共查询到20条相似文献,搜索用时 31 毫秒
1.
Classification and purification of proteins of heterogeneous nuclear ribonucleoprotein particles by RNA-binding specificities. 总被引:87,自引:36,他引:51
下载免费PDF全文
![点击此处可从《Molecular and cellular biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Several proteins of heterogeneous nuclear ribonucleoprotein (hnRNP) particles display very high binding affinities for different ribonucleotide homopolymers. The specificity of some of these proteins at high salt concentrations and in the presence of heparin allows for their rapid one-step purification from HeLa nucleoplasm. We show that the hnRNP C proteins are poly(U)-binding proteins and compare their specificity to that of the previously described cytoplasmic poly(A)-binding protein. These findings provide a useful tool for the classification and purification of hnRNP proteins from various tissues and organisms and indicate that different hnRNP proteins have different RNA-binding specificities. 相似文献
2.
Incomplete RNA splicing is a key feature of the retroviral life cycle. This is in contrast to the processing of most cellular pre-mRNAs, which are usually spliced to completion. In Rous sarcoma virus, splicing control is achieved in part through a cis-acting RNA element termed the negative regulator of splicing (NRS). The NRS is functionally divided into two parts termed NRS5' and NRS3', which bind a number of splicing factors. The U1 and U11 small nuclear ribonucleoproteins interact with sequences in NRS3', whereas NRS5' binds several proteins including members of the SR [corrected] family of proteins. Among the proteins that specifically bind NRS5' is a previously unidentified 55-kDa protein (p55). In this report we describe the isolation and identification of p55. The p55 binding site was localized by UV cross-linking to a 31-nucleotide segment, and a protein that binds specifically to it was isolated by RNA affinity selection and identified by mass spectrometry as hnRNP H. Antibodies against hnRNP H immunoprecipitated cross-linked p55 and induced a supershift of a p55-containing complex formed in HeLa nuclear extract. Furthermore, UV cross-linking and electrophoretic mobility shift assays indicated that recombinant hnRNP H specifically interacts with the p55 binding site, confirming that hnRNP H is p55. The possible roles of hnRNP H in NRS function are discussed. 相似文献
3.
Structure of nuclear ribonucleoprotein: identification of proteins in contact with poly(A)+ heterogeneous nuclear RNA in living HeLa cells 总被引:21,自引:3,他引:18
下载免费PDF全文
![点击此处可从《The Journal of cell biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
《The Journal of cell biology》1981,90(2):380-384
The processing of heterogeneous nuclear RNA into messenger RNA takes place in special nuclear ribonucleoprotein particles known as hnRNP. We report here the identification of proteins tightly complexed with poly(A)+ hnRNA in intact HeLa cells, as revealed by a novel in situ RNA- protein cross-linking technique. The set of cross-linked proteins includes the A, B, and C "core" hnRNP proteins, as well as the greater than 42,000 mol wt species previously identified in noncross-linked hnRNP. These proteins are shown to be cross-linked by virtue of remaining bound to the poly(A)+ hnRNA in the presence of 0.5% sodium dodecyl sulfate, 0.5 M NaCl, and 60% formamide, during subsequent oligo(dT)-cellulose chromatography, and in isopycnic banding in Cs2SO4 density gradients. These results establish that poly(A)+ hnRNA is in direct contact with a moderately complex set of nuclear proteins in vivo. This not only eliminates earlier models of hnRNP structure that were based upon the concept of a single protein component but also suggests that these proteins actively participate in modulating hnRNA structure and processing in the cell. 相似文献
4.
Hans-Erich Wilk Klaus P. Schaefer Paul F. Agris Andra M. Boak Shirley A. Kovacs 《Molecular and cellular biochemistry》1991,106(1):55-66
Precursor mRNA is complexed with proteins in the cell nucleus to form heterogeneous nuclear ribonucleoprotein (hnRNP), and these hnRNPs are found associated in vivo with small nuclear RNPs (snRNPs) for the processing of pre-mRNA. In order to better characterize the ATP-independent initial association of U1 snRNP with hnRNP, an important early event in assembly of the spliceosome complex, we have determined some of the components essential to an in vitro reassociation of U1 snRNP with hnRNP. U1 snRNP reassociated in vitro with 40S hnRNP particles from HeLa cells and, similar to the in vivo hnRNP/U1 snRNP association, the in vitro interaction was sensitive to high salt concentrations. U1 snRNP also associated with in vitro reconstituted hnRNP in which bacteriophage MS2 RNA, which lacks introns, was used as the RNA component. Purified snRNA alone would not associate with the MS2 RNA-reconstituted hnRNP, however, intact U1 snRNP did interact with protein-free MS2 RNA. This indicates that the U1 snRNP proteins are required for the hnRNP/U1 snRNP association, but hnRNP proteins are not. Thus, the initial, ATP-independent association of U1 snRNP with hnRNP seems to be mediated by U1 snRNP protein(s) associating with hnRNA without requiring a splice-site sequence. This complex may then be further stabilized by intron-specific interactions and hnRNP proteins, as well as by other snRNPs. 相似文献
5.
hnRNP A2/B1 binds specifically to single stranded vertebrate telomeric repeat TTAGGGn. 总被引:18,自引:3,他引:15
下载免费PDF全文
![点击此处可从《Nucleic acids research》网站下载免费的PDF全文](/ch/ext_images/free.gif)
We have previously isolated a protein from mouse liver nuclei that specifically binds to single stranded (TTAGGG)n repeats. TTAGGG is the telomeric repeats of mammals and we therefore named the new protein single stranded telomere binding protein (sTBP). Further studies now identify sTBP as heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 on the basis of amino acid sequence determination and antibody reactivity. A2 and B1 form a major part of the protein component of hnRNP particles and are abundant nuclear proteins. Unexpectedly, A2/B1 has a high specificity for binding to the RNA equivalent of TTAGGG, UUAGGG, but under the same conditions does not appear to have a strong affinity for a number of other RNA species. 相似文献
6.
RNA binding specificity of hnRNP proteins: a subset bind to the 3'' end of introns. 总被引:69,自引:8,他引:61
下载免费PDF全文
![点击此处可从《The EMBO journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The binding of hnRNP proteins to pre-mRNAs in nuclear extracts, and as isolated proteins, was studied by using monoclonal antibody immunopurification of hnRNP proteins bound to RNase T1-generated fragments. Several major hnRNP proteins, A1, C and D, bind specifically to the 3' end of introns within a region containing the conserved polypyrimidine stretch between the branch site and the 3' splice site. Mutations which alter the conserved 3' splice site dinucleotide AG strongly impair or abolish the binding of the A1 protein as well as of an anti-Sm reactive component(s) to this region. The A1, C and D proteins do not bind efficiently to fragments of either bacterial RNA or the intronless spliced product (mRNA). The binding of these proteins at the 3' end of the intron does not require addition to the extract of exogenous ATP, but remains after ATP addition. These findings demonstrate that several hnRNP proteins have RNA binding specificities on pre-mRNA, and suggest a model for hnRNP particle structure and assembly. 相似文献
7.
Nucleic acid binding characteristics of group A/B hnRNP proteins 总被引:2,自引:0,他引:2
P Minoo T E Martin R M Riehl 《Biochemical and biophysical research communications》1991,176(2):747-755
hnRNP proteins are primarily defined by their specific sedimentational, reconstitutional, and extraction properties and are presumed to be RNA binding. However, it is not clear whether all these proteins have RNA binding capabilities. Recently, using two monoclonal antibodies, fA12 and AC88, we reported that the abundance of a subclass of the highly basic A/B hnRNP proteins was specifically down regulated during terminal differentiation of human and murine cells in vitro. In this report we have examined the nucleic acid binding characteristics of this subclass and other members of the A/B hnRNP proteins in vitro. All members of class A/B hnRNP proteins appear to have similar but not identical nucleic acid binding characteristics. However, the subclass of proteins recognized by AC88 and fA12 exhibit stronger binding affinities and are shown to be highly selective in their binding to RNA vs DNA in vitro. These proteins also preferentially bind poly(U) RNA, suggesting that in vivo they may bind effectively to uridine rich motifs critical in pre-mRNA processing. 相似文献
8.
9.
10.
hnRNP G: sequence and characterization of a glycosylated RNA-binding protein. 总被引:16,自引:0,他引:16
下载免费PDF全文
![点击此处可从《Nucleic acids research》网站下载免费的PDF全文](/ch/ext_images/free.gif)
M Soulard V Della Valle M C Siomi S Piol-Roma P Codogno C Bauvy M Bellini J C Lacroix G Monod G Dreyfuss 《Nucleic acids research》1993,21(18):4210-4217
The autoantigen p43 is a nuclear protein initially identified with autoantibodies from dogs with a lupus-like syndrome. Here we show that p43 is an RNA-binding protein, and identify it as hnRNP G, a previously described component of heterogeneous nuclear ribonucleoprotein complexes. We demonstrate that p43/hnRNP G is glycosylated, and identify the modification as O-linked N-acetylglucosamine. A full-length cDNA clone for hnRNP G has been isolated and sequenced, and the predicted amino acid sequence for hnRNP G shows that it contains one RNP-consensus RNA binding domain (RBD) at the amino terminus and a carboxyl domain rich in serines, arginines and glycines. The RBD of human hnRNP G shows striking similarities with the RBDs of several plant RNA-binding proteins. 相似文献
11.
12.
Polypyrimidine tract-binding protein and heterogeneous nuclear ribonucleoprotein A1 bind to human T-cell leukemia virus type 2 RNA regulatory elements. 总被引:7,自引:2,他引:5
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
A C Black J Luo C Watanabe S Chun A Bakker J K Fraser J P Morgan J D Rosenblatt 《Journal of virology》1995,69(11):6852-6858
Efficient expression of human T-cell leukemia virus (HTLV) and human immunodeficiency virus structural proteins requires Rx and Rev proteins, respectively. Decreased expression of Gag and Env appears to be due, in part, to intragenic RNA sequences, termed cis-acting repressive sequences (CRS), and may be mediated by binding of specific cellular factors. We demonstrated previously that two cellular proteins, p60CRS and p40CRS, interact with HTLV type 2.5' long terminal repeat CRS RNA and that the interaction of both proteins with CRS RNA correlates with function (A. C. Black, C. T. Ruland, J. Luo, A. Bakker, J. K. Fraser, and J. D. Rosenblatt, Virology 200:29-41, 1994). By radioimmunoprecipitation of HeLa nuclear proteins UV cross-linked to CRS RNAs with murine monoclonal antibodies, we now show that p40CRS is heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and p60CRS is polypyrimidine tract-binding protein or hnRNP I. These immunoprecipitation results were confirmed by an immunobinding assay with hnRNP I and hnRNP AI antibodies and by cross-competition electrophoretic mobility shift experiments. In addition, we mapped a putative hnRNP A1 binding site in U5 RNA and demonstrated that p40CRS (hnRNP A1) binding to that site correlates with CRS function. Since both hnRNP I and hnRNP A1 have been shown to influence splicing and potentially other steps in RNA processing, the binding of both hnRNP I and hnRNP A1 to HTLV RNA regulatory elements may alter retrovirus RNA processing and may be involved in regulation by Rex. 相似文献
13.
Cellular senescence is a terminal growth phase characteristic of normal human diploid fibroblasts. Altered gene expression during cellular senescence is numerous compared to that of younger proliferative cells in culture. We have previously reported that the levels and activities of hnRNP A1 and A2 RNA binding proteins are decreased in senescent human fibroblasts. Both proteins are multifunctional and may influence the expression of mRNA isoforms during development. In this study, we tested whether overexpression of either protein could modulate the mRNA isoforms of the INK4a locus, specifically p14(ARF) and p16(INK4a). Both INK4a mRNA isoforms have been shown to be growth suppressors and deletions of this locus allow cells to escape cellular senescence. We have found that increasing the ratio of either hnRNP A1 or A2 over that of splicing factor SF2/ASF results in the preferential generation of the p14(ARF) isoform. Overexpression of A1 or A2 RNA binding proteins also appear to increase the steady state mRNA levels of both isoforms, suggesting that in addition to alternative splicing, A1 and A2 may effect p14(ARF) and p16(INK4a) mRNA stability. A constitutive decrease in the ratio of hnRNP A1 or A2 to SF2/ASF in senescent fibroblasts is typically accompanied by an increase in the level of p16(INK4a) isoform. Our studies suggest that hnRNP A1 and A2 may exert an important role during replicative senescence by altering expression of cell cycle regulatory proteins through mRNA metabolism. 相似文献
14.
Ngoc T. Vu Margaret A. Park Jacqueline C. Shultz Rachel W. Goehe L. Alexis Hoeferlin Michael D. Shultz Sarah A. Smith Kristen W. Lynch Charles E. Chalfant 《The Journal of biological chemistry》2013,288(12):8575-8584
Caspase-9 has two splice variants, pro-apoptotic caspase-9a and anti-apoptotic caspase-9b, which are regulated by RNA trans-factors associated with exon 3 of caspase-9 pre-mRNA (C9/E3). In this study, we identified hnRNP U as an RNA trans-factor associated with C9/E3. Down-regulation of hnRNP U led to a decrease in the caspase-9a/9b mRNA ratio, demonstrating a novel enhancing function. Importantly, hnRNP U bound specifically to C9/E3 at an RNA cis-element previously reported as the binding site for the splicing repressor, hnRNP L. Phosphorylated hnRNP L interfered with hnRNP U binding to C9/E3, and our results demonstrate the importance of the phosphoinositide 3-kinase/AKT pathway in modulating the association of hnRNP U to C9/E3. Taken together, these findings show that hnRNP U competes with hnRNP L for binding to C9/E3 to enhance the inclusion of the four-exon cassette, and this splice-enhancing function is blocked by the AKT pathway via phosphorylation of hnRNP L. 相似文献
15.
16.
17.
18.