Project description:We report that 2’-O-methylation levels at subset of positions in human ribosomal RNA are variable between cell types and conditions, and that the degree of methylation at distinct sites is responsive to key cellular pathways. MYC overexpression results in increased methylation at a particular rRNA site (18S:C174). We find that this methylation is important for modulating translation of distinct mRNAs, leading to phenotypic changes including modulation of cell proliferation rate.
Project description:We report that 2’-O-methylation levels at subset of positions in human ribosomal RNA are variable between cell types and conditions, and that the degree of methylation at distinct sites is responsive to key cellular pathways. MYC overexpression results in increased methylation at a particular rRNA site (18S:C174). We find that this methylation is important for modulating translation of distinct mRNAs, leading to phenotypic changes including modulation of cell proliferation rate. Thus, differential rRNA 2’-O-methylation can give rise to ribosomes with specialized function.
Project description:The PI3K-Akt-mTOR signaling pathway is a master regulator of RNA translation. Pharmacological inhibition of this pathway preferentially and coordinately suppresses, in a 4EBP1/2-dependent manner, translation of mRNAs encoding ribosomal proteins. However, it remains unclear whether mTOR-4EBP1/2 is the exclusive translational regulator of this group of genes, and furthermore, systematic searches for novel translational modulators have been immensely challenging due to difficulties in scaling existing RNA translation profiling assays. Here, we developed a rapid and highly scalable approach for gene-specific quantitation of RNA translation, termed Targeted Profiling of RNA Translation (TPRT). We applied this technique in a chemical screen for novel translational modulators, and identified numerous preclinical and clinical therapeutic compounds, with diverse nominal targets, that preferentially suppress translation of ribosomal proteins. Surprisingly, some of these compounds act in a manner that bypasses canonical regulation by mTOR-4EBP1/2. Instead, these compounds exert their translational effects in a manner that is dependent upon GCN2-eIF2α, a central signaling axis within the integrated stress response. Furthermore, we were also able to identify metabolic perturbations that also suppress ribosomal protein translation in an mTOR-independent manner. Together, we describe a novel translational assay that is directly applicable to large-scale RNA translation studies, and that enabled us to identify a non-canonical, mTOR-independent mode for translational regulation of ribosomal proteins.
Project description:Diamond Blackfan Anemia (DBA) is associated with developmental defects and profound anemia. Mutations in genes encoding a ribosomal protein of the small (e.g. Rps19) or large (e.g. Rpl11) ribosomal subunit are found in over half of these patients. The mutations cause ribosomal haploinsufficiency, which reduces overall translation efficiency of cellular mRNAs. We reduced expression of *Rps19* or *Rpl11* in mouse erythroblasts and investigated mRNA polyribosome association, which revealed deregulated translation initiation of specific transcripts. Among these were *Bag1*, encoding a Hsp70 co-chaperone, and *Csde1*, encoding an RNA binding protein, both expressed at increased levels in erythroblasts. Their translation initiation is cap-independent and starts from an internal ribosomal entry site (IRES), which appeared sensitive to knock down of Rps19 or Rpl11. Mouse embryos lacking Bag1 die at embryonic day E13.5 with reduced erythroid colony forming cells in the fetal liver, and low Bag1 expression impairs erythroid differentiation in vitro. Reduced expression of Csde1 impairs proliferation and differentiation of erythroid blasts. Protein but not mRNA expression of *BAG1* and *CSDE1* was reduced in erythroblasts cultured from DBA patients. Our data suggest that impaired IRES-mediated translation of mRNAs expressed at increased levels in erythroblasts contributes to the erythroid phenotype of DBA. 3 biological replicates of erythroblasts treated with different shRNA were used for polyribosomal sucrose gradients; RNA was extracted from gradients in 2 samples - mRNA associated with polyribosomes (poly) and the rest (sub).
Project description:Ribosomes are often seen as monolithic machines produced from uniformly regulated genes. However, in yeast most ribosomal proteins come from duplicated genes. Here, we demonstrate that gene duplication may serve as an adaption mechanism modulating the global proteome through the differential expression of ribosomal proteins paralogs after exposure to stress. Our data indicate that the yeast paralog pair of the ribosomal protein L7/uL30 produces two differentially acetylated proteins. Under normal conditions most ribosomes incorporate the hypo-acetylated major form favoring the translation of genes with short open reading frames. Exposure to drugs, on the other hand, increases the production of ribosomes carrying the hyper-acetylated minor paralog that increases translation of long reading frames. Many of these long genes encode cell wall proteins that increase drug resistance in a programed change in translation equilibrium. Together the data reveal a mechanism of translation control through the differential fates of near-identical ribosomal protein isoforms.
Project description:Translational regulation plays a critical role in controlling the environmental responses of diverse bacterial species. In addition to existing, well-established regulatory pathways, the alteration of ribosome function by specific posttranslational modification represents a potential further mechanism for translational control. Although numerous ribosomal proteins undergo diverse posttranslational modifications, in most cases the functional and physiological significance of these changes remains unclear. The datasets presented here support our discovery that the widespread ribosomal modification protein RimK functions as a previously uncharacterized global controller of bacterial mRNA translation. RimK modification of the ribosomal protein RpsF changes both the stability and function of the bacterial ribosome and alters the composition of the bacterial proteome. In addition to multiple ribosomal proteins, rimK deletion in the biocontrol bacterium Pseudomonas fluorescens leads to significantly reduced levels of the important translational regulators Hfq and RsmE. This in turn leads to increased production of ABC transporters, stress response proteins and non-ribosomal peptide synthetases. Deletion of rimK compromises wheat rhizosphere colonization by P. fluorescens and significantly reduces virulence in the phytopathogen Pseudomonas syringae. Critically, the expression of P. fluorescens rimK is not constitutive, but varies throughout wheat rhizosphere colonisation, peaking during initial niche colonisation and declining in the established rhizosphere. Differential modification of the ribosome through temporal control of RimK expression represents a novel regulatory mechanism by which Pseudomonas fine-tunes its proteome to appropriately respond to the surrounding environment.