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.
Highlights
In addition to encoding proteins, mRNAs harbor information required for controlling posttranscriptional regulatory pathways, such as processing, translation, degradation, and cellular localization [1, 2]
E. coli represents an interesting case because unlike in eukaryotes [5] the prokaryotic mRNAs usually do not contain 3’ regulatory untranslated sequences (Daniel Dar and Rotem Sorek, personal communcation), suggesting that they might be recognized through their relatively short 5’ untranslated regions or through their open reading frames. mRNAs that encode integral membrane proteins (IMPs) across evolution are usually translated at distinct membrane locations, and our studies in E. coli have suggested a step through which IMP-encoding mRNAs (MPRs)
Our results show that U-rich RNAs, including endogenous MPRs, interact with a group of cold shock proteins, which are normally expressed in E. coli at a physiological temperature
Summary
In addition to encoding proteins, mRNAs harbor information required for controlling posttranscriptional regulatory pathways, such as processing, translation, degradation, and cellular localization [1, 2]. MRNAs utilize various protein-interaction determinants (structural, sequence-specific, or nonspecific) [3], mostly in 3’ untranslated regions, unique exceptions have been described [e.g. In this regard, E. coli represents an interesting case because unlike in eukaryotes [5] the prokaryotic mRNAs usually do not contain 3’ regulatory untranslated sequences (Daniel Dar and Rotem Sorek, personal communcation), suggesting that they might be recognized through their relatively short 5’ untranslated regions or through their open reading frames. The question of how such a large and diverse group of transcripts can be recognized has not yet been resolved This question is even more challenging if the recognition determinants reside within open reading frames
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