Nucleotide Sequence at the Binding Site for Coat Protein on RNA of Bacteriophage R17

Bernardi, Alberto De; Spahr, Pierre-François

doi:10.1073/pnas.69.10.3033

Cited by 145 publications

(78 citation statements)

References 31 publications

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“…RaPID uses a novel fusion protein (MS2-CP-GFP-SBP) that possesses three relevant functional domains: (1) an amino terminal MS2-CP moiety that shows high affinity to the MS2L RNA aptamer (Bernardi and Spahr 1972;Valegard et al 1994); (2) a GFP reporter that allows for visualization of its intracellular localization in vivo and for protein detection in blots using anti-GFP antibodies; and (3) a carboxy-terminal SBP epitope that allows for specific high-affinity interactions with streptavidin-conjugated matrices (Keefe et al 2001). Together, the CP and GFP domains allow for visualization of the intracellular localization of MS2L-tagged mRNAs of interest (Fig.…”

Section: Discussionmentioning

confidence: 99%

A novel mRNA affinity purification technique for the identification of interacting proteins and transcripts in ribonucleoprotein complexes

Slobodin

Gerst²

2010

RNA

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Intracellular mRNA targeting and localized translation are potential determinants for protein localization. To facilitate targeting, mRNAs possess specific cis-acting sequence motifs that are recognized by trans-acting RNA-binding proteins (RBPs). While many mRNAs are trafficked, our knowledge of the RBPs involved and presence of additional transcripts within these ribonucleoprotein (RNP) complexes is limited. To facilitate the identification of RBPs and transcripts that bind to specific mRNAs, we developed RNA-binding protein purification and identification (RaPID), a novel technique that allows for the affinity purification of MS2 aptamer-tagged mRNAs and subsequent detection of bound RBPs and transcripts using massspectometry and RT-PCR, respectively. RaPID effectively isolated specific mRNAs from both yeast and mammalian cells, and identified known mRNA-RBP interactions (e.g., ASH1-She2; b-Actin-IMP1). By isolating tagged OXA1 mRNA using RaPID, we could identify a yeast COPI subunit (i.e., Sec27) as a candidate interacting protein. This finding was strengthened by the observation that a portion of OXA1 mRNA was delocalized in a sec27-1 temperature-sensitive mutant at restrictive temperatures. Finally, RaPID could also be used to show biochemically the coexistence of different RNA species within the same RNP complex (e.g., coprecipitation of the yeast SRO7, WSC2, SEC3, and IST2 mRNAs with ASH1 mRNA) for the first time.

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Section: Discussionmentioning

confidence: 99%

A novel mRNA affinity purification technique for the identification of interacting proteins and transcripts in ribonucleoprotein complexes

Slobodin

Gerst²

2010

RNA

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“…For example, the bacteriophage R17 coat protein recognizes a hairpin containing the ribosome binding site for the phage replicase (22). Also, viral RdRps replicating full-length products from linear templates have been shown to require various structures on the 3Ј end of the RNA template to initiate accurate synthesis (23)(24)(25)(26).…”

Section: Discussionmentioning

confidence: 99%

Sequence-specific recognition of a subgenomic RNA promoter by a viral RNA polymerase

Siegel

Adkins

Kao

1997

Proc. Natl. Acad. Sci. U.S.A.

109

129

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RNA templates of 33 nucleotides containing the brome mosaic virus (BMV) core subgenomic promoter were used to determine the promoter elements recognized by the BMV RNA-dependent RNA polymerase (RdRp) to initiate RNA synthesis. Nucleotides at positions ؊17, ؊14, ؊13, and ؊11 relative to the subgenomic initiation site must be maintained for interaction with the RdRp. Changes to every other nucleotide at these four positions allow predictions for the base-specific functional groups required for RdRp recognition. RdRp contact of the nucleotide at position ؊17 was suggested with a template competition assay. Comparison of the BMV subgenomic promoter to those from other plant and animal alphaviruses shows a remarkable degree of conservation of the nucleotides required for BMV subgenomic RNA synthesis. We show that the RdRp of the plant-infecting BMV is capable of accurately, albeit inefficiently, initiating RNA synthesis from the subgenomic promoter of the animalinfecting Semliki Forest virus. The sequence-specific recognition of RNA by the BMV RdRp is analogous to the recognition of DNA promoters by DNA-dependent RNA polymerases.Viral RNA replication, a process fundamental to pathogenicity, requires specific recognition of RNA features by proteins. RNA-dependent RNA polymerase (RdRp) is a complex composed of viral and cellular proteins that directs viral RNA synthesis from infecting RNA templates (1). Many viral RdRp proteins have been sequenced and analyzed (2); however, a comprehensive mechanism describing RNA synthesis is lacking. Consequently, general knowledge of RdRps is significantly less than that of other RNA and DNA polymerases.To investigate the mechanism of RNA-directed RNA synthesis, we study brome mosaic virus (BMV), type member of the bromovirus group of plant viruses in the alphavirus-like superfamily of (ϩ)-strand RNA viruses (3). The BMV genome is comprised of three RNAs designated 1, 2, and 3, and a subgenomic RNA4 which is initiated from (Ϫ)-strand RNA3. Enriched BMV RdRp preparations from infected barley can, in a highly specific manner, synthesize (Ϫ)-strand RNA from (ϩ)-strand templates and subgenomic (ϩ)-strand products from (Ϫ)-strand templates (4-6).We have developed a system to study BMV subgenomic RNA initiation from minimal templates, designated proscripts, because they contain the promoter and template for (ϩ)-strand RNA synthesis. We have shown that the 20 nt 3Ј of the subgenomic initiation nucleotide, recognized as the subgenomic core promoter (7,8), are sufficient to direct (ϩ)-strand RNA synthesis (6), permitting the design of proscripts focusing only on the core promoter.In this paper, we have used a functional assay to determine that nucleotides Ϫ17, Ϫ14, Ϫ13, and Ϫ11 relative to the subgenomic initiation site are required for RNA synthesis from the subgenomic core promoter. Moreover, at least one of these nucleotides, Ϫ17, was found likely to be contacted by the BMV RdRp. The resolution achieved in this study allows us to predict the base moieties contacted by RdRp and dem...

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“…Although translational autoregulation has been described in prokaryotic systems (2,5), TS mRNA represents the first eukaryotic mRNA whose regulation is controlled in such a fashion. Furthermore, we demonstrated that incubation of TS protein with either the nucleotide substrate dUMP or the inhibitor 5-fluoro-dUMP repressed its inhibitory effect on TS mRNA translation.…”

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confidence: 99%

Identification of a thymidylate synthase ribonucleoprotein complex in human colon cancer cells.

Chu¹,

Voeller²,

Jones³

et al. 1994

Mol. Cell. Biol.

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Translation of thymidylate synthase (TS) mRNA is controlled by its own protein product, TS, in an autoregulatory manner. Direct binding of TS protein to two different cis-acting elements on the TS mRNA is associated with this translational regulation. In this study, an immunoprecipitation-reverse transcription-PCR technique was used to identify a TS ribonucleoprotein (RNP) complex in cultured human colon cancer cells. Using antibodies specific for TS protein, we show that TS is complexed in vivo with its own TS RNA. Furthermore, evidence demonstrating a direct interaction between the mRNA of the nuclear oncogene c-myc and TS protein is presented.Recently, there has been an increased interest in the characterization of translational regulatory mechanisms. There are a number of eukaryotic mRNAs whose expression is controlled at the level of translation (18). The regulated synthesis of the iron storage protein ferritin by iron represents one of the best-studied examples of this form of regulation (25). A stem-loop structure located within the 5' untranslated region (UTR) of ferritin mRNA, termed the iron-responsive element (IRE), represents the cis-acting element to which the IRE-binding protein binds (16,23). Recent studies have demonstrated that the redox state in the cell is an important determinant of the binding affinity of this protein to the IRE (15,17).Using an in vitro translation system, we showed that translation of human thymidylate synthase (TS) mRNA is regulated by its own protein product, TS, in a negative autoregulatory manner (7). Although translational autoregulation has been described in prokaryotic systems (2, 5), TS mRNA represents the first eukaryotic mRNA whose regulation is controlled in such a fashion. Furthermore, we demonstrated that incubation of TS protein with either the nucleotide substrate dUMP or the inhibitor 5-fluoro-dUMP repressed its inhibitory effect on TS mRNA translation. during the cell cycle is primarily regulated at the transcriptional level (1,19,30), there is now recent evidence suggesting control at the level of translation (22). These findings, taken together, offer supportive evidence for the model of TS translational autoregulation.The purpose of the present study was to identify a TS ribonucleoprotein (RNP) complex in a cultured cell system. With the use of specific antisera to TS, we show that TS protein is complexed with its corresponding TS RNA in human colon cancer cells. In addition, we present evidence demonstrating a specific interaction between the mRNA of the nuclear oncogene c-myc and TS protein. MATERIALS AND METHODSCell culture. The characteristics of the human colon cancer cell line H630 have been previously described (32). The resistant H630-R1O subline was selected in vitro for resistance to 5-FU by exposure of the parent H630 cell line to stepwise increases in 5-FU and was maintained in medium containing 10 p,. Cell lines were grown in 75-cm2 plastic tissue culture flasks (Falcon Labware, Oxnard, Calif.) in growth medium containing RPMI 1640 with 1...

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Nucleotide Sequence at the Binding Site for Coat Protein on RNA of Bacteriophage R17

Cited by 145 publications

References 31 publications

A novel mRNA affinity purification technique for the identification of interacting proteins and transcripts in ribonucleoprotein complexes

A novel mRNA affinity purification technique for the identification of interacting proteins and transcripts in ribonucleoprotein complexes

Sequence-specific recognition of a subgenomic RNA promoter by a viral RNA polymerase

Identification of a thymidylate synthase ribonucleoprotein complex in human colon cancer cells.

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