Gene- and Species-Specific Hox mRNA Translation by Ribosome Expansion Segments.
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ABSTRACT:
SUBMITTER: Leppek K
PROVIDER: S-EPMC7769145 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Ribosomes have been suggested to directly control gene regulation, however regulatory roles for ribosomal RNA (rRNA) remain largely unexplored. Expansion segments (ESs) consist of multitudes of tentacle-like rRNA structures extending from the core ribosome in eukaryotes. ESs are remarkably variable in sequence and size across eukaryotic evolution with a largely unknown function. In characterizing ribosome binding to a regulatory element within a Homeobox (Hox) 5’ UTR, we unexpectedly identify a modular stem-loop within this element that binds to a single ES, ES9S. Here, we described the raw data of the relative quantification proteomic analysis using tandem mass tag mass spectrometry of the ribonucleoprotein (RNP) complexes enriched from affinity pulldown of the 4xS1m-aptamer fusion constructs with the lysate components of C3H/10T1/2 cells and mouse embryo. 4xS1m pulldown is performed by combining mouse Hoxa9 mRNA elements (P3 and P4) with C3H/10T1/2 cell lysates, and by combining a9 IRES180 and an unrelated viral IRES with E11.5 FVB mouse embryo lysates. The aptamer alone served as a negative control. RNPs in the eluate were released by RNAse A treatment and were subjected to TMT 6plex labelling.
2020-11-19 | PXD021678 | Pride
Project description:Historically, the ribosome has been viewed as a complex ribozyme with constitutive rather than regulatory capacity in mRNA translation. Here we identify mutations of the Ribosomal Protein L38 (Rpl38) gene in mice exhibiting surprising tissue-specific patterning defects, including pronounced homeotic transformations of the axial skeleton. In Rpl38 mutant embryos, global protein synthesis is unchanged; however the translation of a select subset of Homeobox mRNAs is perturbed. Our data reveal that RPL38 facilitates 80S complex formation on these mRNAs as a regulatory component of the ribosome to confer transcript-specific translational control. We further show that Rpl38 expression is markedly enriched in regions of the embryo where loss-of-function phenotypes occur. Unexpectedly, a ribosomal protein (RP) expression screen reveals dynamic regulation of individual RPs within the vertebrate embryo. Collectively, these findings suggest that RP activity may be highly regulated to impart a new layer of specificity in the control of gene expression and mammalian development.
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Project description:The secondary structure of ribosomal RNA (rRNA) is largely conserved across all kingdoms of life. However, eukaryotes have evolved extra blocks of rRNA sequences, relative to those of prokaryotes, called expansion segments (ES). A thorough characterization of the potential roles of ES remains to be done, possibly because of limitations in the availability of robust systems to study rRNA mutants. We sought to systematically investigate the potential functions, if any, of the ES in 25S rRNA of Saccharomyces cerevisiae by deletion mutagenesis. We deleted 14 of the 16 different eukaryote-specific ES in yeast 25S rRNA individually and assayed their phenotypes. Our results show that all but two of the ES tested are necessary for optimal growth and are required for production of 25S rRNA, suggesting that ES play roles in ribosome biogenesis. Further, we classified expansion segments into groups that participate in early nucleolar, middle, and late nucleoplasmic steps of ribosome biogenesis, by assaying their pre-rRNA processing phenotypes. This study is the first of its kind to systematically identify the functions of eukaryote-specific expansion segments by showing that they play roles in specific steps of ribosome biogenesis. The catalog of phenotypes we identified, combined with previous investigations of the roles ribosomal proteins in large subunit biogenesis, leads us to infer that assembling ribosomes are composed of distinct RNA and protein structural neighborhood clusters that participate in specific steps of ribosome biogenesis.
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Project description:Regulation of gene expression at the translational level allows rapid adaptation of cellular proteomes to quickly changing environmental conditions and is thus central for prokaryotic organisms. Small non-coding RNAs (sRNAs) have been reported to effectively orchestrate translation control in bacteria and archaea mainly by targeting mRNAs by partial base complementarity. Here we report an unprecedented mechanism how sRNAs are capable of modulating protein biosynthesis in the halophilic archaeon Haloferax volcanii. By analyzing the ribosome-associated ncRNAs (rancRNAs) under different stress conditions we identified an intergenic sRNA, termed rancRNA_s194, that is primarily expressed during exponential growth under all tested conditions. By interaction with the ribosome rancRNA_s194 inhibits peptide bond formation and protein synthesis in vitro but appears to target a specific mRNA in vivo. The respective knock-out strain shows a reduced lag phase in media containing xylose as sole carbon source and outcompetes the wildtype cells under these conditions. Mass spectrometry, polysome profiling and mRNA binding competition experiments suggest that rancRNA_s194 prevents the cstA mRNA from being efficiently translated by H. volcanii ribosomes. These findings enlarge the regulatory repertoire of archaeal sRNAs in modulating post-transcriptional gene expression.
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Project description:In eukaryotic cells, protein synthesis is compartmentalized; mRNAs encoding secretory/membrane proteins are translated on endoplasmic reticulum (ER)-bound ribosomes, whereas mRNAs encoding cytosolic proteins are translated on free ribosomes. mRNA partitioning between the two compartments occurs via positive selection: free ribosomes engaged in the translation of signal sequence-encoding mRNAs are trafficked from the cytosol to the ER. After translation termination, ER-bound ribosomes are thought to dissociate, thereby completing a cycle of mRNA partitioning. At present, the physiological basis for termination-coupled ribosome release is unknown. To gain insight into this process, we examined ribosome and mRNA partitioning during the unfolded protein response, key elements of which include suppression of the initiation stage of protein synthesis and polyribosome breakdown. We report that unfolded protein response (UPR)-elicited polyribosome breakdown resulted in the continued association, rather than release, of ER-bound ribosomes. Under these conditions, mRNA translation in the cytosol was suppressed, whereas mRNA translation on the ER was sustained. Furthermore, mRNAs encoding key soluble stress proteins (XBP-1 and ATF-4) were translated primarily on ER-bound ribosomes. These studies demonstrate that ribosome release from the ER is termination independent and identify new and unexpected roles for the ER compartment in the translational response to induction of the unfolded protein response.
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Project description:The well established phenomenon of ribosome drop-off plays crucial roles in translational accuracy and nutrient starvation responses during protein translation. When cells are under stress conditions, such as amino acid starvation or aminoacyl-tRNA depletion due to a high level of recombinant protein expression, ribosome drop-off can substantially affect the efficiency of protein expression. Here we introduce a mathematical model that describes the effects of ribosome drop-off on the ribosome density along the mRNA and on the concomitant protein synthesis rate. Our results show that ribosome premature termination may lead to non-intuitive ribosome density profiles, such as a ribosome density which increases from the 5' to the 3' end. Importantly, the model predicts that the effects of ribosome drop-off on the translation rate are mRNA-specific, and we quantify their resilience to drop-off, showing that the mRNAs which present ribosome queues are much less affected by ribosome drop-off than those which do not. Moreover, among those mRNAs that do not present ribosome queues, resilience to drop-off correlates positively with the elongation rate, so that sequences using fast codons are expected to be less affected by ribosome drop-off. This result is consistent with a genome-wide analysis of S. cerevisiae, which reveals that under favourable growth conditions mRNAs coding for proteins involved in the translation machinery, known to be highly codon biased and using preferentially fast codons, are highly resilient to ribosome drop-off. Moreover, in physiological conditions, the translation rate of mRNAs coding for regulatory, stress-related proteins, is less resilient to ribosome drop-off. This model therefore allows analysis of variations in the translational efficiency of individual mRNAs by accounting for the full range of known ribosome behaviours, as well as explaining mRNA-specific variations in ribosome density emerging from ribosome profiling studies.
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Project description:Control of mRNA translation is a crucial regulatory mechanism used by bacteria to respond to their environment. In the soil bacterium Pseudomonas fluorescens, RimK modifies the C-terminus of ribosomal protein RpsF to influence important aspects of rhizosphere colonisation through proteome remodelling. In this study, we show that RimK activity is itself under complex, multifactorial control by the co-transcribed phosphodiesterase trigger enzyme (RimA) and a polyglutamate-specific protease (RimB). Furthermore, biochemical experimentation and mathematical modelling reveal a role for the nucleotide second messenger cyclic-di-GMP in coordinating these activities. Active ribosome regulation by RimK occurs by two main routes: indirectly, through changes in the abundance of the global translational regulator Hfq and directly, with translation of surface attachment factors, amino acid transporters and key secreted molecules linked specifically to RpsF modification. Our findings show that post-translational ribosomal modification functions as a rapid-response mechanism that tunes global gene translation in response to environmental signals.
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Project description:Zfrp8/PDCD2 is a highly conserved protein essential for stem cell maintenance in both flies and mammals. It is also required in fast proliferating cells such as cancer cells. Our previous studies suggested that Zfrp8 functions in the formation of mRNP (mRNA ribonucleoprotein) complexes and also controls RNA of select Transposable Elements (TEs). Here we show that in Zfrp8/PDCD2 knock down (KD) ovaries, specific mRNAs and TE transcripts show increased nuclear accumulation. We also show that Zfrp8/PDCD2 interacts with the (40S) small ribosomal subunit through direct interaction with RpS2 (uS5). By studying the distribution of endogenous and transgenic fluorescently tagged ribosomal proteins we demonstrate that Zfrp8/PDCD2 regulates the cytoplasmic levels of components of the small (40S) ribosomal subunit, but does not control nuclear/nucleolar localization of ribosomal proteins. Our results suggest that Zfrp8/PDCD2 functions at late stages of ribosome assembly and may regulate the binding of specific mRNA-RNPs to the small ribosomal subunit ultimately controlling their cytoplasmic localization and translation.
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