Ontology highlight
ABSTRACT:
PROVIDER: PRJNA785998 | ENA |
REPOSITORIES: ENA
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Project description:Ribosome-associated quality control (RQC) represents a rescue pathway in eukaryotic cells triggered upon translational stalling. Collided ribosomes are recognized for subsequent dissociation followed by degradation of nascent peptides. However, endogenous RQC-inducing sequences and the mechanism underlying the ubiquitin-dependent ribosome dissociation remain poorly understood. Here, we identified the SDD1 mRNA from S. cerevisiae as an endogenous RQC substrate and reveal its mRNA and nascent peptide dependent stalling mechanism by mutational and cryo-EM analyses. In vitro translation of SDD1 mRNA enabled the reconstitution of Hel2-dependent poly-ubiquitination of collided di- and preferentially tri-ribosomes. Distinct trisome architecture was visualized by cryo-EM and provides the structural basis for more efficient recognition by Hel2 over disomes. Subsequently, the Slh1 helicase subunit of the RQC trigger (RQT) complex preferentially dissociates the first stalled poly-ubiquitinated ribosome in an ATP-dependent manner. Together, these findings provide fundamental mechanistic insights into RQC and its physiological role in maintaining cellular protein homeostasis.
2020-01-27 | GSE131214 | GEO
Project description:The genetic code is an abstraction of how mRNA codons and tRNA anticodons molecularly interact during protein synthesis; the stability and regulation of this interaction remains largely unexplored. Here, we quantitatively characterized the expression of m
2014-08-08 | E-MTAB-2326 | biostudies-arrayexpress
Project description:Precise control of protein synthesis by engineering sequence elements in 5’ untranslated region (5’UTR) remains a fundamental challenge. To accelerate our understanding of cis-regulatory code embedded in 5’UTR, we devised massively parallel reporter assays from a synthetic mRNA library composed of over one million 5’UTR variants. A completely randomized 10-nucleotide sequence preceding an upstream open reading frame (uORF) and downstream GFP leads to a broad range of mRNA translatability and stability in mammalian cells. While efficient translation protects mRNA from degradation, uORF translation triggers mRNA decay in a UPF1-dependent manner. We also identified translational inhibitory elements in 5’UTR with G-quadruplex as a mark for mRNA decay in the P-body. Unexpectedly, an unstructured A-rich element in 5’UTR, while enabling cap-independent translation, destabilizes mRNAs in the absence of translation. Our results not only expose diverse sequence features of 5’UTR in controlling mRNA translatability, but also reveal ribosome-dependent and -independent mRNA surveillance pathways.
2020-06-01 | GSE145046 | GEO
Project description:The control of mRNA stability plays a central role in regulating gene expression patterns. While much is known about the roles of 5´ and 3´ untranslated regions in the mRNA stability control, the impact of protein-coding sequences on mRNA stability had been obscure. Recently, several groups reported that codon composition in the ORF affects mRNA deadenylation and degradation rates in a translation-dependent manner. Hence, codons define not only the amino acid sequences to be synthesized but also the stability of mRNAs. However, how 61 codons differently affect mRNA stability remains unclear. Besides, aberrant stalling of the ribosome induces ribosome quality control (RQC) and No-go decay. The relationship between the two co-translational mRNA decay pathways is not systematically analyzed. To precisely characterize the effects of 61 codons on mRNA stability, we developed a simplified reporter system that allows detection of the effect of every single codon on mRNA stability in zebrafish embryos. Using this system, we show that the effect of codons on mRNA stability is partially but significantly correlated with the translation elongation rate and tRNA abundance. Interestingly, the codon effect is still maintained in zebrafish embryos lacking Znf598, an essential mediator of RQC and NGD. Znf598-dependent NGD targets a particular type of ribosome stalling but has limited impact on endogenous mRNA stability. Our study thus defines two related co-translational mRNA decay pathways during animal development.
2020-05-05 | GSE133392 | GEO
Project description:Ribosome-associated quality control (RQC) represents a rescue pathway in eukaryotic cells triggered upon translational stalling. Collided ribosomes are recognized for subsequent dissociation followed by targeting of the faulty nascent peptides for degradation. However, endogenous RQC inducing sequences and the mechanism underlying the ubiquitin-dependent ribosome dissociation remain poorly understood. Here, we identified the SDD1 mRNA from S. cerevisiae as an endogenous RQC substrate and reveal its mRNA and nascent peptide dependent stalling mechanism by mutational and cryo-EM analyses. In vitro translation of SDD1 mRNA enabled the reconstitution of Hel2-dependent poly-ubiquitination of colliding di- and preferentially tri-ribosomes. Their distinct architecture is visualized by cryo-EM and provides the structural basis for more efficient recognition by Hel2 over disomes. Subsequently, the Slh1/Rqt2 helicase subunit of the RQC trigger (RQT) complex preferentially dissociates the first stalled poly-ubiquitinated ribosome in an ATP dependent manner. Together, these findings provide fundamental mechanistic insights into RQC and its physiological role in dealing with endogenous substrates to maintain cellular protein homeostasis.
2020-02-24 | PXD017196 | Pride
Project description:Early vertebrate embryogenesis is characterized by extensive post-transcriptional regulation during the maternal-to-zygotic transition. The N6-methyladenosine (m6A) modifications on mRNA has been shown to affect both translation and stability of transcripts. Here we investigate the m6A topology during early vertebrate embryogenesis and its association with RNA stability, translation efficiency and effect on miR-430 degradation kinetics. Notably, we find a strong association of m6A with cytoplasmic polyadenylation and translational efficiency prior to zygotic genome activation. Genes required for zygotic genome activation such as nanog and pou5f3 display dynamic m6A levels. After zygotic genome activation m6A is associated with improved stability and dampens the effect of miR-430 mediated degradation. Through sequence analyses we identified enrichment of motifs for RNA binding proteins involved in translational regulation and RNA degradation. We propose a role for m6A in multiple mRNA regulatory mechanisms, for the first time in an in vivo system and improve our understanding of the combinatorial code behind the complex post transcriptional regulation of reprogramming during early vertebrate development.
2017-05-01 | GSE89815 | GEO
Project description:The contributions of phosphorylation-mediated signaling networks to colon cancer metastasis are poorly defined. To better understand the role of constitutive signaling alterations in cancer progression, the global phosphoproteomes of the SW480 and SW620 cell lines were compared by LC-MS/MS. These patient-matched cell lines were derived from a primary colon tumor and a lymph node metastasis, respectively. Network analysis of the significantly altered phosphosites revealed differential regulation in cellular adhesion, mitosis, and mRNA translational machinery. Among the latter group were sites on phosphoproteins with known roles in mRNA biogenesis and splicing, transport through the nuclear pores, initiating translation, as well as mRNA stability and degradation. Though alterations in these processes have been associated with oncogenic transformation, control of mRNA stability has typically not been associated with cancer progression. Notably, the single phosphosite with the greatest relative change in SW620 was Ser2 on eIF2S2, suggesting that SW620 cells translate faster or with greater efficiency than SW480 cells. These broad changes in the regulation of translation also occur without over-expression of eIF4E. Altogether, this work shows that metastatic cells exhibit constitutive changes to the phosphoproteome, and that mRNA stability and translational efficiency may be important targets of deregulation during cancer progression.
2017-04-01 | MSV000080864 | MassIVE
Project description:In the process of translation, ribosomes first bind to mRNAs (translation initiation) and then move along the mRNA (elongation) to synthesize proteins. Elongation pausing is deemed highly relevant to co-translational folding of nascent peptides and the functionality of protein products, which positioned the evaluation of elongation speed as one of the central questions in the field of translational control. By employing three types of RNA-seq methods, we experimentally and computationally resolved elongation speed at individual gene level and under physiological condition in human cells. We proposed the elongation velocity index (EVI) as a relative measure and successfully distinguished slow-translating genes from the background translatome. The proteins encoded by the low-EVI genes are more stable than the proteome background. In normal cell and lung cancer cell comparisons, we found that the relatively slow-translating genes are relevant to the maintenance of malignant phenotypes. In addition, we identified cell-specific slow-translating codons, which may serve as a causal factor of elongation deceleration. We sequenced mRNA, translating mRNA (RNC-mRNA) and ribosome footprints in normally growing HeLa cells.
2016-02-09 | E-GEOD-46613 | biostudies-arrayexpress
Project description:Several studies have reported that functionally related genes exhibit similar codon usage, which has been shown to impact protein production by the control of mRNA decay or translation. However, codon-mediated control and their relationship to growth conditions have been intensely debated and remain unclear. Here, we investigate the post-transcriptional gene expression control mechanisms employed in cells in proliferation or quiescence. These investigations show that in proliferative conditions we see that subsets of mRNAs with increased mRNA stability are associated with enhanced translation. However, this is not the case in quiescence, where mRNA stability is not linked to changes in translation. In addition, we observe that while G/C-ending codons are more frequently used in both conditions, there is a shift towards A/U-ending codons in proliferation, and this is accompanied by corresponding changes in tRNAs and translational output.
2022-08-01 | GSE179642 | GEO
Project description:Translation and mRNA degradation are intimately connected, yet the mechanisms that regulate them are not fully understood. Here we examine the regulation of translation and mRNA stability in mouse embryonic stem cells (ESCs) and during differentiation. In contrast to previous reports, we found that transcriptional changes account for most of the molecular changes during ESC differentiation. Within ESCs translation level and mRNA stability are positively correlated. The RNA-binding protein DDX6 has been implicated in processes involving both translational repression and mRNA destabilization; in yeast DDX6 connects codon optimality and mRNA stability and in mammals DDX6 is involved in microRNA-mediated repression. We generated DDX6 KO ESCs and found that while there was minimal connection between codon usage and stability changes, the loss of DDX6 leads to the translational depression of microRNA targets. Surprisingly, the translational derepression of microRNA targets occurs without affecting mRNA stability. Furthermore, DDX6 KO ESCs share overlapping phenotypes and global molecular changes with ESCs that completely lack all microRNAs. Together our results demonstrate that the loss of DDX6 decouples the two forms of microRNA induced repression and emphasize that translational repression by microRNAs is underappreciated.
2018-07-25 | GSE112760 | GEO