scholarly journals Identification of Ebp1 as a component of cytoplasmic bcl-2 mRNP (messenger ribonucleoprotein particle) complexes

2006 ◽  
Vol 396 (1) ◽  
pp. 99-107 ◽  
Author(s):  
Sudeep K. Bose ◽  
Tapas K. Sengupta ◽  
Sumita Bandyopadhyay ◽  
Eleanor K. Spicer
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Ribonucleoprotein Particle ◽  
Mobility Shift ◽  
Cell Extracts ◽  
Mrna Binding Proteins ◽  
Messenger Ribonucleoprotein ◽  
Co Precipitation ◽  
Mrna Binding

The 3′-UTR (untranslated region) of bcl-2 mRNA contains an ARE (AU-rich element) that potentially regulates the stability of bcl-2 mRNA in a cell specific fashion. Previous studies have demonstrated that multiple proteins interact with bcl-2 mRNA in HL-60 (human leukaemia-60) cells, potentially contributing to the overexpression of Bcl-2 protein. Treatment of HL-60 cells with taxol or okadaic acid has been shown to induce destabilization of bcl-2 mRNA, which was associated with decreased binding of trans-acting factors to bcl-2 mRNA. Nucleolin has been identified as one of the bcl-2 mRNA-binding proteins [Sengupta, Bandyopadhyay, Fernandes and Spicer (2004) J. Biol. Chem. 279, 10855–10863]. In an effort to identify additional bcl-2 mRNA-binding proteins, two polypeptides of approx. 45 kDa and 60 kDa were isolated from HL-60 cells by AREbcl-2 (transcripts that contain bcl-2 AREs) RNA affinity chromatography. These proteins were identified as the human proliferation associated protein, Ebp1, and human DRBP76 (double stranded RNA-binding protein 76) respectively, by MALDI (matrix-assisted laser-desorption ionization)-MS. RNA electrophoretic mobility shift assays indicated that recombinant Ebp1 binds to AREbcl-2 RNA but not to the group 1 ARE present in GM-CSF (granulocyte macrophage-colony stimulating factor) mRNA in vitro. Antibody supershift assays demonstrated that Ebp1 is present in protein–AREbcl-2 RNA complexes formed with cytosolic HL-60 extracts. The interaction of Ebp1 with bcl-2 mRNA in HL-60 cells was also demonstrated by RNA co-immunoprecipitation assays. This interaction was not detected in extracts of taxol-treated HL-60 cells. Immunoprecipitation assays further revealed that Ebp1 co-precipitates with nucleolin from HL-60 cytoplasmic extracts. The observation that co-precipitation was decreased when extracts were treated with RNase suggests that Ebp1 and nucleolin are present in the same bcl-2 mRNP (messenger ribonucleoprotein particle) complexes. RNA-decay assays further demonstrated that Ebp1 decreased the rate of decay of β-globin–AREbcl-2 transcripts in HL-60 cell extracts. Collectively, these results indicate a novel function for Ebp1 in contributing to the regulation of bcl-2 expression in HL-60 cells.

scholarly journals The endoplasmic reticulum-associated mRNA-binding proteins ERBP1 and ERBP2 interact in bloodstream-form Trypanosoma brucei

2019 ◽  
Author(s):  
Kathrin Bajak ◽  
Kevin Leiss ◽  
Christine Clayton ◽  
Esteban Erben
Keyword(s):  
Endoplasmic Reticulum ◽  
Ribosomal Protein ◽  
Trypanosoma Brucei ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Bloodstream Form ◽  
Control Of Gene Expression ◽  
Mrna Binding Proteins ◽  
Mrna Binding

AbstractKinetoplastids rely heavily on post-transcriptional mechanisms for control of gene expression, and on RNA-binding proteins that regulate mRNA splicing, translation and decay. Trypanosoma brucei ERBP1 (Tb927.10.14150) and ERBP2 (Tb927.9.9550) were previously identified as mRNA binding proteins that lack canonical RNA-binding domains. We here show that ERBP1 is associated with the endoplasmic reticulum, like ERBP2, and that the two proteins interact in vivo. Loss of ERBP1 from bloodstream-form T. brucei initially resulted in a growth defect but proliferation was restored after more prolonged cultivation. Results from a pull-down of tagged ERBP1 suggest that it preferentially binds to ribosomal protein mRNAs. The ERBP1 sequence resembles that of Saccharomyces cerevisiae Bfr1, which also localises to the endoplasmic reticulum and binds to ribosomal protein mRNAs. However, unlike Bfr1, ERBP1 does not bind to mRNAs encoding secreted proteins, and it is also not recruited to stress granules after starvation.

scholarly journals An Interaction between Two RNA Binding Proteins, Nab2 and Pub1, Links mRNA Processing/Export and mRNA Stability

2007 ◽  
Vol 27 (18) ◽  
pp. 6569-6579 ◽  
Author(s):  
Luciano H. Apponi ◽  
Seth M. Kelly ◽  
Michelle T. Harreman ◽  
Alexander N. Lehner ◽  
Anita H. Corbett ◽  
...  
Keyword(s):  
Mrna Stability ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Tail Length ◽  
Functional Link ◽  
Mrna Binding Protein ◽  
Mrna Binding

ABSTRACT mRNA stability is modulated by elements in the mRNA transcript and their cognate RNA binding proteins. Poly(U) binding protein 1 (Pub1) is a cytoplasmic Saccharomyces cerevisiae mRNA binding protein that stabilizes transcripts containing AU-rich elements (AREs) or stabilizer elements (STEs). In a yeast two-hybrid screen, we identified nuclear poly(A) binding protein 2 (Nab2) as being a Pub1-interacting protein. Nab2 is an essential nucleocytoplasmic shuttling mRNA binding protein that regulates poly(A) tail length and mRNA export. The interaction between Pub1 and Nab2 was confirmed by copurification and in vitro binding assays. The interaction is mediated by the Nab2 zinc finger domain. Analysis of the functional link between these proteins reveals that Nab2, like Pub1, can modulate the stability of specific mRNA transcripts. The half-life of the RPS16B transcript, an ARE-like sequence-containing Pub1 target, is decreased in both nab2-1 and nab2-67 mutants. In contrast, GCN4, an STE-containing Pub1 target, is not affected. Similar results were obtained for other ARE- and STE-containing Pub1 target transcripts. Further analysis reveals that the ARE-like sequence is necessary for Nab2-mediated transcript stabilization. These results suggest that Nab2 functions together with Pub1 to modulate mRNA stability and strengthen a model where nuclear events are coupled to the control of mRNA turnover in the cytoplasm.

scholarly journals Expression of IGF-II mRNA-binding proteins (IMPs) in gonads and testicular cancer

Reproduction ◽  
2005 ◽  
Vol 130 (2) ◽  
pp. 203-212 ◽  
Author(s):  
Niels A Hammer ◽  
Thomas v O Hansen ◽  
Anne Grete Byskov ◽  
Eva Rajpert-De Meyts ◽  
Marie Louise Grøndahl ◽  
...  
Keyword(s):  
Expression Pattern ◽  
Translational Control ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Developmental Expression ◽  
Human Testis ◽  
Mrna Binding Proteins ◽  
Mrna Binding

Insulin-like growth factor-II mRNA-binding proteins 1, 2 and 3 (IMP1, IMP2 and IMP3) belong to a family of RNA-binding proteins implicated in mRNA localization, turnover and translational control. We examined their expression pattern during development of murine and human testis and ovaries. In the mouse, IMPs were expressed in male and female gonadal cells at embryonic day 12.5 (E12.5). From E16.5, IMP1 and IMP3 became restricted to the developing germ cells, whereas IMP2 expression persisted in the interstitial cells. In mature mouse and human ovaries, IMP1, IMP2 and IMP3 were detected in resting and growing oocytes and in the granulosa cells. In testis, IMP1 and IMP3 were found mainly in the spermatogonia, whereas IMP2 was expressed in the immature Leydig cells. Moreover, all three IMPs were detected in human semen. The developmental expression pattern of IMP1 and IMP3 in the human testis prompted us to examine their possible involvement in testicular neoplasia. IMPs were detected primarily in germ-cell neoplasms, including preinvasive testicular carcinoma in situ, classical and spermatocytic seminoma, and nonseminomas, with particularly high expression in undifferentiated embryonal carcinoma. The relative expression of IMP1, IMP2 and IMP3 varied among tumor types and only IMP1 was detected in all carcinoma in situ cells. Thus IMPs, and in particular IMP1, may be useful auxiliary markers of testicular neoplasia.

scholarly journals The endoplasmic reticulum-associated mRNA-binding proteins ERBP1 and ERBP2 interact in bloodstream-form Trypanosoma brucei

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e8388 ◽  
Author(s):  
Kathrin Bajak ◽  
Kevin Leiss ◽  
Christine E. Clayton ◽  
Esteban Erben
Keyword(s):  
Endoplasmic Reticulum ◽  
Ribosomal Protein ◽  
Trypanosoma Brucei ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Bloodstream Form ◽  
Control Of Gene Expression ◽  
Mrna Binding Proteins ◽  
Mrna Binding

Kinetoplastids rely heavily on post-transcriptional mechanisms for control of gene expression, and on RNA-binding proteins that regulate mRNA splicing, translation and decay. Trypanosoma brucei ERBP1 (Tb927.10.14150) and ERBP2 (Tb927.9.9550) were previously identified as mRNA binding proteins that lack canonical RNA-binding domains. We show here that ERBP1 is associated with the endoplasmic reticulum, like ERBP2, and that the two proteins interact in vivo. Loss of ERBP1 from bloodstream-form T. brucei initially resulted in a growth defect but proliferation was restored after more prolonged cultivation. Pull-down analysis of tagged ERBP1 suggests that it preferentially binds to ribosomal protein mRNAs. The ERBP1 sequence resembles that of Saccharomyces cerevisiae Bfr1, which also localises to the endoplasmic reticulum and binds to ribosomal protein mRNAs. However, unlike Bfr1, ERBP1 does not bind to mRNAs encoding secreted proteins, and it is also not recruited to stress granules after starvation.

scholarly journals Modulation of Viral Programmed Ribosomal Frameshifting and Stop Codon Readthrough by the Host Restriction Factor Shiftless

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1230
Author(s):  
Sawsan Napthine ◽  
Chris H. Hill ◽  
Holly C. M. Nugent ◽  
Ian Brierley
Keyword(s):  
Rna Binding ◽  
Mutational Analysis ◽  
Stop Codon ◽  
Leukemia Virus ◽  
Size Exclusion ◽  
Human Immunodeficiency Virus Replication ◽  
Mobility Shift ◽  
Ribosomal Frameshifting ◽  
Stop Codon Readthrough

The product of the interferon-stimulated gene C19orf66, Shiftless (SHFL), restricts human immunodeficiency virus replication through downregulation of the efficiency of the viral gag/pol frameshifting signal. In this study, we demonstrate that bacterially expressed, purified SHFL can decrease the efficiency of programmed ribosomal frameshifting in vitro at a variety of sites, including the RNA pseudoknot-dependent signals of the coronaviruses IBV, SARS-CoV and SARS-CoV-2, and the protein-dependent stimulators of the cardioviruses EMCV and TMEV. SHFL also reduced the efficiency of stop-codon readthrough at the murine leukemia virus gag/pol signal. Using size-exclusion chromatography, we confirm the binding of the purified protein to mammalian ribosomes in vitro. Finally, through electrophoretic mobility shift assays and mutational analysis, we show that expressed SHFL has strong RNA binding activity that is necessary for full activity in the inhibition of frameshifting, but shows no clear specificity for stimulatory RNA structures.

scholarly journals Identification of mRNAs Associated with αCP2-Containing RNP Complexes

2003 ◽  
Vol 23 (19) ◽  
pp. 7055-7067 ◽  
Author(s):  
Shelly A. Waggoner ◽  
Stephen A. Liebhaber
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Cell Function ◽  
Rna Binding Proteins ◽  
Translation Control ◽  
Viral Mrnas ◽  
Posttranscriptional Gene Regulation ◽  
Direct Binding

ABSTRACT Posttranscriptional controls in higher eukaryotes are central to cell differentiation and developmental programs. These controls reflect sequence-specific interactions of mRNAs with one or more RNA binding proteins. The α-globin poly(C) binding proteins (αCPs) comprise a highly abundant subset of K homology (KH) domain RNA binding proteins and have a characteristic preference for binding single-stranded C-rich motifs. αCPs have been implicated in translation control and stabilization of multiple cellular and viral mRNAs. To explore the full contribution of αCPs to cell function, we have identified a set of mRNAs that associate in vivo with the major αCP2 isoforms. One hundred sixty mRNA species were consistently identified in three independent analyses of αCP2-RNP complexes immunopurified from a human hematopoietic cell line (K562). These mRNAs could be grouped into subsets encoding cytoskeletal components, transcription factors, proto-oncogenes, and cell signaling factors. Two mRNAs were linked to ceroid lipofuscinosis, indicating a potential role for αCP2 in this infantile neurodegenerative disease. Surprisingly, αCP2 mRNA itself was represented in αCP2-RNP complexes, suggesting autoregulatory control of αCP2 expression. In vitro analyses of representative target mRNAs confirmed direct binding of αCP2 within their 3′ untranslated regions. These data expand the list of mRNAs that associate with αCP2 in vivo and establish a foundation for modeling its role in coordinating pathways of posttranscriptional gene regulation.

scholarly journals SSMART: Sequence-structure motif identification for RNA-binding proteins

2018 ◽  
Author(s):  
Alina Munteanu ◽  
Neelanjan Mukherjee ◽  
Uwe Ohler
Keyword(s):  
Binding Sites ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Sequence Motif ◽  
Primary Sequence ◽  
Sequence Structure ◽  
Rna Targets

AbstractMotivationRNA-binding proteins (RBPs) regulate every aspect of RNA metabolism and function. There are hundreds of RBPs encoded in the eukaryotic genomes, and each recognize its RNA targets through a specific mixture of RNA sequence and structure properties. For most RBPs, however, only a primary sequence motif has been determined, while the structure of the binding sites is uncharacterized.ResultsWe developed SSMART, an RNA motif finder that simultaneously models the primary sequence and the structural properties of the RNA targets sites. The sequence-structure motifs are represented as consensus strings over a degenerate alphabet, extending the IUPAC codes for nucleotides to account for secondary structure preferences. Evaluation on synthetic data showed that SSMART is able to recover both sequence and structure motifs implanted into 3‘UTR-like sequences, for various degrees of structured/unstructured binding sites. In addition, we successfully used SSMART on high-throughput in vivo and in vitro data, showing that we not only recover the known sequence motif, but also gain insight into the structural preferences of the RBP.AvailabilitySSMART is freely available at https://ohlerlab.mdc-berlin.de/software/SSMART_137/[email protected]

scholarly journals Humoral Autoimmune Responses to Insulin-Like Growth Factor II mRNA-Binding Proteins IMP1 and p62/IMP2 in Ovarian Cancer

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Xinxin Liu ◽  
Hua Ye ◽  
Liuxia Li ◽  
Wenjie Li ◽  
Yi Zhang ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Growth Factor ◽  
Binding Proteins ◽  
Immunofluorescence Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Insulin Like Growth Factor ◽  
Mrna Binding Proteins ◽  
Normal Individuals ◽  
Factor Ii ◽  
Mrna Binding

Ovarian cancer is one of the leading causes of cancer-related deaths among women. There is an urgent need of better approaches for the identification of appropriate biomarkers in the early detection of ovarian cancer. The aim of this study was to elucidate the significance of autoantibodies against insulin-like growth factor II mRNA-binding proteins (IMPs) in patients with ovarian cancer. In this study, autoantibody responses to two members (IMP1 and p62/IMP2) of IMPs were evaluated by enzyme-linked immunosorbent assay (ELISA), western blotting, and indirect immunofluorescence assay in sera from patients with ovarian cancer and normal human individuals. The results have demonstrated that both IMP1 and p62/IMP2 can induce relatively higher frequency of autoantibody responses in patients with ovarian cancer (26.5% and 29.4%) compared to normal individuals(P<0.01). Our preliminary data suggest that IMP1 and p62/IMP2 can stimulate autoimmune responses in ovarian cancer, and anti-IMP1 and anti-p62/IMP2 autoantibodies could be used as potential biomarkers in immunodiagnosis of ovarian cancer.

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