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:Multiple processes exist in a cell to ensure continuousproduction of essential proteins either through cap-dependent or cap-independent translation processes. Viruses depend on the host translation machinery for viral protein synthesis. Therefore, viruses have evolved clever strategies to utilize the host translation machinery. Earlier studies have shown that genotype1 hepatitis E virus (g1-HEV) utilizes both cap-dependent and cap-independent translation machineries for its replication and proliferation. Cap-independent translation in g1-HEV is driven by an eighty seven nucleotide-long RNA element which acts as a noncanonical, internal ribosome entry site like (IRESl) element. Here, we have identified the RNA-protein interactome of the HEV IRESl element and characterized the functional significance of some of its components. Our study reveals indispensable roles of host ribosomal proteinRPL5 and DHX9 (RNA helicase A) in mediating efficient translation from the IRESlelement and establish the function of HEV IRESl as a bonafide internal ribosome entry site.
Project description:Programmed ribosomal frameshifting is the key event during translation of the SARS-CoV-2 RNA genome allowing synthesis of the viral RNA-dependent RNA polymerase and downstream viral proteins. Here we present the cryo-EM structure of the mammalian ribosome in the process of translating viral RNA paused in a conformation primed for frameshifting. We observe that the viral RNA adopts a pseudoknot structure lodged at the mRNA entry channel of the ribosome to generate tension in the mRNA that leads to frameshifting. The nascent viral polyprotein that is being synthesized by the ribosome paused at the frameshifting site forms distinct interactions with the ribosomal polypeptide exit tunnel. We use biochemical experiments to validate our structural observations and to reveal mechanistic and regulatory features that influence the frameshifting efficiency. Finally, a compound previously shown to reduce frameshifting is able to inhibit SARS-CoV-2 replication in infected cells, establishing coronavirus frameshifting as target for antiviral intervention.
Project description:We conducted an in vivo genome-wide CRISPR activation screen to identify genes that accelerate distal metastasis by breast cancer patient-derived circulating tumor cells (CTCs) following direct intravascular inoculation in mice. Regulators of translation and ribosomal proteins were prominent among these, and expression of RPL15, a component of the large ribosome subunit, was sufficient to increase metastatic growth in multiple organs. RPL15 overexpression selectively increases translation of other ribosomal proteins and cell cycle regulators. Unsupervised analysis of single-cell RNA sequencing of freshly-isolated CTCs from breast cancer patients identifies a subset with strong ribosomal and protein translation signatures, correlated with increased proliferative markers, epithelial markers and poor clinical outcome. Thus, ribosome protein expression identifies an aggressive subset of CTCs, whose therapeutic targeting may suppress metastatic progression.