MRNA regions where 80S ribosomes pause during translation elongation in vivo interact with protein uS19, a component of the decoding site
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ABSTRACT:
PROVIDER: PRJNA526909 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR8723631.fastq.gz | Fastqsanger.gz | |||
| SRR8723632.fastq.gz | Fastqsanger.gz | |||
| SRR8723633.fastq.gz | Fastqsanger.gz | |||
| SRR8723634.fastq.gz | Fastqsanger.gz | |||
| SRR8723635.fastq.gz | Fastqsanger.gz |
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Project description:80S-ribosome bound uS19 translatome-wide crosslinking sites in human mRNAs revealed by PAR-CLIP
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Project description:In eukaryotic ribosomes, the conserved protein uS19, formerly known as S15, extends with its C-terminal tail to the decoding site. The cross-linking of uS19 to the A site codon has been detected using synthetic mRNAs bearing 4-thiouridine (s4U) residues. Here, we showed that the A-site tRNA prevents this cross-linking and that the P site codon does not contact uS19. Next, we focused on determining uS19-mRNA interactions in vivo by applying the photoactivatable-ribonucleoside enhancing cross-linking and immunoprecipitation method to a stable HEK293 cell line producing FLAG-tagged uS19 and grown in a medium containing s4U. We found that when translation was stopped by cycloheximide, uS19 was efficiently cross-linked to mRNA regions with a high frequency of Glu, Lys and, more rarely, Arg codons. The results indicate that the complexes, in which the A site codon is not involved in the formation of the mRNA-tRNA duplex, are present among the cycloheximide-arrested 80S complexes, which implies pausing of elongating ribosomes at the above mRNA regions. Thus, our findings demonstrate that the human ribosomal protein uS19 interacts with mRNAs during translation elongation and highlight the regions of mRNAs where ribosome pausing occurs, bringing new structural and functional insights into eukaryotic translation in vivo.
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Project description:eIF3 is a multi-subunit complex thought to execute numerous functions in canonical translation initiation, including mRNA recruitment to the 40S ribosome, scanning for the start codon, and inhibition of 60S subunit joining 1–3. eIF3 was also found to interact with 40S and 60S ribosomal proteins and translation elongation factors 4, but a direct involvement in translation elongation has never been demonstrated. Using selective ribosome profiling, we made the unexpected observation that eIF3 remains bound to post-initiation 80S ribosomes, followed by release after translation of ~50 codons. Furthermore, eIF3 deficiency reduces early ribosomal elongation speed, particularly on mRNAs encoding proteins associated with membrane-associated functions, resulting in defective synthesis of their encoded proteins and abnormal mitochondrial and lysosomal physiology. Accordingly, heterozygous eIF3e+/- knockout mice accumulate giant mitochondria in skeletal muscle and show a progressive decline in muscle strength with age. Hence, in addition to its canonical role in translation initiation, eIF3 interacts with 80S ribosomes to enhance, at the level of early elongation, the synthesis of proteins with membrane-associated functions, an activity that is critical for normal muscle health.
2020-06-15 | GSE149697 | GEO
Project description:Terminal oligopyrimidine motif-containing (TOP) mRNAs encode all ribosomal proteins in mammals and are regulated to tune ribosome synthesis to cell state. Previous studies implicate LARP1 in 40S- or 80S-ribosome complexes that repress and stabilize TOP mRNAs. However, a mechanistic understanding of how LARP1 and TOP mRNAs interact with ribosomes to coordinate TOP mRNA outcomes is lacking. Here, we show that LARP1 senses the cellular supply of ribosomes by directly binding non-translating 80S ribosomes. Cryo-EM structures reveal a previously uncharacterized domain of LARP1 bound to and occluding the 40S mRNA channel and mutations at the LARP1-ribosome interface block formation of the 40S/80S-LARP1-TOP complexes. Free cytosolic ribosomes induce sequestration of TOP mRNAs in repressed 80S-LARP1-TOP complexes independent of alterations in mTOR signaling. Together, this work demonstrates a ribosome-sensing function of LARP1 that allows it to tune ribosome protein synthesis to the availability of free ribosomes.
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2024-06-23 | PXD051211 | Pride
Project description:Cells can respond to stalled ribosomes by sensing ribosome collisions and employing quality control pathways. How ribosome stalling is resolved without collisions, however, has remained elusive. Here, focusing on non-colliding stalling exhibited by decoding-defective ribosomes, we identified Fap1 as a stalling sensor triggering 18S non-functional rRNA decay via poly-ubiquitination of uS3. Ribosome profiling revealed an enrichment of Fap1 at the translation initiation site but also association with elongating individual ribosomes. Cryo-EM structures of Fap1-bound ribosomes elucidated Fap1 probing the mRNA simultaneously at both the entry and exit channels suggesting a mRNA stasis sensing activity, and Fap1 sterically hinders formation of canonical collided di-ribosomes. Our findings indicate that individual stalled ribosomes are the potential signal for ribosome dysfunction, leading to accelerated turnover of the ribosome itself.
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Project description:Fully assembled ribosomes exist in two populations: polysomes and monosomes. While the former has been studied extensively, to what extent translation occurs on monosomes and its importance for overall translational output remains controversial. Here, we used ribosome profiling to examine the translational status of 80S monosomes in Saccharomyces cerevisiae. We found that the vast majority of 80S monosomes are elongating, not initiating. Further, most mRNAs exhibit some degree of monosome occupancy, with monosomes predominating on nonsense-mediated decay (NMD) targets, upstream open reading frames (uORFs), canonical ORFs shorter than ~590 nucleotides and ORFs for which the total time required to complete elongation is substantially shorter than that required for initiation. Importantly, mRNAs encoding low-abundance regulatory proteins tend to be enriched in the monosome fraction. Our data highlight the importance of monosomes for the translation of highly regulated mRNAs. We examined the translational status of single 80S ribosomes using ribosome profiling, and compared these monosome footprints to both polysome ribosome footprints and general ribosome profiling. RNASeq libraries were also prepared from the overall sample input.
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Project description:Mass spectrometry data of 80S ribosomes in nine mouse tissues and different developmental stages of testes
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Project description:Translation is initiated by binding of the eIF4F complex to the 5' cap of the mRNA, which is followed by scanning of the initiation codon by scanning ribosomes. Here we demonstrate that the ASC-1 complex (ASCC), which was previously shown to promote the dissociation of colliding 80S ribosomes, associates with the scanning ribosomes to regulate translation initiation. Sel-TCP-seq analysis revealed that ASCC3, a subunit of ASCC with a helicase domain, localizes predominantly to the 5' untranslated region of mRNAs. Knockdown of ASCC3 resulted in reduced translation efficiency associated with reduced 43S preinitiation complex (PIC) loading and a reduced speed of scanning ribosomes. In addition, depletion of the ubiquitin ligase ZNF598, a sensor of collided 80S ribosomes, also reduces the PIC loading and speed of scanning ribosomes. Our results have thus revealed that ASCC is required not only for dissociation of colliding 80S ribosomes, but also for efficient translation initiation by scanning ribosomes.
2023-03-31 | GSE186502 | GEO
Project description:Recent studies have revealed that the mRNA translation is punctuated by ribosomal pauses through the body of transcripts. However, little is known about its physiological significance and regulatory aspects. Here we present a multi-dimensional ribosome profiling approach to quantify the dynamics of initiation and elongation of 80S ribosomes across the entire transcriptome in mammalian cells. We show that a subset of transcripts have a significant pausing of 80S ribosome around the start codon, creating a major barrier to the commitment of translation elongation. Intriguingly, genes encoding ribosome proteins themselves exhibit an exceptionally high initiation pausing on their transcripts. Our studies also reveal that the initiation pausing is dependent on the 5’ untranslated region (5’ UTR) of mRNAs and subject to the regulation of mammalian target of rapamycin complex 1 (mTORC1). Thus, the initiation pausing of 80S ribosome represents a novel regulatory step in translational control mediated by nutrient signaling pathway. Untreated TSC2 WT MEFs, TSC2 KO MEFs and TSC2 WT MEFs, TSC2 KO MEFs treated with 20nM rapamycin for 30 minutes or 3hours were harvested for ribosme profiling. The fraction samples were pooled into three groups based on velocity sedimentation: single ribosome fraction (Small group), fractions with 2 ~ 4 ribosomes (Medium group), and the one with ≥5 ribosomes (Large group). RNA were extracted from the whole cell lysis and each fraction group.
2011-11-12 | E-GEOD-25331 | biostudies-arrayexpress