Search PubMed⌕ Search

Biomedical subjects

W van Venrooij

Publications and source records attributed to W van Venrooij.

8 recordsLinked to original sources
PubMed1995

Molecular characterization of the spliceosomal proteins U1A and U2B" from higher plants.

In addition to their role in pre-mRNA splicing, the human spliceosomal proteins U1A and U2B" are important models of how RNP motif-containing proteins execute sequence-specific RNA binding. Genes encoding U1A and U2B" have been isolated from potato and thereby provide the only evolutionary comparison available for both proteins and represent the only full-length genes encoding plant spliceosomal proteins to have been cloned and characterized. In vitro RNA binding experiments revealed the ability of potato U2B" to interact with human U2A' to enhance sequence-specific binding and to distinguish cognate RNAs of either plant or animal origin. A comparison of the sequence of U1A and U2B" proteins indicated that multiple residues which could affect RNP motif conformation probably govern the specific distinction in RNA binding by these proteins. Since human U1A modulates polyadenylation in vertebrates, the possibility that plant U1A might be exploited in the characterization of this process in plants was examined. However, unlike vertebrate U1A, neither U1A from potato nor Arabidopsis bound their own mRNA and no evidence for binding to upstream efficiency elements in polyadenylation signals was obtained, suggesting that plant U1A is not involved in polyadenylation.

Amino Acid Sequence↗
PubMed1990

Assignment of seven genes to distinct intervals on the midportion of human chromosome 19q surrounding the myotonic dystrophy gene region.

Hybridization studies using a panel of somatic cell hybrids with subchromosomal segments of 19q have localized the genes encoding hormone-sensitive lipase (LIPE), carcinoembryonic antigen (CEA), and small nuclear ribonucleoprotein polypeptide A (SNRPA) to various regions of 19q13.1; the cellular receptor for poliovirus sensitivity (PVS) to 19q13.2; and the genes coding for prostate-specific antigen (APS), human pancreatic kallikrein (KLK1), and small nuclear ribonucleoprotein 70-kD polypeptide (SNRP70) to 19q13.3----qter. Our results exclude several of these genes from being seriously considered as a candidate for the myotonic dystrophy gene on 19q.

Animals↗
PubMed1986

Protein composition of nuclear matrix preparations from HeLa cells: an immunochemical approach.

Procedures for the isolation of HeLa S3 nuclear matrices were re-examined with special emphasis on the use of various nucleases and detergents as well as on the ionic strength of the final salt extraction. The protein composition of the resulting nuclear matrix preparations was analysed by one- and two-dimensional gel electrophoresis and found to be extremely reproducible. By means of co-electrophoresis several typical cytoskeletal proteins (actin, vimentin and cytokeratins) and heterogeneous nuclear RNA (hnRNA)-associated core proteins (hnRNP) were shown to be present in such nuclear matrix preparations. The nature of some other protein components was elucidated using two-dimensional immunoblotting and immunofluorescence. For this purpose mouse monoclonal antibodies to cytoskeletal components (vimentin, cytokeratins), small nuclear RNP (70 X 10(3) Mr protein of U1-RNP), hnRNP (C1/C2) and the pore-complex lamina (lamins A, B and C) were used next to human autoimmune sera obtained from patients with connective tissue diseases and directed against the residual nucleoli and the internal fibrillar mass. These antibodies enabled us to identify a number of proteins present specifically in the nuclear matrix and to show that part of the cytoskeletal proteins are still present in the isolated structures.

Cell Nucleus↗
PubMed1985

Autoantibodies to ribonucleoprotein particles containing U2 small nuclear RNA.

Autoantibodies exclusively precipitating U1 and U2 small nuclear ribonucleoprotein (snRNP) particles [anti-(U1,U2)RNP] were detected in sera from four patients with autoimmune disorders. When tested by immunoblotting, these sera recognized up to four different protein antigens in purified mixtures of U1-U6 RNP particles. With purified antibody fractions eluted from individual antigen bands on nitrocellulose blots, each anti-(U1,U2)RNP serum precipitated U2 RNP by virtue of the recognition of a U2 RNP-specific B" antigen (mol. wt. 28 500). Antibodies to the U2 RNP-specific A' protein (mol. wt. 31 000) were found in only one serum. The B" antigen differs slightly in mol. wt. from the U1-U6 RNA-associated B/B' antigens and can be separated from this doublet by two-dimensional gel electrophoresis, due to its more acidic pI. In immunoprecipitation assays, the purified anti-B" antibody specificity also reacts with U1 RNPs which is due to cross-reactivity of the antibody with the U1 RNA-specific A protein, as demonstrated by immunoblotting using proteins from isolated U1 RNPs as antigenic material. Thus the A antigen not only bears unique antigenic sites for anti-A antibodies contained in anti-(U1)RNP sera, it also shares epitopes with the U2 RNP-specific B" antigen.

Antibody Specificity↗
PubMed1982

Sequence dependent interaction of hnRNP proteins with late adenoviral transcripts.

Irradiation with ultraviolet light was used to induce covalent linkage between hnRNA and its associated proteins in intact HeLa cells, late after infection with adenovirus type 2. Covalently linked hnRNA-protein complexes, containing polyadenylated adenoviral RNA, were isolated and their protein moiety characterized. Host 42,000 Mr hnRNP proteins proved to be the major proteins crosslinked to viral hnRNA. To investigate their possible involvement in RNA processing, the localization of these cross-linked polypeptides on adenoviral late transcripts was determined. Sequences of RNA around the attachment sites of the protein were isolated. After in vitro labeling they were hybridized to Southern blots of adeno DNA fragments. The hybridization patterns revealed that the 42,000 Mr polypeptides can be linked to adenoviral transcripts over the entire length of the RNA, corresponding to 16.2-91.5 m.u. of the viral genome. Fine mapping within the Hind III B region (16.8-31.5 m.u.) established, however, that the localization of the cross-linked polypeptides was not random in all parts of the transcript. Sequences around the third leader and the 3' part of the i-leader were overrepresented, whereas the regions encoding VA I and VA II RNA and the late region 1 mRNA bodies were underrepresented in the cross-linked RNA. Using genomic DNA fragments and a cDNA clone containing the tripartite leader it appeared that leader and intervening sequences were represented about equally in cross-linked RNA fragments. Although these results do not support the notion that introns or exons are specifically interacting with one RNP protein, they demonstrate that the 42,000 hnRNP proteins are non randomly positioned on the RNA sequence.

Adenoviruses, Human↗
PubMed1982

On the localization and transport of specific adenoviral mRNA-sequences in the late infected HeLa cell.

In the nucleus of HeLa cells late after infection with adenovirus type 2 mRNA-sequences which are processed via RNA splicing are attached to the nuclear matrix (Mariman et al., 1982). Although the mRNA, which codes for polypeptide IX, is not formed via splicing, about 70% of the non-polyadenylated pre-mRNA and the polyadenylated pIX mRNA are bound to the matrix structure, indicating that polyadenylation is performed while the RNA is associated with the matrix. Binding to the nuclear matrix seems to be a common property of all mRNA-sequences in the nucleus. At the late stage of infection most of the newly synthesized mRNAs which appear in the cytoplasm are viral specific (Beltz & Flint, 1979). Kinetic analysis of the newly synthesized poly(A)-containing mRNA on sucrose gradients reveals that 7-12 S messengers appear more rapidly in the cytoplasm than messengers larger than 13 S. More specifically, the nuclear exit time of the pIX-mRNA, which is the major 9 S adenoviral messenger late after infection, was determined to be about 4 min, while messengers transcribed from the late region 3 need more than 16 min to arrive in the cytoplasm. In the cytoplasm about 70% of the mRNA is bound to the cytoskeletal framework, while 30% remains as free mRNP. Analysis of the mRNA in both fractions reveals that L3-, E1B- and pIX-specific polyadenylated mRNA preferably exist as cytoskeleton-bound mRNA. However, significant differences occur in the partition of specific messengers over free and cytoskeletal RNA fractions.

Adenoviruses, Human↗
PubMed1982

Detection of a cellular polypeptide associated with adenovirus-coded VA RNA using in vitro labeling of proteins cross-linked to RNA.

Ultraviolet light induced RNA-protein cross-linking for identification of polypeptides interacting with RNA in intact cells (Wagenmakers et al. 1980), is limited by the intensity of the label in the proteins or in residual nucleotides remaining attached to the proteins after RNase treatment of the RNA-protein complexes. Here we report a method, where th cross-linked RNA-protein complexes are treated with RNase T1 and the T1-oligonucleotides covalently linked to the proteins are labeled in the 5' terminus using gamma-32P-ATP and T4 polynucleotide kinase. The cross-linked proteins can then readily be identified owing to the incorporated 32P label. As examples, proteins associated with polyadenylated mRNA, hnRNA and adenoviral VA RNA were identified. A protein with a molecular weight of approximately 50,000 is found associated with adenovirus-coded VA RNA. This was confirmed by binding assays, in which labeled VAI RNA is incubated with proteins from uninfected and adenovirus infected HeLa cells immobilized on nitrocellulose sheets.

Adenoviruses, Human↗