Evidence for a cytoplasmic pool of ribosome-free mRNAs encoding inner membrane proteins in Escherichia coli

Benhalevy, Daniel; Biran, Ido; Bochkareva, Elena; Sorek, Rotem; Bibi, Eitan

doi:10.1371/journal.pone.0183862

Cited by 12 publications

(17 citation statements)

References 54 publications

(75 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In E. coli , mRNAs encoding IMPs are considered to reach the IM in a translation- and SRP-dependent manner ( 54 , 55 ) or via a mechanism in which the ribosome-mRNA complex can localize to the IM independently of translation ( 56 ) or by combining the two different pathways ( 57 ). We found that the SRP suppressors not only decreased the translation rate ( Fig.…”

Section: Discussionmentioning

confidence: 99%

Signal Recognition Particle Suppressor Screening Reveals the Regulation of Membrane Protein Targeting by the Translation Rate

Zhao

Cui

et al. 2021

mBio

View full text Add to dashboard Cite

The signal recognition particle (SRP) is conserved in all living organisms, and it cotranslationally delivers proteins to the inner membrane or endoplasmic reticulum. Recently, SRP loss was found not to be lethal in either the eukaryote Saccharomyces cerevisiae or the prokaryote Streptococcus mutans. In Escherichia coli, the role of SRP in mediating inner membrane protein (IMP) targeting has long been studied. However, the essentiality of SRP remains a controversial topic, partly hindered by the lack of strains in which SRP is completely absent. Here we show that the SRP was nonessential in E. coli by suppressor screening. We identified two classes of extragenic suppressors—two translation initiation factors and a ribosomal protein—all of which are involved in translation initiation. The translation rate and inner membrane proteomic analyses were combined to define the mechanism that compensates for the lack of SRP. The primary factor that contributes to the efficiency of IMP targeting is the extension of the time window for targeting by pausing the initiation of translation, which further reduces translation initiation and elongation rates. Furthermore, we found that easily predictable features in the nascent chain determine the specificity of protein targeting. Our results show why the loss of the SRP pathway does not lead to lethality. We report a new paradigm in which the time delay in translation initiation is beneficial during protein targeting in the absence of SRP. IMPORTANCE Inner membrane proteins (IMPs) are cotranslationally inserted into the inner membrane or endoplasmic reticulum by the signal recognition particle (SRP). Generally, the deletion of SRP can result in protein targeting defects in Escherichia coli. Suppressor screening for loss of SRP reveals that pausing at the translation start site is likely to be critical in allowing IMP targeting and avoiding aggregation. In this work, we found for the first time that SRP is nonessential in E. coli. The time delay in initiation is different from the previous mechanism that only slows down the elongation rate. It not only maximizes the opportunity for untranslated ribosomes to be near the inner membrane but also extends the time window for targeting translating ribosomes by decreasing the speed of translation. We anticipate that our work will be a starting point for a more delicate regulatory mechanism of protein targeting.

show abstract

Section: Discussionmentioning

confidence: 99%

Signal Recognition Particle Suppressor Screening Reveals the Regulation of Membrane Protein Targeting by the Translation Rate

Zhao

Cui

et al. 2021

mBio

View full text Add to dashboard Cite

show abstract

“…Whether the protein signal sequence or solely the act of translation of its mRNA is required is so far unknown. Another study also reported, based on cell-fractionation, RNA-seq, and qPCR, that mRNAs of inner membrane proteins are enriched at the membrane and depletion of the SRP receptor FtsY reduced the amounts of all mRNAs at the membrane (15). However, mRNAs encoding inner membrane proteins were also found in the soluble ribosome-free fraction, which might represent a stage prior to their translation and targeting to the membrane.…”

Section: Localization Of Messenger Rnasmentioning

confidence: 98%

“…Similar approaches have also been used to investigate RNA localization. Particularly, when cell fractionation methods are combined with high-throughput RNA sequencing, the relative distribution of transcripts between the membrane and the cytoplasm can be estimated at the whole transcriptome level (15). Compared to fractionation-based approaches, visualization of RNAs by light microscopy techniques provides the most direct information on subcellular localization of individual RNA species.…”

Section: Approaches To Study Rna Localizationmentioning

confidence: 99%

“…For example, overexpression of the cold-shock protein CspE increased the fraction of membrane protein mRNAs in the ribosome-free fraction and their amount on the membrane and positively affected their translation, indicating potential regulation of subcellular RNA localization (15). Moreover, the 98-aa protein ComN, a post-transcriptional regulator of competence gene expression, localizes to the division site and cell poles via direct interaction with DivIVA, a key protein involved in cell pole differentiation in B. subtilis (66).…”

Section: Localization Of Messenger Rnasmentioning

confidence: 99%

See 1 more Smart Citation

RNA Localization in Bacteria

Fei

Sharma

2018

Microbiol Spectr

View full text Add to dashboard Cite

Diverse mechanisms and functions of post-transcriptional regulation by small regulatory RNAs (sRNAs) and RNA binding proteins (RBPs) have been described in bacteria. In contrast, little is known about the spatial organization of RNAs in bacterial cells. In eukaryotes, subcellular localization and transport of RNAs play important roles in diverse physiological processes, such as embryonic patterning, asymmetric cell division, epithelial polarity, and neuronal plasticity. It is now clear that bacterial RNAs also can accumulate at distinct sites in the cell. However, due the small size of bacterial cells, localized RNA and translation are more challenging to study in bacterial cells, and the underlying molecular mechanisms of transcript localization are less understood. Here we review the emerging examples of RNAs localized to specific subcellular locations in bacteria, with indications that subcellular localization of transcripts might be important for regulatory processes. Diverse mechanisms for bacterial RNA localization have been suggested, including close association to their genomic site of transcription, or to the localizations of their protein products in translation-dependent or independent processes. We also provide an overview of the state-of-the-art of technologies to visualize and track bacterial RNAs, ranging from hybridization-based approaches in fixed cells to in vivo imaging approaches using fluorescent protein reporters and/or RNA aptamers in single living bacterial cells. We conclude with a discussion of open questions in the field and ongoing technological developments regarding RNA imaging in eukaryotic systems that might likewise provide novel insights into RNA localization in bacteria.

show abstract

“…Interestingly, E. coli CspC and CspE preferentially bind U-rich artificial transcripts and endogenous membrane mRNAs ( Benhalevy et al, 2015 ). Moreover, CspE overexpression causes the accumulation of ribosome-free transcripts encoding membrane proteins in the cytoplasm and positively affects their translation in the membrane ( Benhalevy et al, 2017 ). This suggests that, via CspE mediation, these transcripts avoid CTT until they reach the membrane, where they are locally translated.…”

Section: Mechanisms Potentially Enabling Uttmentioning

confidence: 99%

Coupled Transcription-Translation in Prokaryotes: An Old Couple With New Surprises

Irastortza-Olaziregi

Amster‐Choder

2021

Front. Microbiol.

View full text Add to dashboard Cite

Coupled transcription-translation (CTT) is a hallmark of prokaryotic gene expression. CTT occurs when ribosomes associate with and initiate translation of mRNAs whose transcription has not yet concluded, therefore forming “RNAP.mRNA.ribosome” complexes. CTT is a well-documented phenomenon that is involved in important gene regulation processes, such as attenuation and operon polarity. Despite the progress in our understanding of the cellular signals that coordinate CTT, certain aspects of its molecular architecture remain controversial. Additionally, new information on the spatial segregation between the transcriptional and the translational machineries in certain species, and on the capability of certain mRNAs to localize translation-independently, questions the unanimous occurrence of CTT. Furthermore, studies where transcription and translation were artificially uncoupled showed that transcription elongation can proceed in a translation-independent manner. Here, we review studies supporting the occurrence of CTT and findings questioning its extent, as well as discuss mechanisms that may explain both coupling and uncoupling, e.g., chromosome relocation and the involvement of cis- or trans-acting elements, such as small RNAs and RNA-binding proteins. These mechanisms impact RNA localization, stability, and translation. Understanding the two options by which genes can be expressed and their consequences should shed light on a new layer of control of bacterial transcripts fate.

show abstract

Evidence for a cytoplasmic pool of ribosome-free mRNAs encoding inner membrane proteins in Escherichia coli

Cited by 12 publications

References 54 publications

Signal Recognition Particle Suppressor Screening Reveals the Regulation of Membrane Protein Targeting by the Translation Rate

Signal Recognition Particle Suppressor Screening Reveals the Regulation of Membrane Protein Targeting by the Translation Rate

RNA Localization in Bacteria

Coupled Transcription-Translation in Prokaryotes: An Old Couple With New Surprises

Contact Info

Product

Resources

About