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Model Uracil-Rich RNAs and Membrane Protein mRNAs Interact Specifically with Cold Shock Proteins in Escherichia coli.

Benhalevy D, Bochkareva ES, Biran I, Bibi E - PLoS ONE (2015)

Bottom Line: This is implied from the emerging concept that MPRs are specifically recognized and delivered to membrane-bound ribosomes in a translation-independent manner.MPRs might be recognized through uracil-rich segments that encode hydrophobic transmembrane helices.Our results suggest that the evolutionarily conserved cold shock proteins may have a role, possibly as promiscuous chaperons, in the biogenesis of MPRs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.

ABSTRACT
Are integral membrane protein-encoding mRNAs (MPRs) different from other mRNAs such as those encoding cytosolic mRNAs (CPRs)? This is implied from the emerging concept that MPRs are specifically recognized and delivered to membrane-bound ribosomes in a translation-independent manner. MPRs might be recognized through uracil-rich segments that encode hydrophobic transmembrane helices. To investigate this hypothesis, we designed DNA sequences encoding model untranslatable transcripts that mimic MPRs or CPRs. By utilizing in vitro-synthesized biotinylated RNAs mixed with Escherichia coli extracts, we identified a highly specific interaction that takes place between transcripts that mimic MPRs and the cold shock proteins CspE and CspC, which are normally expressed under physiological conditions. Co-purification studies with E. coli expressing 6His-tagged CspE or CspC confirmed that the specific interaction occurs in vivo not only with the model uracil-rich untranslatable transcripts but also with endogenous MPRs. Our results suggest that the evolutionarily conserved cold shock proteins may have a role, possibly as promiscuous chaperons, in the biogenesis of MPRs.

No MeSH data available.


Related in: MedlinePlus

Characterization of CspE-, CspC-, and CspC/E deleted cells.(A) PCR analysis of the cspE and cspC in E. coli ΔcspE, ΔcspC, or ΔcspCΔcspE::kanR. (B) Western blot analysis with anti-CspE antibodies of total extracts from wild type and the deleted E. coli strains. (C) (D)In vitro synthesized biotinylated Ra or Rb were incubated with S300 fractions of wild type E. coli or E. coli ΔcspE, or E. coli ΔcspE ΔcspC in the presence of 2 mM Mg2+. The RNA-protein complexes were purified by streptavidin beads, eluted with SDS buffer and separated by tris-tricine SDS-PAGE. The gels were stained with Instant blue.
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pone.0134413.g005: Characterization of CspE-, CspC-, and CspC/E deleted cells.(A) PCR analysis of the cspE and cspC in E. coli ΔcspE, ΔcspC, or ΔcspCΔcspE::kanR. (B) Western blot analysis with anti-CspE antibodies of total extracts from wild type and the deleted E. coli strains. (C) (D)In vitro synthesized biotinylated Ra or Rb were incubated with S300 fractions of wild type E. coli or E. coli ΔcspE, or E. coli ΔcspE ΔcspC in the presence of 2 mM Mg2+. The RNA-protein complexes were purified by streptavidin beads, eluted with SDS buffer and separated by tris-tricine SDS-PAGE. The gels were stained with Instant blue.

Mentions: In an attempt to search for interesting phenotypes in the absence of CspE and/or CspC, we studied E. coli(ΔcspE) E. coliΔcspC) and a double-deletion E. coli(ΔcspEΔcspC) strain (see methods). Initially the strains were evaluated both by PCR (Fig 5A) and Western blotting with anti-CspE antibodies (Fig 5B). Growth experiments in rich media showed that all the strains grow as well as wild type E. coli at 37°C (data not shown). Next, we repeated the co-purification experiments utilizing in vitro-synthesized biotinylated Ra and Rb and extracts from the deleted cells. In the ΔcspE strain, a small protein was co-purified only with Ra, and the mass spectrometry results revealed that it is CspC, as expected (Fig 5C, left panel, star). Similarly, in the ΔcspEΔcspC strain a third small protein was found to poorly interact only with U-rich Ra, and it was identified by mass spectrometry as CspA (Fig 5C, right panel, star).


Model Uracil-Rich RNAs and Membrane Protein mRNAs Interact Specifically with Cold Shock Proteins in Escherichia coli.

Benhalevy D, Bochkareva ES, Biran I, Bibi E - PLoS ONE (2015)

Characterization of CspE-, CspC-, and CspC/E deleted cells.(A) PCR analysis of the cspE and cspC in E. coli ΔcspE, ΔcspC, or ΔcspCΔcspE::kanR. (B) Western blot analysis with anti-CspE antibodies of total extracts from wild type and the deleted E. coli strains. (C) (D)In vitro synthesized biotinylated Ra or Rb were incubated with S300 fractions of wild type E. coli or E. coli ΔcspE, or E. coli ΔcspE ΔcspC in the presence of 2 mM Mg2+. The RNA-protein complexes were purified by streptavidin beads, eluted with SDS buffer and separated by tris-tricine SDS-PAGE. The gels were stained with Instant blue.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4520561&req=5

pone.0134413.g005: Characterization of CspE-, CspC-, and CspC/E deleted cells.(A) PCR analysis of the cspE and cspC in E. coli ΔcspE, ΔcspC, or ΔcspCΔcspE::kanR. (B) Western blot analysis with anti-CspE antibodies of total extracts from wild type and the deleted E. coli strains. (C) (D)In vitro synthesized biotinylated Ra or Rb were incubated with S300 fractions of wild type E. coli or E. coli ΔcspE, or E. coli ΔcspE ΔcspC in the presence of 2 mM Mg2+. The RNA-protein complexes were purified by streptavidin beads, eluted with SDS buffer and separated by tris-tricine SDS-PAGE. The gels were stained with Instant blue.
Mentions: In an attempt to search for interesting phenotypes in the absence of CspE and/or CspC, we studied E. coli(ΔcspE) E. coliΔcspC) and a double-deletion E. coli(ΔcspEΔcspC) strain (see methods). Initially the strains were evaluated both by PCR (Fig 5A) and Western blotting with anti-CspE antibodies (Fig 5B). Growth experiments in rich media showed that all the strains grow as well as wild type E. coli at 37°C (data not shown). Next, we repeated the co-purification experiments utilizing in vitro-synthesized biotinylated Ra and Rb and extracts from the deleted cells. In the ΔcspE strain, a small protein was co-purified only with Ra, and the mass spectrometry results revealed that it is CspC, as expected (Fig 5C, left panel, star). Similarly, in the ΔcspEΔcspC strain a third small protein was found to poorly interact only with U-rich Ra, and it was identified by mass spectrometry as CspA (Fig 5C, right panel, star).

Bottom Line: This is implied from the emerging concept that MPRs are specifically recognized and delivered to membrane-bound ribosomes in a translation-independent manner.MPRs might be recognized through uracil-rich segments that encode hydrophobic transmembrane helices.Our results suggest that the evolutionarily conserved cold shock proteins may have a role, possibly as promiscuous chaperons, in the biogenesis of MPRs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.

ABSTRACT
Are integral membrane protein-encoding mRNAs (MPRs) different from other mRNAs such as those encoding cytosolic mRNAs (CPRs)? This is implied from the emerging concept that MPRs are specifically recognized and delivered to membrane-bound ribosomes in a translation-independent manner. MPRs might be recognized through uracil-rich segments that encode hydrophobic transmembrane helices. To investigate this hypothesis, we designed DNA sequences encoding model untranslatable transcripts that mimic MPRs or CPRs. By utilizing in vitro-synthesized biotinylated RNAs mixed with Escherichia coli extracts, we identified a highly specific interaction that takes place between transcripts that mimic MPRs and the cold shock proteins CspE and CspC, which are normally expressed under physiological conditions. Co-purification studies with E. coli expressing 6His-tagged CspE or CspC confirmed that the specific interaction occurs in vivo not only with the model uracil-rich untranslatable transcripts but also with endogenous MPRs. Our results suggest that the evolutionarily conserved cold shock proteins may have a role, possibly as promiscuous chaperons, in the biogenesis of MPRs.

No MeSH data available.


Related in: MedlinePlus