RNA Splicing Protein Research Articles

Altered Expression of RNA Splicing Proteins in Alzheimer's Disease Patients: Evidence from Two Microarray Studies

Background/Aims: Dysregulation of pre-mRNA splicing from an altered expression of RNA splice-regulatory proteins may act as the convergence point underlying aberrant gene expression changes in Alzheimer's disease (AD). Methods: Two microarray datasets from a control/AD postmortem brain cohort of 31 subjects - 9 controls and 22 AD subjects (National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database) - were used. Results: Between the two microarray studies, the expression of six splice-regulatory protein genes showed concordant changes in AD. These genes were then correlated with gene expression changes of transcripts reported to be altered in AD. Amyloid beta (A4) precursor protein and tropomyosin receptor kinase B transcripts were found to correlate significantly with the same splice-regulatory proteins in the two studies. Conclusion: This study highlights a susceptibility network that can potentially link a number of susceptibility genes.

Transcriptional changes and developmental abnormalities in a zebrafish model of myotonic dystrophy type 1

Myotonic dystrophy type I (DM1) is a multi-system, autosomal dominant disorder caused by expansion of a CTG repeat sequence in the 3′UTR of the DMPK gene. The size of the repeat sequence correlates with age at onset and disease severity, with large repeats leading to congenital forms of DM1 associated with hypotonia and intellectual disability. In models of adult DM1, expanded CUG repeats lead to an RNA toxic gain of function, mediated at least in part by sequestering specific RNA splicing proteins, most notably muscleblind-related (MBNL) proteins. However, the impact of CUG RNA repeat expression on early developmental processes is not well understood. To better understand early developmental processes in DM1, we utilized the zebrafish, Danio rerio, as a model system. Direct injection of (CUG)91 repeat-containing mRNA into single-cell embryos induces toxicity in the nervous system and muscle during early development. These effects manifest as abnormal morphology, behavioral abnormalities and broad transcriptional changes, as shown by cDNA microarray analysis. Co-injection of zebrafish mbnl2 RNA suppresses (CUG)91 RNA toxicity and reverses the associated behavioral and transcriptional abnormalities. Taken together, these findings suggest that early expression of exogenously transcribed CUG repeat RNA can disrupt normal muscle and nervous system development and provides a new model for DM1 research that is amenable to small-molecule therapeutic development.

Identification of SFPQ as novel interacting partner of HIV-1 Integrase and its functional characterization

Background HIV-1 requires the support of its appropriate host for the survival and propagation like any other parasite. These host cell factors have been reported to be involved in different stages of viral life cycle. The nuclear import and infection maintenance of HIV-1 Integrase (IN) in human cells is dependent upon the integration efficiency of the proviral DNA and stability of viral RNA. In our study we identified a new host cell interacting factor for HIV-1 IN, SFPQ-a RNA splicing protein, by cross-linking, pull down and mass spectrometry.

Identification of Differentially Expressed Proteins in Curcumin-Treated Prostate Cancer Cell Lines

Due to high prevalence and slow progression of prostate cancer, primary prevention appears to be attractive strategy for its eradication. During the last decade, curcumin (diferuloylmethane), a natural compound from the root of turmeric (Curcuma longa), was described as a potent chemopreventive agent. Curcumin exhibits anti-inflammatory, anticarcinogenic, antiproliferative, antiangiogenic, and antioxidant properties in various cancer cell models. This study was designed to identify proteins involved in the anticancer activity of curcumin in androgen-dependent (22Rv1) and -independent (PC-3) human prostate cancer cell lines using two-dimensional difference in gel electrophoresis (2D-DIGE). Out of 425 differentially expressed spots, we describe here the MALDI-TOF-MS analysis of 192 spots of interest, selected by their expression profile. This approach allowed the identification of 60 differentially expressed proteins (32 in 22Rv1 cells and 47 in PC-3 cells). Nineteen proteins are regulated in both cell lines. Further bioinformatic analysis shows that proteins modulated by curcumin are implicated in protein folding (such as heat-shock protein PPP2R1A; RNA splicing proteins RBM17, DDX39; cell death proteins HMGB1 and NPM1; proteins involved in androgen receptor signaling, NPM1 and FKBP4/FKBP52), and that this compound could have an impact on miR-141, miR-152, and miR-183 expression. Taken together, these data support the hypothesis that curcumin is an interesting chemopreventive agent as it modulates the expression of proteins that potentially contribute to prostate carcinogenesis.

Systematic identification of common functional modules related to heart failure with different etiologies

The development of heart failure (HF) is a complex process that can be initiated by multiple etiologies. Identifying common functional modules associated with HF is a challenging task. Here, we developed a systems method to identify these common functional modules by integrating multiple expression profiles, protein interactions from four species, gene function annotations, and text information. We identified 1439 consistently differentially expressed genes (CDEGs) across HF with different etiologies by applying three meta-analysis methods to multiple HF-related expression profiles. Using a weighted human interaction network constructed by combining interaction data from multiple species, we extracted 60 candidate CDEG modules. We further evaluated the functional relevance of each module by using expression, interaction network, functional annotations, and text information together. Finally, five functional modules with significant biological relevance were identified. We found that almost half of the genes in these modules are hubs in the weighted network, and that these modules can accurately classify HF patients from healthy subjects. We also identified many significantly enriched biological processes that contribute to the pathophysiology of HF, including two new ones, RNA splicing and vesicle-mediated protein transport. In summary, we proposed a novel framework to analyze common functional modules related to HF with different etiologies. Our findings provide important insights into the complex mechanism of HF. Further biological experimentations should be required to validate these novel biological processes.

Proteome-wide Detection of Abl1 SH3-binding Peptides by Integrating Computational Prediction and Peptide Microarray

Protein-protein interactions are essential for regulating almost all aspects of cellular functions. Many of these interactions are mediated by weak and transient protein domain-peptide binding, but they are often under-represented in high throughput screening of protein-protein interactions using techniques such as yeast two-hybrid and mass spectrometry. On the other hand, computational predictions and in vitro binding assays are valuable in providing clues of in vivo interactions. We present here a systematic approach that integrates computer modeling and a peptide microarray technology to identify binding peptides of the SH3 domain of the tyrosine kinase Abl1 in the human proteome. Our study provides a comprehensive list of candidate interacting partners for the Abl1 protein, among which the presence of numerous methyltransferases and RNA splicing proteins may suggest a novel function of Abl1 in chromatin remodeling and RNA processing. This study illustrates a powerful approach for integrating computational and experimental methods to detect protein interactions mediated by domain-peptide recognition.

Genetic variation in the alternative splicing regulator RBM20 is associated with dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a leading cause of heart failure and death. The etiology of DCM is genetically heterogeneous. We sought to define the prevalence of mutations in the RNA splicing protein RBM20 in a large cohort with DCM and to determine whether genetic variation in RBM20 is associated with clinical outcomes. Subjects included in the Genetic Risk Assessment of Defibrillator Events (GRADE) study were aged at least 18 years, had an ejection fraction of ≤30%, and an implantable cardioverter-defibrillator (ICD). The coding region and splice junctions of RBM20 were screened in subjects with DCM; 2 common polymorphisms in RBM20, rs942077 and rs35141404, were genotyped in all GRADE subjects. A total of 1465 subjects were enrolled in the GRADE study, and 283 with DCM were screened for RBM20 mutations. The mean age of subjects with DCM was 58 ± 13 years, 64% were males, and the mean follow-up time was 24.2 ± 17.1 months after ICD placement. RBM20 mutations were identified in 8 subjects with DCM (2.8%). Mutation carriers had a similar survival, transplantation rate, and frequency of ICD therapy compared with nonmutation carriers. Three of 8 subjects with RBM20 mutations (37.5%) had atrial fibrillation (AF), whereas 19 subjects without mutations (7.4%) had AF (P = .02). Among all GRADE subjects, rs35141404 was associated with AF (minor allele odds ratio = 0.62; 95% confidence interval = 0.44-0.86; P = .006). In the subset of GRADE subjects with DCM, rs35141404 was associated with AF (minor allele odds ratio = 0.58; P = .047). Mutations in RBM20 were observed in approximately 3% of subjects with DCM. There were no differences in survival, transplantation rate, and frequency of ICD therapy in mutation carriers.

ATM germline mutations in Spanish early‐onset breast cancer patients negative for BRCA1/BRCA2 mutations

Heterozygous carriers of ATM (ataxia telangiectasia mutated gene) mutations have increased risk of breast cancer (BC). We have estimated the prevalence of mutations in the ATM gene among Spanish patients with early-onset BC. Forty-three patients diagnosed with BC before the age of 46 years, and negative for BRCA1 and BRCA2 mutations, were analysed for the presence of ATM mutations. A total of 34 ATM sequence variants were detected: 1 deleterious mutation, 10 unclassified variants and 23 polymorphisms. One patient (2.3%) carried the ATM deleterious mutation (3802delG that causes ataxia telangiectasia in the homozygous state) and 13 patients carried the 10 ATM unclassified variants. The truncating mutation 3802delG and eight of the rare variants were not detected in a control group of 150 individuals. Different bioinformatic sequence analysis tools were used to evaluate the effects of the unclassified ATM changes on RNA splicing and function protein. This in silico analysis predicted that the missense variants 7653 T>C and 8156 G>A could alter the splicing by disrupting an exonic splicing enhancer motif and the 3763 T>G, 6314 G>C, and 8156 G>A variants would affect the ATM protein function. These are the initial results concerning the prevalence of germline mutations in the ATM gene among BC cases in a Spanish population, and they suggest that ATM mutations can confer increased susceptibility to early-onset BC.

Germline mutations in the breast cancer susceptibility gene PTEN are rare in high-risk non-BRCA1/2 French Canadian breast cancer families

Cowden syndrome is a disease associated with an increase in breast cancer susceptibility. Alleles in PTEN and other breast cancer susceptibility genes would be responsible for approximately 25% of the familial component of breast cancer risk, BRCA1 and BRCA2 being the two major genes responsible for this inherited risk. In order to evaluate the proportion of high-risk French Canadian non-BRCA1/BRCA2 breast/ovarian cancer families potentially harboring a PTEN germline mutation, the whole coding and flanking intronic sequences were analyzed in a series of 98 breast cancer cases. Although no germline mutation has been identified in the coding region, our study led to the identification of four intronic variants. Further investigations were performed to analyze the effect of these variants, alone and/or in combination, on splicing and PTEN protein levels. Despite suggestive evidence emerging from in silico analyses, the presence of these intronic variants do not seem to alter RNA splicing or PTEN protein levels. In addition, as loss of PTEN or part of it has been reported, Western blot analysis has also been performed. No major deletion could be identified in our cohort. Therefore, assuming a Poisson distribution for the frequency of deleterious mutation in our cohort, if the frequency of such deleterious mutation was 2%, we would have had a 90% or greater chance of observing at least one such mutation. These results suggest that PTEN germline mutations are rare and are unlikely to account for a significant proportion of familial breast cancer cases in the French Canadian population.

Structure and Interactions of the First Three RNA Recognition Motifs of Splicing Factor Prp24

The essential Saccharomyces cerevisiae pre-messenger RNA splicing protein 24 (Prp24) has four RNA recognition motifs (RRMs) and facilitates U6 RNA base-pairing with U4 RNA during spliceosome assembly. Prp24 is a component of the free U6 small nuclear ribonucleoprotein particle (snRNP) but not the U4/U6 bi-snRNP, and so is thought to be displaced from U6 by U4/U6 base-pairing. The interaction partners of each of the four RRMs of Prp24 and how these interactions direct U4/U6 pairing are not known. Here we report the crystal structure of the first three RRMs and the solution structure of the first two RRMs of Prp24. Strikingly, RRM 2 forms extensive inter-domain contacts with RRMs 1 and 3. These contacts occupy much of the canonical RNA-binding faces (β-sheets) of RRMs 1 and 2, but leave the β-sheet of RRM 3 exposed. Previously identified substitutions in Prp24 that suppress mutations in U4 and U6 spliceosomal RNAs cluster primarily in the β-sheet of RRM 3, but also in a conserved loop of RRM 2. RNA binding assays and chemical shift mapping indicate that a large basic patch evident on the surface of RRMs 1 and 2 is part of a high affinity U6 RNA binding site. Our results suggest that Prp24 binds free U6 RNA primarily with RRMs 1 and 2, which may remodel the U6 secondary structure. The β-sheet of RRM 3 then influences U4/U6 pairing through interaction with an unidentified ligand.

The proteome survey of an electricity‐generating organ (Torpedo californica electric organ)

Torpedo californica is a species in class Chondrichthyes. Electric rays have evolved the electric organ, which is similar to the mammalian neuromuscular junction (NMJ). Here, we took a combined cDNA sequencing and proteomic approach to define the molecular constituents of the T. californica electric organ. For soluble proteins, 2-DE was used and 224 protein spots were mapped. Plasma membrane fractions were analyzed using the shotgun approach (LC-MS/MS). A Torpedo cDNA library was constructed and 607 cDNA clones were sequenced. Identification of electric organ proteins was done using cross-species comparisons, and a custom database was constructed from cDNA translations. We unambiguously identified 121 proteins and transcripts, 103 of which were novel additions to the existing databases of Torpedo fish. Fifteen proteins of known function, but not previously associated with either the electroplaque or NMJ, were present at high abundance. These included the heat shock and oxidative stress proteins, annexin V (calelectrin), and plectin 1. Most interesting were the unambiguous matches to 11 human ORFs of unknown function, including four potential RNA splicing proteins, a vacuolar sorting protein, and a tetraspanin containing protein. This analysis identified proteins that may play a role in the higher vertebrate neuromuscular junction or other electrical synapses.

Functional genomic analysis of commercial baker's yeast during initial stages of model dough-fermentation

Gene expression profiles of baker's yeast during initial dough-fermentation were investigated using liquid fermentation (LF) media to obtain insights at the molecular level into rapid adaptation mechanisms of baker's yeast. Results showed that onset of fermentation caused drastic changes in gene expression profiles within 15 min. Genes involved in the tricarboxylic acid (TCA) cycle were down-regulated and genes involved in glycolysis were up-regulated, indicating a metabolic shift from respiration to fermentation. Genes involved in ethanol production ( PDC genes and ADH1), in glycerol synthesis ( GPD1 and HOR2), and in low-affinity hexose transporters ( HXT1 and HXT3) were up-regulated at the beginning of model dough-fermentation. Among genes up-regulated at 15 min, several genes classified as transcription were down-regulated within 30 min. These down-regulated genes are involved in messenger RNA splicing and ribosomal protein biogenesis and in transcriptional regulator ( SRB8, MIG1). In contrast, genes involved in amino acid metabolism and in vitamin metabolism, such as arginine biosynthesis, riboflavin biosynthesis, and thiamin biosynthesis, were subsequently up-regulated after 30 min. Interestingly, the genes involved in the unfolded protein response (UPR) pathway were also subsequently up-regulated. Our study presents the first overall description of the transcriptional response of baker's yeast during dough-fermentation, and will thus help clarify genomic responses to various stresses during commercial fermentation processes.

Identification of a family of DNA-binding proteins with homology to RNA splicing factors

We describe a unique family of human proteins that are capable of binding to the cAMP regulatory element (CRE) and that are homologous to RNA splicing proteins. A human cDNA was isolated that encodes a protein with a distinctive combination of modular domain structures: 2 leucine-zipper-like domains, a DNA-binding zinc-finger-like domain, an RNA-binding zinc-finger-like domain, and 2 coiled-coil protein-protein interaction domains. It also has a serine-arginine-rich domain, commonly found in proteins involved in RNA splicing. The protein was discovered using the CRE as bait in a yeast 1-hybrid assay. It was then shown to bind specifically to the CRE in vitro using gel shift assays. We have named the protein CRE-associated protein (CREAP). We show that it is widely expressed in human tissues but is highly expressed in several fetal tissues and in several regions of the adult brain. CREAP is closely related to 2 human proteins of unknown function. CREAP shows significant homology with a small nuclear ribonucleoprotein of yeast, Luc7p, involved in 5' splice site recognition. The 3 human CREAP proteins form a unique family with the potential to act as transcription factors that link to RNA processing.

CHD1 associates with NCoR and histone deacetylase as well as with RNA splicing proteins

CHD1 is one of a family of nuclear proteins containing two chromodomains, a SWI/SNF-like helicase/ATPase domain and a DNA binding domain. We found that CHD1 co-immunoprecipitates with histone deacetylase (HDAC) activity and that CHD1 also associates with NCoR, a transcriptional corepressor, in yeast two-hybrid and in vitro pull-down assays. NCoR is known to associate with HDACs to effect its repressive activity, suggesting that the predicted chromatin remodeling activity of CHD1 plays a role in this repression. Yeast two-hybrid assays also showed that CHD1 interacts with splicing proteins mKIAA0164, Srp20, and SAF-B. Splicing assays show that CHD1 overexpression can affect alternative splicing. These results suggest that CHD1 may function in both chromatin mediated transcriptional repression and RNA splicing.

Expression, Cellular Localization, and Enzymatic Activities of RNA Helicase II/Guβ

RNA helicase II/Gu (RH-II/Gu) is a nucleolar DEAD-box protein that unwinds double-stranded RNA and introduces secondary structure to a single-stranded RNA. We recently identified its paralogue, RH-II/Guβ, in contrast to the original RH-II/Guα. Their similar intron–exon structures on chromosome 10 suggest gene duplication. To determine functional differences, their expression, localization, and enzymatic activities were compared. RH-II/Guα is expressed two- to threefold more than RH-II/Guβ in most tissues. Both proteins localize to nucleoli, suggesting roles in ribosomal RNA production, but RH-II/Guβ also localizes to nuclear speckles containing splicing factor SC35, suggesting possible involvement in pre-mRNA splicing. The C-terminus responsible for nuclear speckle localization of RH-II/Guβ contains an arginine–serine-rich domain present in some RNA splicing proteins. In vitro assays show weaker ATPase and RNA helicase activities of RH-II/Guβ. RH-II/Guα unwinds RNA substrate with a 21- or 34-nt duplex and 5′ overhangs, but RH-II/Guβ unwinds only the shorter duplex. Although RH-II/Guβ has no RNA folding activity, it catalyzes formation of an RNA complex with unidentified structure, which is not observed when assayed with a mixture of the two enzymes. Instead, the presence of RH-II/Guβ stimulates RH-II/Guα unwinding activity. Our data suggest distinct and complex regulation of expression of the two paralogues with nonredundant gene products.

Characterization of a putative murine mitochondrial transporter homology of hMRS3/4

We report here a novel murine gene, Mrs3/4, encoding a putative mitochondrial transporter homologous to the yeast mitochondrial RNA splicing protein 3 and 4 (yMRS3&4) and its human counterpart hMRS3/4. By analyses of radiation hybrids, Mrs3/4 was mapped to mouse Chromosome (Chr) 19 between D19Mit66 and D19Mit24 with a distance of 22.4 cR to D19Mit66, a region that is syntenic to human Chromosome 10q24, where the human gene is located. Structural analysis shows that these proteins belong to the mitochondrial carrier family (MCF), which is characterized by three repeats with two transmembrane domains in each repeat. The murine Mrs3/4 gene has two splicing forms similar to the human homolog. The long form corresponds to the 341-amino acid (aa) protein with six transmembrane domains, and the short form corresponds to the 177-aa protein with three transmembrane domains. Both forms have well-conserved signature sequences of MCF. Targeting experiments showed that both forms have mitochondrial localization. Northern blot analyses showed that Mrs3/4 is ubiquitously expressed as a 1.8-kb transcript with a relative abundance in the heart, liver, kidney, and testis. In conclusion, the reported Mrs3/4 gene shows a strong conservation, mitochondrial protein localization, and a ubiquitous expression, which suggests that it has an important role in mammalian cells, most likely as an ion transporter across the mitochondrial inner membrane.

Characterization of a novel human putative mitochondrial transporter homologous to the yeast mitochondrial RNA splicing proteins 3 and 4

We report here a novel human gene, hMRS3/ 4, encoding a putative mitochondrial transporter structurally and functionally homologous to the yeast mitochondrial RNA splicing proteins 3 and 4. These proteins belong to the family of mitochondrial carrier proteins (MCF) and are likely to function as solute carriers. hMRS3/ 4 spans ∼10 kb of genomic DNA on chromosome 10q24 and consists of four exons that encode a 364-aa protein with six transmembrane domains. A putative splice variant, encoding a 177-aa protein with three transmembrane domains, was also identified. hMRS3/4 has a well-conserved signature sequence of MCF and is targeted into the mitochondria. When expressed in yeast, hMRS3/ 4 efficiently restores the mitochondrial functions in mrs3 omrs4 o knock-out mutants. Ubiquitous expression in human tissues and a well-conserved structure and function suggest an important role for hMRS3/4 in human cells.

Molecular Cloning and Expression Analysis of a Putative Nuclear Protein, SR-25

We cloned a full-length mouse cDNA and its human homologue encoding a novel protein designated as “SR-25.” In Northern blot analysis, SR-25 mRNA was expressed in all organs tested, and relatively abundant in testis and thymus. Deduced amino acid sequences of mouse SR-25 and human SR-25 showed 77.7% identity. SR-25 has a serine-arginine repeat (SR repeat) and two types of amino acid clusters: a serine cluster and a highly basic cluster. Based on the presence of many nuclear localizing signals and a similarity to RNA splicing proteins, SR-25 is strongly suggested to be a nuclear protein and may contribute to RNA splicing.

Protein Fate in Neurodegenerative Proteinopathies: Polyglutamine Diseases Join the (Mis)Fold

Protein misfolding is now recognized to be a central feature of neurodegenerative diseases. Alzheimer disease (AD), Parkinson disease (PD), amyotrophic lateral sclerosis (ALS), prion diseases, and the polyglutamine (polygln) diseases all represent proteinopathies—diseases in which a particular protein or set of proteins misfolds and aggregates. In the past decade, mutations have been identified in an increasing number of genes underlying specific forms of neurodegeneration—for example, genes encoding amyloid precursor protein (APP) and presenilins 1 and 2 in AD, parkin and alpha-synuclein in PD, Cu/Zn superoxide dismutase in ALS, tau in frontotemporal dementia, prion protein in Creutzfeldt-Jacob and Gerstmann-Straussler diseases, and at least eight different proteins in the polygln diseases (Hardy and GwinnHardy 1998; Price et al. 1998). Identification of these mutations was a first step in understanding the disease process; next, it will be necessary to explain how specific changes in disease proteins may alter the neuron’s physiology and to further question how neuronal responses to the mutant protein influence the disease process. This review examines the fate of mutant protein in neurons and the potential role of posttranslational events in pathogenesis, with particular focus on the polygln diseases. Posttranslational events have already been implicated

A series of mutations in the dihydropteridine reductase gene resulting in either abnormal RNA splicing or DHPR protein defects. Mutations in brief no. 244. Online.

Five novel mutations are described which result in the rare hyperphenylalaninemia DHPR-deficiency. Three of these are located at different intron/exon boundaries within the DHPR gene, and disrupt the maturation of the DHPR transcript such that little full-length mRNA can be detected by RT-PCR. Each mutation alters a conserved nucleotide within the splice site consensus sequence, and results in the skipping of an exon and, in one case, the activation of an inappropriate splicing signal. Two further mutations are missense mutations resulting in a non-conservative amino acid change within the DHPR protein (L14P and G17V) and are associated with a severe phenotype.