Abstract
Translation-independent mRNA localization represents an emerging concept in cell biology. In Escherichia coli, mRNAs encoding integral membrane proteins (MPRs) are targeted to the membrane where they are translated by membrane associated ribosomes and the produced proteins are inserted into the membrane co-translationally. In order to better understand aspects of the biogenesis and localization of MPRs, we investigated their subcellular distribution using cell fractionation, RNA-seq and qPCR. The results show that MPRs are overrepresented in the membrane fraction, as expected, and depletion of the signal recognition particle-receptor, FtsY reduced the amounts of all mRNAs on the membrane. Surprisingly, however, MPRs were also found relatively abundant in the soluble ribosome-free fraction and their amount in this fraction is increased upon overexpression of CspE, which was recently shown to interact with MPRs. CspE also conferred a positive effect on the membrane-expression of integral membrane proteins. We discuss the possibility that the effects of CspE overexpression may link the intriguing subcellular localization of MPRs to the cytosolic ribosome-free fraction with their translation into membrane proteins and that the ribosome-free pool of MPRs may represent a stage during their targeting to the membrane, which precedes translation.
Highlights
Integral membrane proteins (IMPs) are usually translated by membrane bound ribosomes
We study the subcellular distribution of mRNAs in E. coli by cell fractionation for the first time
The quantitative PCR (qPCR) analysis of the distribution of randomly selected transcripts shows that at a steady state, 10–20% of the total mRNAs migrated to the gradient pellet, 30–45% migrated with 70S ribosomes, 30–60% migrated to the ribosome-free fraction, and only a minority of mRNA seems to exist in other fractions that were not analyzed (Fig 1A, fractions 4–7 and 11–12)
Summary
Integral membrane proteins (IMPs) are usually translated by membrane bound ribosomes. We proposed that MPRs in E. coli, and possibly elsewhere, might be selectively recognized through features derived from their high uracil content in long segments throughout their coding sequence (~60 nucleotidelong) [14]. To investigate this hypothesis, we previously searched for uracil-rich RNA-binding proteins [15]. Our results revealed that overexpression of the cold shock protein CspE increased the MPRs pool in the ribosome-free fraction and their amount on the membrane and positively affected their translation into integral membrane proteins
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
