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

Area of Science:

• Molecular Biology
• Cell Biology
• Genetics

Background:

• Integral membrane protein-encoding mRNAs (MPRs) may be recognized and delivered to membrane-bound ribosomes independently of translation.
• Uracil-rich segments encoding hydrophobic transmembrane helices are hypothesized to mediate MPR recognition.

Purpose of the Study:

• To investigate the hypothesis that uracil-rich segments in MPRs mediate specific interactions.
• To identify proteins involved in the recognition and potential biogenesis of MPRs.

Main Methods:

• Design of DNA sequences encoding model untranslatable transcripts mimicking MPRs and cytosolic mRNA-encoding transcripts (CPRs).
• In vitro binding assays using synthesized biotinylated RNAs and Escherichia coli extracts.
• Co-purification studies with E. coli expressing 6His-tagged cold shock proteins CspE and CspC.

Main Results:

• A specific interaction was identified between MPR-mimicking transcripts and cold shock proteins CspE and CspC in vitro.
• In vivo co-purification confirmed the interaction between CspE/CspC and both model MPR transcripts and endogenous MPRs.
• Cold shock proteins bind specifically to uracil-rich segments characteristic of MPRs.

Conclusions:

• Evolutionarily conserved cold shock proteins (CspE and CspC) specifically interact with integral membrane protein-encoding mRNAs (MPRs).
• These proteins may act as promiscuous chaperones involved in the biogenesis of MPRs.
• The findings suggest a novel mechanism for MPR targeting and processing.