A cDNA clone for a polyadenylated RNA-binding protein of Xenopus laevis oocytes hybridizes to four developmentally regulated mRNAs

1985 ◽  
Vol 5 (10) ◽  
pp. 2697-2704
Author(s):  
L J Lorenz ◽  
J D Richter
Keyword(s):  
Xenopus Laevis ◽  
Cdna Clone ◽  
Rna Binding ◽  
Binding Protein ◽  
Single Gene ◽  
In Vitro Translation ◽  
Xenopus Laevis Oocytes ◽  
Cdna Insert ◽  
Alternative Processing

Xenopus laevis oocytes contain a unique group of proteins which decrease during oogenesis, bind poly(A) RNA, and possibly play a role in the regulation of translation. A monoclonal antibody generated against one of these proteins was used to screen an expression vector cDNA library. A cDNA clone was isolated and confirmed to code for the binding protein by in vitro translation of hybrid-selected RNA followed by immunoprecipitation. This cDNA, when used in RNA gel blots, hybridized to four transcripts of 2.0, 1.7 (two transcripts of similar size), and 1.2 kilobases. All of the transcripts decreased in amount during oogenesis and were not evident in somatic cells. In addition, the fraction of the transcripts associated with polysomes decreased during oogenesis. Digestion of the cDNA insert with PstI generated two fragments of 220 and 480 base pairs which, when used as probes in an RNA gel blot, hybridized to unique as well as common transcripts. Genomic Southern blots suggested the presence of a single gene, indicating that these transcripts arose by alternative processing.

scholarly journals A cDNA clone for a polyadenylated RNA-binding protein of Xenopus laevis oocytes hybridizes to four developmentally regulated mRNAs.

1985 ◽  
Vol 5 (10) ◽  
pp. 2697-2704 ◽  
Author(s):  
L J Lorenz ◽  
J D Richter
Keyword(s):  
Xenopus Laevis ◽  
Cdna Clone ◽  
Rna Binding ◽  
Binding Protein ◽  
Single Gene ◽  
In Vitro Translation ◽  
Xenopus Laevis Oocytes ◽  
Cdna Insert ◽  
Alternative Processing

Xenopus laevis oocytes contain a unique group of proteins which decrease during oogenesis, bind poly(A) RNA, and possibly play a role in the regulation of translation. A monoclonal antibody generated against one of these proteins was used to screen an expression vector cDNA library. A cDNA clone was isolated and confirmed to code for the binding protein by in vitro translation of hybrid-selected RNA followed by immunoprecipitation. This cDNA, when used in RNA gel blots, hybridized to four transcripts of 2.0, 1.7 (two transcripts of similar size), and 1.2 kilobases. All of the transcripts decreased in amount during oogenesis and were not evident in somatic cells. In addition, the fraction of the transcripts associated with polysomes decreased during oogenesis. Digestion of the cDNA insert with PstI generated two fragments of 220 and 480 base pairs which, when used as probes in an RNA gel blot, hybridized to unique as well as common transcripts. Genomic Southern blots suggested the presence of a single gene, indicating that these transcripts arose by alternative processing.

Characterization and purification of lupus antigen La, and RNA-binding protein

1985 ◽  
Vol 5 (3) ◽  
pp. 586-590
Author(s):  
A M Francoeur ◽  
E K Chan ◽  
J I Garrels ◽  
M B Mathews
Keyword(s):  
Hela Cell ◽  
Gel Electrophoresis ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
In Vitro Translation ◽  
Cell Protein ◽  
Two Dimensional Gel Electrophoresis ◽  
La Antigen

HeLa cell La antigen, an RNA-binding protein, was characterized by using two-dimensional gel electrophoresis. Eight isoelectric forms (pI 6 to 7) were observed, many containing phosphate. An in vitro translation product similar in size and antigenicity was identified. The HeLa cell protein purified by using an assay based on ribonucleoprotein reconstitution with adenovirus VA RNAI also comprised several isoelectric forms.

scholarly journals Heterogeneous Nuclear Ribonucleoprotein (hnRNP) E1 Binds to hnRNP A2 and Inhibits Translation of A2 Response Element mRNAs

2006 ◽  
Vol 17 (8) ◽  
pp. 3521-3533 ◽  
Author(s):  
Linda D. Kosturko ◽  
Michael J. Maggipinto ◽  
George Korza ◽  
Joo Won Lee ◽  
John H. Carson ◽  
...  
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
In Vitro Translation ◽  
Dependent Manner ◽  
Response Element ◽  
Heterogeneous Nuclear Ribonucleoprotein ◽  
Nuclear Ribonucleoprotein ◽  
Hnrnp E1

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 is a trans-acting RNA-binding protein that mediates trafficking of RNAs containing the cis-acting A2 response element (A2RE). Previous work has shown that A2RE RNAs are transported to myelin in oligodendrocytes and to dendrites in neurons. hnRNP E1 is an RNA-binding protein that regulates translation of specific mRNAs. Here, we show by yeast two-hybrid analysis, in vivo and in vitro coimmunoprecipitation, in vitro cross-linking, and fluorescence correlation spectroscopy that hnRNP E1 binds to hnRNP A2 and is recruited to A2RE RNA in an hnRNP A2-dependent manner. hnRNP E1 is colocalized with hnRNP A2 and A2RE mRNA in granules in dendrites of oligodendrocytes. Overexpression of hnRNP E1 or microinjection of exogenous hnRNP E1 in neural cells inhibits translation of A2RE mRNA, but not of non-A2RE RNA. Excess hnRNP E1 added to an in vitro translation system reduces translation efficiency of A2RE mRNA, but not of nonA2RE RNA, in an hnRNP A2-dependent manner. These results are consistent with a model where hnRNP E1 recruited to A2RE RNA granules by binding to hnRNP A2 inhibits translation of A2RE RNA during granule transport.

scholarly journals The 36-kilodalton embryonic-type cytoplasmic polyadenylation element-binding protein in Xenopus laevis is ElrA, a member of the ELAV family of RNA-binding proteins.

1997 ◽  
Vol 17 (11) ◽  
pp. 6402-6409 ◽  
Author(s):  
L Wu ◽  
P J Good ◽  
J D Richter
Keyword(s):  
Xenopus Laevis ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Dominant Negative ◽  
Cytoplasmic Polyadenylation ◽  
Element Binding Protein

The translational activation of several maternal mRNAs in Xenopus laevis is dependent on cytoplasmic poly(A) elongation. Messages harboring the UUUUUAU-type cytoplasmic polyadenylation element (CPE) in their 3' untranslated regions (UTRs) undergo polyadenylation and translation during oocyte maturation. This CPE is bound by the protein CPEB, which is essential for polyadenylation. mRNAs that have the poly(U)12-27 embryonic-type CPE (eCPE) in their 3' UTRs undergo polyadenylation and translation during the early cleavage and blastula stages. A 36-kDa eCPE-binding protein in oocytes and embryos has been identified by UV cross-linking. We now report that this 36-kDa protein is ElrA, a member of the ELAV family of RNA-binding proteins. The proteins are identical in size, antibody directed against ElrA immunoprecipitates the 36-kDa protein, and the two proteins have the same RNA binding specificity in vitro. C12 and activin receptor mRNAs, both of which contain eCPEs, are detected in immunoprecipitated ElrA-mRNP complexes from eggs and embryos. In addition, this in vivo interaction requires the eCPE. Although a number of experiments failed to define a role for ElrA in cytoplasmic polyadenylation, the expression of a dominant negative ElrA protein in embryos results in an exogastrulation phenotype. The possible functions of ElrA in gastrulation are discussed.

scholarly journals Characterization and purification of lupus antigen La, and RNA-binding protein.

1985 ◽  
Vol 5 (3) ◽  
pp. 586-590 ◽  
Author(s):  
A M Francoeur ◽  
E K Chan ◽  
J I Garrels ◽  
M B Mathews
Keyword(s):  
Hela Cell ◽  
Gel Electrophoresis ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
In Vitro Translation ◽  
Cell Protein ◽  
Two Dimensional Gel Electrophoresis ◽  
La Antigen

HeLa cell La antigen, an RNA-binding protein, was characterized by using two-dimensional gel electrophoresis. Eight isoelectric forms (pI 6 to 7) were observed, many containing phosphate. An in vitro translation product similar in size and antigenicity was identified. The HeLa cell protein purified by using an assay based on ribonucleoprotein reconstitution with adenovirus VA RNAI also comprised several isoelectric forms.

In vitro translation of B-cell stimulatory factor-1 in Xenopus laevis oocytes

1986 ◽  
Vol 98 (2) ◽  
pp. 538-543 ◽  
Author(s):  
Melissa A. Brown ◽  
Cynthia Watson ◽  
Junichi Ohara ◽  
William E. Paul
Keyword(s):  
Xenopus Laevis ◽  
B Cell ◽  
In Vitro Translation ◽  
Xenopus Laevis Oocytes ◽  
Stimulatory Factor

scholarly journals Different forms of soluble cytoplasmic mRNA binding proteins and particles in Xenopus laevis oocytes and embryos.

1991 ◽  
Vol 112 (1) ◽  
pp. 1-11 ◽  
Author(s):  
M T Murray ◽  
G Krohne ◽  
W W Franke
Keyword(s):  
Xenopus Laevis ◽  
Translational Regulation ◽  
Rna Binding ◽  
Xenopus Laevis Oocytes ◽  
Stoichiometric Ratio ◽  
Cytoplasmic Proteins ◽  
Rnase Digestion ◽  
Mrna Binding ◽  
Insight Into

To gain insight into the mechanisms involved in the formation of maternally stored mRNPs during Xenopus laevis development, we searched for soluble cytoplasmic proteins of the oocyte that are able to selectively bind mRNAs, using as substrate radiolabeled mRNA. In vitro mRNP assembly in solution was followed by UV-cross-linking and RNase digestion, resulting in covalent tagging of polypeptides by nucleotide transfer. Five polypeptides of approximately 54, 56 60, 70, and 100 kD (p54, p56, p60, p70, and p100) have been found to selectively bind mRNA and assemble into mRNPs. These polypeptides, which correspond to previously described native mRNP components, occur in three different particle classes of approximately 4.5S, approximately 6S, and approximately 15S, as also determined by their reactions with antibodies against p54 and p56. Whereas the approximately 4.5S class contains p42, p60, and p70, probably each in the form of individual molecules or small complexes, the approximately 6S particles appears to consist only of p54 and p56, which occur in a near-stoichiometric ratio suggestive of a heterodimer complex. The approximately 15S particles contain, in addition to p54 and p56, p60 and p100 and this is the single occurring form of RNA-binding p100. We have also observed changes in the in vitro mRNA binding properties of these polypeptides during oogenesis and early embryonic development, in relation to their phosphorylation state and to the activity of an approximately 15S particle-associated protein kinase, suggesting that these proteins are involved in the developmental translational regulation of maternal mRNAs.

Structural and Genetic Aspects of Proline-rich Proteins

1987 ◽  
Vol 66 (2) ◽  
pp. 457-461 ◽  
Author(s):  
A. Bennick
Keyword(s):  
General Structure ◽  
Single Gene ◽  
Conclusive Evidence ◽  
In Vitro Translation ◽  
Binding Studies ◽  
Nmr Studies ◽  
Post Translational Modifications ◽  
Single Precursor ◽  
Made In

Considerable advances have been made in the genetics of salivary proline-rich proteins (PRP). The genes for acidic, basic, and glycosylated PRP have been cloned. They code for precursor proteins that all have an acidic N-terminal followed by proline-rich repeat sequences. Structural studies on secreted proteins have demonstrated that not only acidic but also some basic PRPs have this general structure. It is possible that mRNA for different PRP may have originated from a single gene by differential mRNA splicing, but post-translational cleavages of the primary translation product apparently also occur. In vitro translation of salivary gland mRNA results in a single precursor protein for acidic PRP. Such in vitro translated protein can be cleaved by salivary kallikrein, giving rise to two commonly secreted acidic PRPs, and kallikrein or kallikrein-like enzymes may be responsible for other post-translational cleavages of PRPs. Acidic as well as some basic PRPs are phosphorylated. A protein kinase has been demonstrated in salivary glands which phosphorylates the PRPs and other secreted salivary proteins in a cAMP and Ca2+-calmodulinindependent manner. Knowledge of the conformation of PRPs is limited. There is no conclusive evidence of polyproline-like structure in the proline-rich part of PRPs. Ca2+ binding studies on acidic PRPs indicate that there is interaction between the Ca2+ binding N-terminal end and the proline-rich C-terminal part. This interaction is relieved by modification of arginine side-chains. 1H, 32P, and 43Ca NMR studies have further elucidated the conformation of acidic PRPs in solution. Present evidence shows that salivary PRPs constitute a unique superfamily of proteins which pose a number of interesting questions concerning gene structure, pre- and post-translational modifications, and protein conformation.

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