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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 4 model untranslatable RNAs.(A) Schematic representation of the Ra-Rd encoding genes [see text, S1 Fig]. (B) Uracil content of the model transcripts, utilizing a sliding window of 55 nucleotides as calculated by the software DNA Strider. (C) Wild type E. coli expressing Ra or Rb were disrupted by sonication and cell extracts were fractionated by sucrose density gradient (a representative gradient is shown). The gradient fractions were analyzed for RNA content (A260). The indicated free and ribosomal fractions were pooled. (D) The contents of the indicated R transcripts and endogenous, translatable transcripts encoding PrfA and RpoD as controls, were measured by qPCR in the pooled free and ribosomal fractions. Error bars indicate SEM (n = 3).
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pone.0134413.g001: Characterization of 4 model untranslatable RNAs.(A) Schematic representation of the Ra-Rd encoding genes [see text, S1 Fig]. (B) Uracil content of the model transcripts, utilizing a sliding window of 55 nucleotides as calculated by the software DNA Strider. (C) Wild type E. coli expressing Ra or Rb were disrupted by sonication and cell extracts were fractionated by sucrose density gradient (a representative gradient is shown). The gradient fractions were analyzed for RNA content (A260). The indicated free and ribosomal fractions were pooled. (D) The contents of the indicated R transcripts and endogenous, translatable transcripts encoding PrfA and RpoD as controls, were measured by qPCR in the pooled free and ribosomal fractions. Error bars indicate SEM (n = 3).

Mentions: In order to determine whether U-rich mRNAs bind specific factors in a translation-independent manner, we constructed 4 plasmids, each harboring a different 414-bp-long DNA fragment that encodes a model untranslatable RNA (Ra-Rd, Fig 1A and 1B; S1 Fig). Ra mimics part of the IMP lacY open reading frame, encoding its N-terminal 138 amino acids, which includes 4 transmembrane helices (TMs). Rb mimics part of the cytoplasmic protein lacZ ORF, which includes amino acids 336–473. Similarly, Rc and Rd are derived from genes encoding part of the IMP MelB and the cytoplasmic protein MelA, respectively. All the 4 transcripts are devoid of genuine ribosome binding sites and in addition, they are decorated with stop codons and contain no start codons (S1 Fig). As such, these transcripts are considered untranslatable and allow investigation of their translation-independent interactions (see later).


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 4 model untranslatable RNAs.(A) Schematic representation of the Ra-Rd encoding genes [see text, S1 Fig]. (B) Uracil content of the model transcripts, utilizing a sliding window of 55 nucleotides as calculated by the software DNA Strider. (C) Wild type E. coli expressing Ra or Rb were disrupted by sonication and cell extracts were fractionated by sucrose density gradient (a representative gradient is shown). The gradient fractions were analyzed for RNA content (A260). The indicated free and ribosomal fractions were pooled. (D) The contents of the indicated R transcripts and endogenous, translatable transcripts encoding PrfA and RpoD as controls, were measured by qPCR in the pooled free and ribosomal fractions. Error bars indicate SEM (n = 3).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134413.g001: Characterization of 4 model untranslatable RNAs.(A) Schematic representation of the Ra-Rd encoding genes [see text, S1 Fig]. (B) Uracil content of the model transcripts, utilizing a sliding window of 55 nucleotides as calculated by the software DNA Strider. (C) Wild type E. coli expressing Ra or Rb were disrupted by sonication and cell extracts were fractionated by sucrose density gradient (a representative gradient is shown). The gradient fractions were analyzed for RNA content (A260). The indicated free and ribosomal fractions were pooled. (D) The contents of the indicated R transcripts and endogenous, translatable transcripts encoding PrfA and RpoD as controls, were measured by qPCR in the pooled free and ribosomal fractions. Error bars indicate SEM (n = 3).
Mentions: In order to determine whether U-rich mRNAs bind specific factors in a translation-independent manner, we constructed 4 plasmids, each harboring a different 414-bp-long DNA fragment that encodes a model untranslatable RNA (Ra-Rd, Fig 1A and 1B; S1 Fig). Ra mimics part of the IMP lacY open reading frame, encoding its N-terminal 138 amino acids, which includes 4 transmembrane helices (TMs). Rb mimics part of the cytoplasmic protein lacZ ORF, which includes amino acids 336–473. Similarly, Rc and Rd are derived from genes encoding part of the IMP MelB and the cytoplasmic protein MelA, respectively. All the 4 transcripts are devoid of genuine ribosome binding sites and in addition, they are decorated with stop codons and contain no start codons (S1 Fig). As such, these transcripts are considered untranslatable and allow investigation of their translation-independent interactions (see later).

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