Molecular characterization of a novel fission yeast gene spUAP2 that interacts with the splicing factor spU2AF59

A protein essential for pre-mRNA splicing, the U2 auxiliary factor (U2AF), is composed of a large and small subunit. Previously we cloned and characterized both subunits, spU2AF⁵⁹ and spU2AF²³, from fission yeast. We now report a novel U2AF-associated-protein, spUAP2, which interacts with both subunits. SpUAP2 contains a classical and a degenerate RNA recognition motif (RRM), both of which are required for interaction with spU2AF⁵⁹. Interaction also requires the arginine/serine-rich region and the first RRM of spU2AF⁵⁹. A null allele of the gene for spUAP2 is lethal. Received: 23 May / Accepted: 6 August 1997     

The geminivirus nuclear shuttle protein is a virulence factor that suppresses transmembrane receptor kinase activity

Despite the large number of leucine-rich-repeat (LRR) receptor-like-kinases (RLKs) in plants and their conceptual relevance in signaling events, functional information is restricted to a few family members. Here we describe the characterization of new LRR-RLK family members as virulence targets of the geminivirus nuclear shuttle protein (NSP). NSP interacts specifically with three LRR-RLKs, NIK1, NIK2, and NIK3, through an 80-amino acid region that encompasses the kinase active site and A-loop. We demonstrate that these NSP-interacting kinases (NIKs) are membrane-localized proteins with biochemical properties of signaling receptors. They behave as authentic kinase proteins that undergo autophosphorylation and can also phosphorylate exogenous substrates. Autophosphorylation occurs via an intermolecular event and oligomerization precedes the activation of the kinase. Binding of NSP to NIK inhibits its kinase activity in vitro, suggesting that NIK is involved in antiviral defense response. In support of this, infectivity assays showed a positive correlation between infection rate and loss of NIK1 and NIK3 function. Our data are consistent with a model in which NSP acts as a virulence factor to suppress NIK-mediated antiviral responses.     

Variola virus IL-18 binding protein interacts with three human IL-18 residues that are part of a binding site for human IL-18 receptor alpha subunit

Interleukin-18 (IL-18) plays an important role in host defense against microbial pathogens. Many poxviruses encode homologous IL-18 binding proteins (IL-18BP) that neutralize IL-18 activity. Here, we examined whether IL-18BP neutralizes IL-18 activity by binding to the same region of IL-18 where IL-18 receptor (IL-18R) binds. We introduced alanine substitutions to known receptor binding sites of human IL-18 and found that only the substitution of Leu5 reduced the binding affinity of IL-18 with IL-18BP of variola virus (varvIL-18BP) by more than 4-fold. The substitutions of Lys53 and Ser55, which were not previously known to be part of the receptor binding site but that are spatially adjacent to Leu5, reduced the binding affinity to varvIL-18BP by approximately 100- and 7-fold, respectively. These two substitutions also reduced the binding affinity with human IL-18R alpha subunit (hIL-18Ralpha) by 4- and 2-fold, respectively. Altogether, our data show that varvIL-18BP prevents IL-18 from binding to IL-18R by interacting with three residues that are part of the binding site for hIL-18Ralpha.     

A deep intronic mutation in FGB creates a consensus exonic splicing enhancer motif that results in afibrinogenemia caused by aberrant mRNA splicing, which can be corrected in vitro with antisense oligonucleotide treatment

We previously described a novel homozygous point mutation (FGB c.115-600A>G) located deep within intron 1 of the fibrinogen beta gene (FGB), as a likely cause of afibrinogenemia. While this was the only mutation detected, its pathological mechanism was unclear. Here we show the mutation causes the inclusion of a 50-bp cryptic exon by creating a consensus heptad motif recognized by the spliceosome recruiting protein pre-mRNA splicing factor (SF2)/arginine/serine-rich alternative splicing factor (ASF) splicing factor 2/alternative splicing factor (SF2/ASF). Translation of the aberrant mRNA would result in truncation of the Bbeta chain, preventing fibrinogen synthesis. Selective introduction of a second mutation into the enhancer motif abolished the SF2/ASF binding motif and re-established normal pre-mRNA splicing. Subsequent introduction of antisense phosphorodiamidate morpholino oligonucleotides (PMOs) into transfected cells containing the mutant construct blocked the protein-RNA interaction and successfully restored normal splicing ( approximately 50% at 2 microM and approximately 90% at 10 microM). The molecular characterization of this case has revealed a unique disease mechanism, shown the importance of screening for deep intronic mutations, and provided evidence that antisense gene therapy is potentially practical for the treatment of diseases caused by this class of mutation.     

The nuclear microspherule protein 58 is a novel RNA-binding protein that interacts with fragile X mental retardation protein in polyribosomal mRNPs from neurons

The fragile X syndrome, the leading cause of inherited mental retardation, is due to the inactivation of the fragile mental retardation 1 gene (FMR1) and the subsequent absence of its gene product FMRP. This RNA-binding protein is thought to control mRNA translation and its absence in fragile X cells leads to alteration in protein synthesis. In neurons, FMRP is thought to repress specific mRNAs during their transport as silent ribonucleoparticles (mRNPs) from the cell body to the distant synapses which are the sites of local synthesis of neuro-specific proteins. The mechanism by which FMRP sorts out its different mRNAs targets might be tuned by the intervention of different proteins. Using a yeast two-hybrid system, we identified MicroSpherule Protein 58 (MSP58) as a novel FMRP-cellular partner. In cell cultures, we found that MSP58 is predominantly present in the nucleus where it interacts with the nuclear isoform of FMRP. However, in neurons but not in glial cells, MSP58 is also present in the cytoplasmic compartment, as well as in neurites, where it co-localizes with FMRP. Biochemical evidence is given that MSP58 is associated with polyribosomal poly(A)+ mRNPs. We also show that MSP58, similar to FMRP, is present on polyribosomes prepared from synaptoneurosomes and that it behaves as an RNA-binding protein with a high affinity to the G-quartet structure. We propose that this novel cellular partner for FMRP escorts FMRP-containing mRNP from the nucleus and nucleolus to the somato-dendritic compartment where it might participate in neuronal translation regulation.     

DNA polymorphism and epigenetic marks modulate the affinity of a scaffold/matrix attachment region to the nuclear matrix

Mechanisms that regulate attachment of the scaffold/matrix attachment regions (S/MARs) to the nuclear matrix remain largely unknown. We have studied the effect of simple sequence length polymorphism (SSLP), DNA methylation and chromatin organization in an S/MAR implicated in facioscapulohumeral dystrophy (FSHD), a hereditary disease linked to a partial deletion of the D4Z4 repeat array on chromosome 4q. This FSHD-related nuclear matrix attachment region (FR-MAR) loses its efficiency in myoblasts from FSHD patients. Three criteria were found to be important for high-affinity interaction between the FR-MAR and the nuclear matrix: the presence of a specific SSLP haplotype in chromosomal DNA, the methylation of one specific CpG within the FR-MAR and the absence of histone H3 acetylated on lysine 9 in the relevant chromatin fragment.     

FCRLA is a resident endoplasmic reticulum protein that associates with intracellular Igs, IgM, IgG and IgA

Fc receptor-like A (FCRLA) is an unusual member of the extended Fc receptor family. FCRLA has homology to receptors for the Fc portion of Ig (FCR) and to other FCRL proteins. However, unlike these other family representatives, which are typically transmembrane receptors with extracellular ligand-binding domains, FCRLA has no predicted transmembrane domain or N-linked glycosylation sites and is an intracellular protein. We show by confocal microscopy and biochemical assays that FCRLA is a soluble resident endoplasmic reticulum (ER) protein, but it does not possess the amino acid sequence KDEL as an ER retention motif in its C-terminus. Using a series of deletion mutants, we found that its ER retention is most likely mediated by the amino terminal partial Ig-like domain. We have identified ER-localized Ig as the FCRLA ligand. FCRLA is unique among the large family of Fc receptors, in that it is capable of associating with multiple Ig isotypes, IgM, IgG and IgA. Among hemopoietic cells, FCRLA expression is restricted to the B lineage and is most abundant in germinal center B lymphocytes. The studies reported here demonstrate that FCRLA is more broadly expressed among human B lineage cells than originally reported; it is found at significant levels in resting blood B cells and at varying levels in all B-cell subsets in tonsil.     

TEG-1 CD2BP2 regulates stem cell proliferation and sex determination in the C. elegans germ line and physically interacts with the UAF-1 U2AF65 splicing factor

Background : For a stem cell population to exist over an extended period, a balance must be maintained between self‐renewing (proliferating) and differentiating daughter cells. Within the Caenorhabditis elegans germ line, this balance is controlled by a genetic regulatory pathway, which includes the canonical Notch signaling pathway. Results : Genetic screens identified the gene teg‐1 as being involved in regulating the proliferation versus differentiation decision in the C. elegans germ line. Cloning of TEG‐1 revealed that it is a homolog of mammalian CD2BP2, which has been implicated in a number of cellular processes, including in U4/U6.U5 tri‐snRNP formation in the pre‐mRNA splicing reaction. The position of teg‐1 in the genetic pathway regulating the proliferation versus differentiation decision, its single mutant phenotype, and its enrichment in nuclei, all suggest TEG‐1 also functions as a splicing factor. TEG‐1, as well as its human homolog, CD2BP2, directly bind to UAF‐1 U2AF65, a component of the U2 auxiliary factor. Conclusions : TEG‐1 functions as a splicing factor and acts to regulate the proliferation versus meiosis decision. The interaction of TEG‐1 CD2BP2 with UAF‐1 U2AF65, combined with its previously described function in U4/U6.U5 tri‐snRNP, suggests that TEG‐1 CD2BP2 functions in two distinct locations in the splicing cascade. Developmental Dynamics 241:505–521, 2012.© 2012 Wiley Periodicals, Inc.     

Decay-accelerating factor is a component of subendothelial extracellular matrix in vitro,and is augmented by activation of endothelial protein kinase C

The vasculature is protected from complement activation by regulatory molecules expressed on endothelial cells. However, complement fixation also occurs on subendothelial extracellular matrix (ECM) in vitro, and is initiated simply by retraction or removal of overlying cells. To investigate mechanisms controlling vascular complement activation, we examined subendothelial ECM for the presence of complement regulatory proteins. Decay-accelerating factor (DAF) was found on both human umbilical vein endothelial cells (HUVEC) and in their ECM; in contrast, membrane cofactor protein was found only on cells. ECM and HUVEC DAF were distinguishable based on several properties. While HUVEC DAF is anchored to cell membranes by a phospholipase C-sensitive glycosylphosphatidylinositol linkage, DAF was removed from ECM only by proteolytic digestion. Cytokines (TNF-α, IL-1β, IL-4) increased HUVEC DAF expression, but had minimal effect on ECM DAF; in contrast, phorbol 12-myristate 13-acetate (PMA) and wheat germ agglutinin markedly increased DAF on both HUVEC and ECM. The effect of PMA was mediated by activation of protein kinase C. The complement regulatory potential of ECM DAF was assessed by evaluating the effect of DAF-neutralizing antibodies on C3 deposition on HUVEC ECM, as well as on HeLa cell ECM, which had a considerably higher DAF content. DAF blockade enhanced C3 deposition on HeLa ECM, but had no effect on HUVEC ECM. As ECM DAF is likely to be immobile, i.e. able to interact only with C3 convertases forming in the immediate vicinity, its ability to regulate complement activation may be particularly density dependent, and contingent on endothelial-dependent up-regulation.     

SYNCRIP (synaptotagmin-binding, cytoplasmic RNA-interacting protein) is a host factor involved in hepatitis C virus RNA replication

Hepatitis C virus (HCV) RNA replication requires viral nonstructural proteins as well as cellular factors. Recently, a cellular protein, synaptotagmin-binding, cytoplasmic RNA-interacting protein (SYNCRIP), also known as NSAP1, was found to bind HCV RNA and enhance HCV IRES-dependent translation. We investigate whether this protein is also involved in the HCV RNA replication. We found that SYNCRIP was associated with detergent-resistant membrane fractions and colocalized with newly-synthesized HCV RNA. Knock-down of SYNCRIP by siRNA significantly decreased the amount of HCV RNA in the cells containing a subgenomic replicon or a full-length viral RNA. Lastly, an in vitro replication assay after immunodepletion of SYNCRIP showed that SYNCRIP was directly involved in HCV RNA replication. These findings indicate that SYNCRIP has dual functions, participating in both RNA replication and translation in HCV life cycle.     

Assessment of the functional impact on the pre‐mRNA splicing process of 28 nucleotide variants associated with Pompe disease in GAA exon 2 and their recovery using antisense technology

Glycogen storage disease II (GSDII), also called Pompe disease, is an autosomal recessive inherited disease caused by a defect in glycogen metabolism due to the deficiency of the enzyme acid alpha‐glucosidase (GAA) responsible for its degradation. So far, more than 500 sequence variants of the GAA gene have been reported but their possible involvement on the pre‐messenger RNA splicing mechanism has not been extensively studied. In this work, we have investigated, by an in vitro functional assay, all putative splicing variants within GAA exon 2 and flanking introns. Our results show that many variants falling in the canonical splice site or the exon can induce GAA exon 2 skipping. In these cases, therefore, therapeutic strategies aimed at restoring protein folding of partially active mutated GAA proteins might not be sufficient. Regarding this issue, we have tested the effect of antisense oligonucleotides (AMOs) that were previously shown capable of rescuing splicing misregulation caused by the common c.‐32‐13T>G variant associated with the childhood/adult phenotype of GSDII. Interestingly, our results show that these AMOs are also quite effective in rescuing the splicing impairment of several exonic splicing variants, thus widening the potential use of these effectors for GSDII treatment.     

The Caenorhabditis elegans DDX-23, a homolog of yeast splicing factor PRP28, is required for the sperm-oocyte switch and differentiation of various cell types

DEAD/H-box proteins are involved in various aspects of RNA metabolism. Here we report the developmental function of a DEAD-box protein, DDX-23, in Caenorhabditis elegans, which has significant homology with the yeast splicing factor PRP28. We found by RNAi and mutant analyses that DDX-23 is essential for both embryonic and post-embryonic development, and required for differentiation of the majority of somatic tissues. When the germline function of ddx-23 was inhibited, hermaphrodite animals showed a reduced number of germ cells and failed to switch from spermatogenesis to oogenesis. These phenotypes were similar to those of the mutants of the three DEAH-box proteins (MOG-1, MOG-4, and MOG-5) whose yeast orthologs are involved in the pre-mRNA splicing pathway. We speculate that DDX-23 functions with the three MOG proteins in the same pathway to regulate tissue differentiation, robust germline proliferation, and the sperm/oocyte switch through modulations of ribonucleoprotein complexes.     

Regulation of human IL-18 gene expression: interaction of PU.1 with GC-box binding protein is involved in human IL-18 expression in myeloid cells

Interleukin-18 (IL-18) is a pro-inflammatory cytokine which participates in host defense against a variety of infections as well as in chronic inflammation including autoimmune diseases. However, little is known about human IL-18 regulation at the gene level. We have previously demonstrated that sodium butyrate, a bacterial fermentation product, induces IL-18 production via the proximal region of the promoter. In this study we investigated the molecular mechanisms for basal and sodium butyrate-induced expression of IL-18 in human myeloid cells. Two regulatory regions, a consensus binding site for PU.1 and a GC-rich region, are required for basal IL-18 promoter activity in human myeloid cells. PU.1 bound to the PU.1 consensus binding site in electrophoretic mobility shift assays, and overexpression of PU.1 led to activation of the IL-18 promoter through this site. Mutation analysis revealed that the GC-rich region, but not PU.1 site, participates in sodium butyrate-induced transactivation. Furthermore, DNA pull-down experiments and the critical spacing of the two binding sites suggest that formation of a protein complex involving both cis elements and the respective binding proteins might be crucial for human IL-18 expression.     

Hepatocyte nuclear factor 3 alpha belongs to a gene family in mammals that is homologous to the Drosophila homeotic gene fork head.

By analysis of cDNA clones that cross-hybridized with a portion of the cDNA encoding the recently described rat protein hepatocyte nuclear factor 3 alpha (HNF-3 alpha, previously called HNF-3A), we now describe two additional members, HNF-3 beta and HNF-3 gamma, of this gene family. A 110-amino-acid region in the DNA-binding domain of this family is not only very highly conserved in rodents (HNF-3 alpha, -3 beta, and -3 gamma are identical in 93 of 110 amino acids in this region) but also in Drosophila where the homeotic gene fork head has 88 of the 93 residues that are identical in the three rat genes. The HNF-3 family in rodents is expressed in cells that derive from the lining of the primitive gut; some of the embryonic Drosophila cells in which fork head is expressed also give rise to gut and salivary glands. Thus, it appears that this gene family, the DNA-binding portion of which is unlike that of any previously recognized DNA-binding proteins, may contribute to differentiation of cells in internal organs in both vertebrates and invertebrates.