Ribosome profiling reveals multiple roles of SecA in cotranslational protein export
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ABSTRACT:
PROVIDER: PRJNA769692 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR16249166.fastq.gz | Fastqsanger.gz | |||
| SRR16249167.fastq.gz | Fastqsanger.gz | |||
| SRR16249168.fastq.gz | Fastqsanger.gz | |||
| SRR16249169.fastq.gz | Fastqsanger.gz | |||
| SRR16249170.fastq.gz | Fastqsanger.gz |
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Project description:SecA, an ATPase known to posttranslationally translocate secretory proteins across the bacterial plasma membrane, also interacts with ribosomes. Here, we used a combination of ribosome profiling methods to investigate the cotranslational actions of SecA in vivo. SecA scans translating ribosomes at the plasma membrane and cotranslationally engages large periplasmic loops on inner membrane proteins. In coordination with the proton motive force, SecA resolves the cytoplasmic accumulation of periplasmic loops during cotranslational protein translocation. SecA also associates with a subset of secretory proteins at an early stage of translation and mediates their cotranslational transport, whereas the chaperone trigger factor (TF) delays SecA engagement on other secretory proteins to impose a posttranslational mode of translocation. The hydrophobicity of signal sequence is a determinant of nascent protein triage between SecA and TF. Our results elucidate the principles of SecA-driven cotranslational protein translocation and reveal a hierarchical network of protein export pathways in bacteria.
2022-05-16 | GSE185572 | GEO
Project description:SecA, an ATPase known to posttranslationally translocate secretory proteins across the bacterial plasma membrane, also binds ribosomes, but the role of SecA's ribosome interaction has been unclear. Here, we used a combination of ribosome profiling methods to investigate the cotranslational actions of SecA. Our data reveal the widespread accumulation of large periplasmic loops of inner membrane proteins in the cytoplasm during their cotranslational translocation, which are specifically recognized and resolved by SecA in coordination with the proton motive force (PMF). Furthermore, SecA associates with 25% of secretory proteins with highly hydrophobic signal sequences at an early stage of translation and mediates their cotranslational transport. In contrast, the chaperone trigger factor (TF) delays SecA engagement on secretory proteins with weakly hydrophobic signal sequences, thus enforcing a posttranslational mode of their translocation. Our results elucidate the principles of SecA-driven cotranslational protein translocation and reveal a hierarchical network of protein export pathways in bacteria.
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Project description:Zea mays ribosome export associated1 is differentially expressed in 1 experiment(s);
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Project description:Folding of newly synthesized proteins to the native state is a major challenge within the crowded cellular environment, since non-productive interactions can lead to misfolding, aggregation and degradation1. Cells cope with this challenge by coupling synthesis with polypeptide folding and by employing molecular chaperones to safeguard folding already cotranslationally2. However, little is known about the final step of folding, the assembly of polypeptides into complexes, although most of the cellular proteome forms oligomeric assemblies3. In prokaryotes, a proof-of-concept study showed that assembly of heterodimeric luciferase is an organized cotranslational process, facilitated by spatially confined translation of the subunits encoded on a polycistronic mRNA4. In eukaryotes, however, fundamental differences such as rarity of polycistronic mRNAs and different chaperone constellations raise the question whether assembly is also coordinated with translation. Here we provide a systematic and mechanistic analysis of protein complex assembly in eukaryotes using ribosome profiling. We determined the in vivo nascent subunits interactions of 12 hetero-oligomeric protein complexes of Saccharomyces cerevisiae at near-residue resolution. We find 9 complexes assemble cotranslationally; the 3 complexes that do not show cotranslational interactions are regulated by dedicated assembly chaperones5-7. Cotranslational assembly often occurs uni-directionally, with one fully synthesized subunit engaging its nascent partner subunit(s), thereby counteracting its aggregation propensity. The onset of cotranslational subunit association coincides sharply with full exposure of the nascent interaction domain at the ribosomal tunnel exit. The action of the ribosome-associated Hsp70 chaperone Ssb8 is coordinated with assembly. Ssb transiently engages partially synthesized interaction domains, then dissociates before the onset of partner subunit association, presumably to prevent premature assembly interactions. Our study shows that cotranslational subunit association is a prevalent mechanism for hetero-oligomers assembly in yeast and indicates that translation, folding and assembly of protein complexes are integrated processes in eukaryotes.
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Project description:Cotranslational assembly of protein complexes in eukaryotes revealed by ribosome profiling
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Project description:Suppressed 60S ribosome exportation in maize ribosome export associated 1 mutant triggers inconsistent transcriptional and translational regulations
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Project description:The piRNA pathway is a conserved small RNA-based immune system that protects animal germ cell genomes from the harmful effects of transposon mobilisation. In Drosophila ovaries, most piRNAs originate from dual-strand clusters, which generate piRNAs from both genomic strands. Dual-strand clusters use non-canonical transcription mechanisms. Although transcribed by RNA polymerase II, cluster transcripts lack splicing signatures and polyA tails. mRNA processing is important for general mRNA export mediated by Nuclear export factor 1. Although UAP56, a component of the transcription and export complex, has been implicated in piRNA precursor export, it remains unknown how dual-strand cluster transcripts are specifically targeted for piRNA biogenesis by export from the nucleus to cytoplasmic processing centers. Here we report that dual-strand cluster transcript export requires CG13741/Bootlegger and the Drosophila Nuclear export factor family protein, Nxf3. Bootlegger is specifically recruited to piRNA clusters and in turn brings Nxf3. We find that Nxf3 specifically binds to piRNA precursors and is essential for their export to piRNA biogenesis sites, a process that is critical for germline transposon silencing. Our data shed light on how dual-strand clusters bypass canonical mRNA features to be specifically exported via Nxf3, ensuring proper piRNA production.
2019-07-27 | GSE133528 | GEO
Project description:By developing Akron-Seq, a novel approach that captures native 3' and 5' ends of capped and polyadenylated RNAs respectively, we show that canonical human mRNAs are subject to repeated, cotranslational, ribosome-phased, endonucleolytic cuts in a process that we term ribothrypsis.
2018-01-23 | GSE107838 | GEO
Project description:Nuclear export of mRNA is essential for eukaryotic cells to establish the flow of genetic information in the nucleus to protein synthesis in the cytoplasm. This transport process is highly regulated to ensure efficient and accurate gene expression. Viruses are well known for their ability to manipulate host gene expression. Here, we report that ORF10 of Kaposi’s sarcoma associated herpesvirus (KSHV), a nuclear DNA virus, inhibits mRNA export in a transcript-selective manner to control cellular gene expression. This export inhibitory effect of ORF10 requires the interaction with an RNA export factor, Rae1. Genome-wide analysis by RNA sequencing revealed the subset of cellular mRNAs whose nuclear export is blocked by ORF10. The 3’ untranslated regions (3’ UTRs) of ORF10-targeted transcripts confer their sensitivity to nuclear export inhibition by ORF10. In the context of KSHV replication, the interaction of ORF10 with Rae1 is important for the virus to express viral genes and produce infectious virions. Our results suggest that a nuclear replicating DNA virus can selectively interfere with RNA export through Rae1 to restrict host gene expression for optimal viral replication.
2016-10-18 | GSE82241 | GEO
Project description:As nascent polypeptides exit ribosomes, they are engaged by a series of processing, targeting and folding factors. Here we present a selective ribosome profiling strategy that enables global monitoring of when these factors engage polypeptides in the complex cellular environment. Studies of the Escherichia coli chaperone Trigger Factor (TF) reveal that, while TF can interact with many polypeptides, β-barrel outer membrane proteins are the most prominent substrates. Loss of TF leads to broad outer membrane defects and premature, cotranslational protein translocation. While in vitro studies suggested that TF is prebound to ribosomes waiting for polypeptides to emerge from the exit channel, we find that in vivo TF engages ribosomes only after ~100 amino acids are translated. Moreover, excess TF interferes with cotranslational removal of the N-terminal formyl methionine. Our studies support a triaging model in which proper protein biogenesis relies on the fine-tuned, sequential engagement of processing, targeting ad folding factors. Examination of translation in the Gram-negative bacterium Escherichia coli, as well as an analysis of the interactions between nascent chains and the molecular chaperone Trigger Factor.
2011-12-09 | E-GEOD-33671 | biostudies-arrayexpress