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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

High throughput sequencing of endogenous RNAs that co-purify with 6His-CspE.(A) Wild type E. coli expressing CspE-6His were disrupted in the presence of either 2 mM or 15 mM [Mg2+] (top and bottom panels, respectively) and the total cell extracts were subjected to metal affinity chromatography using Talon resin. RNA was prepared from the total cell extracts and the imidazole-eluted material (see Fig 3) and subjected to high throughput sequencing. The amount of CspE-6His bound MPRs (left panels) and CPRs (right panels) is plotted as a function of the amount of the same mRNAs in the total extract. (B) CspE-binding of all detected mRNAs was calculated as [RPKMCspE-bound / RPKMextract]. The quota of MPRs and CPRs in each 10th percentile along the CspE-association landscape is presented as a moving average plot. (C) This panel shows the CspE-binding values in the presence of 2 mM [Mg2+] for selected MPRs and CPRs that were similarly analyzed by qPCR (see Fig 3D for comparison). (D) An independent experiment that shows the CspE-binding values for selected MPRs and CPRs in the presence of 15 mM [Mg2+].
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pone.0134413.g004: High throughput sequencing of endogenous RNAs that co-purify with 6His-CspE.(A) Wild type E. coli expressing CspE-6His were disrupted in the presence of either 2 mM or 15 mM [Mg2+] (top and bottom panels, respectively) and the total cell extracts were subjected to metal affinity chromatography using Talon resin. RNA was prepared from the total cell extracts and the imidazole-eluted material (see Fig 3) and subjected to high throughput sequencing. The amount of CspE-6His bound MPRs (left panels) and CPRs (right panels) is plotted as a function of the amount of the same mRNAs in the total extract. (B) CspE-binding of all detected mRNAs was calculated as [RPKMCspE-bound / RPKMextract]. The quota of MPRs and CPRs in each 10th percentile along the CspE-association landscape is presented as a moving average plot. (C) This panel shows the CspE-binding values in the presence of 2 mM [Mg2+] for selected MPRs and CPRs that were similarly analyzed by qPCR (see Fig 3D for comparison). (D) An independent experiment that shows the CspE-binding values for selected MPRs and CPRs in the presence of 15 mM [Mg2+].

Mentions: Next, the input and eluted RNAs were analyzed by qPCR using primers specific to various MPRs and CPRs. On average, the results show an interesting pattern of pulled-down MPRs and CPRs (Fig 3E, S2 Fig). Of the few test examples, it is apparent that several MPRs are enrichred in the pulled down material, compared to CPRs, with both 6His-CspE (Fig 3E upper panel) and 6His-CspC (Fig 3E, lower panel). The question why other MPRs, such as SecY are underrepresented is currently being investigated. Preliminary experiments under high [Mg+2] conditions revealed an increased specificity of CspE for MPRs, including secY (data not shown, and Fig 4), in agreement with the previous studies of the effect of [Mg+2] (Fig 2D, 2E and 2F).


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)

High throughput sequencing of endogenous RNAs that co-purify with 6His-CspE.(A) Wild type E. coli expressing CspE-6His were disrupted in the presence of either 2 mM or 15 mM [Mg2+] (top and bottom panels, respectively) and the total cell extracts were subjected to metal affinity chromatography using Talon resin. RNA was prepared from the total cell extracts and the imidazole-eluted material (see Fig 3) and subjected to high throughput sequencing. The amount of CspE-6His bound MPRs (left panels) and CPRs (right panels) is plotted as a function of the amount of the same mRNAs in the total extract. (B) CspE-binding of all detected mRNAs was calculated as [RPKMCspE-bound / RPKMextract]. The quota of MPRs and CPRs in each 10th percentile along the CspE-association landscape is presented as a moving average plot. (C) This panel shows the CspE-binding values in the presence of 2 mM [Mg2+] for selected MPRs and CPRs that were similarly analyzed by qPCR (see Fig 3D for comparison). (D) An independent experiment that shows the CspE-binding values for selected MPRs and CPRs in the presence of 15 mM [Mg2+].
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134413.g004: High throughput sequencing of endogenous RNAs that co-purify with 6His-CspE.(A) Wild type E. coli expressing CspE-6His were disrupted in the presence of either 2 mM or 15 mM [Mg2+] (top and bottom panels, respectively) and the total cell extracts were subjected to metal affinity chromatography using Talon resin. RNA was prepared from the total cell extracts and the imidazole-eluted material (see Fig 3) and subjected to high throughput sequencing. The amount of CspE-6His bound MPRs (left panels) and CPRs (right panels) is plotted as a function of the amount of the same mRNAs in the total extract. (B) CspE-binding of all detected mRNAs was calculated as [RPKMCspE-bound / RPKMextract]. The quota of MPRs and CPRs in each 10th percentile along the CspE-association landscape is presented as a moving average plot. (C) This panel shows the CspE-binding values in the presence of 2 mM [Mg2+] for selected MPRs and CPRs that were similarly analyzed by qPCR (see Fig 3D for comparison). (D) An independent experiment that shows the CspE-binding values for selected MPRs and CPRs in the presence of 15 mM [Mg2+].
Mentions: Next, the input and eluted RNAs were analyzed by qPCR using primers specific to various MPRs and CPRs. On average, the results show an interesting pattern of pulled-down MPRs and CPRs (Fig 3E, S2 Fig). Of the few test examples, it is apparent that several MPRs are enrichred in the pulled down material, compared to CPRs, with both 6His-CspE (Fig 3E upper panel) and 6His-CspC (Fig 3E, lower panel). The question why other MPRs, such as SecY are underrepresented is currently being investigated. Preliminary experiments under high [Mg+2] conditions revealed an increased specificity of CspE for MPRs, including secY (data not shown, and Fig 4), in agreement with the previous studies of the effect of [Mg+2] (Fig 2D, 2E and 2F).

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