65 and UAP56 are required for prespliceosome (PS) formation. We tested the predictions that the yeast UAP56 homolog, SUB2, is required for the same step and functions collaboratively with MUD2, the yeast homolog of U2AF65. Unexpectedly, sub2-1 extracts accumulate PS-like complexes. Moreover, deletion of MUD2 exacerbates the cs phenotype of sub2 alleles yet suppresses both the ts sub2-1 and the lethal Deltasub2 phenotypes. We propose that Sub2 functionally interacts with Mud2 both before and after PS formation. In the absence of Mud2, Sub2 function becomes dispensable.