Structural and mechanistic insights into the function of Leishmania ribosome lacking a single pseudouridine modification.

Leishmania is the causative agent of cutaneous and visceral diseases affecting millions of individuals worldwide. Pseudouridine (Ψ), the most abundant modification on rRNA, changes during the parasite life cycle. Alterations in the level of a specific Ψ in helix 69 (H69) affected ribosome function. To decipher the molecular mechanism of this phenotype, we determine the structure of ribosomes lacking the single Ψ and its parental strain at ∼2.4-3 Å resolution using cryo-EM. Our findings demonstrate the significance of a single Ψ on H69 to its structure and the importance for its interactions with helix 44 and specific tRNAs. Our study suggests that rRNA modification affects translation of mRNAs carrying codon bias due to selective accommodation of tRNAs by the ribosome. Based on the high-resolution structures, we propose a mechanism explaining how the ribosome selects specific tRNAs.

Mutations in RPS19 may affect ribosome function and biogenesis in Diamond Blackfan anemia.

Ribosomes, the cellular organelles translating the genetic code to proteins, are assemblies of RNA chains and many proteins (RPs) arranged in precise fine-tuned interwoven structures. Mutated ribosomal genes cause ribosomopathies, including Diamond Blackfan anemia (DBA, a rare heterogeneous red-cell aplasia connected to ribosome malfunction) or failed biogenesis. Combined bioinformatical, structural, and predictive analyses of potential consequences of possibly expressed mutations in eS19, the protein product of the highly mutated RPS19, suggest that mutations in its exposed surface could alter its positioning during assembly and consequently prevent biogenesis, implying a natural selective strategy to avoid malfunctions in ribosome assembly. A search for RPS19 pseudogenes indicated > 90% sequence identity with the wild-type, hinting at its expression in cases of absent or truncated gene products.

Cryo-EM structure of the ancient eukaryotic ribosome from the human parasite Giardia lamblia.

Giardiasis is a disease caused by the protist Giardia lamblia. As no human vaccines have been approved so far against it, and resistance to current drugs is spreading, new strategies for combating giardiasis need to be developed. The G. lamblia ribosome may provide a promising therapeutic target due to its distinct sequence differences from ribosomes of most eukaryotes and prokaryotes. Here, we report the cryo-electron microscopy structure of the G. lamblia (WB strain) ribosome determined at 2.75 Å resolution. The ribosomal RNA is the shortest known among eukaryotes, and lacks nearly all the eukaryote-specific ribosomal RNA expansion segments. In contrast, the ribosomal proteins are typically eukaryotic with some species-specific insertions/extensions. Most typical inter-subunit bridges are maintained except for one missing contact site. Unique structural features are located mainly at the ribosome's periphery. These may be exploited as target sites for the design of new compounds that inhibit selectively the parasite's ribosomal activity.

Genome instability drives epistatic adaptation in the human pathogen Leishmania.

How genome instability is harnessed for fitness gain despite its potential deleterious effects is largely elusive. An ideal system to address this important open question is provided by the protozoan pathogen Leishmania, which exploits frequent variations in chromosome and gene copy number to regulate expression levels. Using ecological genomics and experimental evolution approaches, we provide evidence that Leishmania adaptation relies on epistatic interactions between functionally associated gene copy number variations in pathways driving fitness gain in a given environment. We further uncover posttranscriptional regulation as a key mechanism that compensates for deleterious gene dosage effects and provides phenotypic robustness to genetically heterogenous parasite populations. Finally, we correlate dynamic variations in small nucleolar RNA (snoRNA) gene dosage with changes in ribosomal RNA 2'-O-methylation and pseudouridylation, suggesting translational control as an additional layer of parasite adaptation. Leishmania genome instability is thus harnessed for fitness gain by genome-dependent variations in gene expression and genome-independent compensatory mechanisms. This allows for polyclonal adaptation and maintenance of genetic heterogeneity despite strong selective pressure. The epistatic adaptation described here needs to be considered in Leishmania epidemiology and biomarker discovery and may be relevant to other fast-evolving eukaryotic cells that exploit genome instability for adaptation, such as fungal pathogens or cancer.

ATR signalling mediates the prosurvival function of phospho-NPM against PIDDosome mediated cell death.

We uncover a novel non-canonical function of ATR kinase in the control of PIDDosome activation, and show that under normal cellular conditions involving no replication stress, ATR kinase controls the phosphorylation of cellular NPM via pChk1 as well as the two phosphatases, PPM1D and PP1ß. We show that pNPM triggers the dissociation of NPM from PIDD, preventing the cell from undergoing caspase 2 mediated cell death via PIDDosome, thereby acting as an endogenous negative regulator of PIDDosome activation. pChk1 interaction with NPM is abrogated following ATR kinase inhibition, leading to the drop in nucleoplasmic/chromatin pNPM level, inducing PIDD. Consistent with this mechanism, the phosphomimic mutants of Chk1 and NPM become refractory to ATR/pChk1 kinase inhibition by avoiding PIDDosome signalling.

ATR kinase regulates its attenuation via PPM1D phosphatase recruitment to chromatin during recovery from DNA replication stress signalling.

In eukaryotes, in response to replication stress, DNA damage response kinase, ATR is activated, whose signalling abrogation leads to cell lethality due to aberrant fork remodelling and excessive origin firing. Here we report that inhibition of ATR kinase activity specifically during replication stress recovery results in persistent ATR signalling, evidenced by the presence of ATR-dependent phosphorylation marks (gamma H2AX, pChk1 and pRad17) and delayed cell cycle re-entry. Further, such disruption of ATR signalling attenuation leads to double-strand breaks, fork collapse and thereby 'replication catastrophe'. PPM1D phosphatase, a nucleolar localized protein, relocates to chromatin during replication stress and reverts back to nucleolus following stress recovery, under the control of ATR kinase action. Inhibition of ATR kinase activity, specifically during post replication stress, triggers dislodging of the chromatin-bound PPM1D from nucleus to cytoplasm followed by its degradation, thereby leading to persistence of activated ATR marks in the nuclei. Chemical inhibition of PPM1D activity or SiRNA mediated depletion of the protein during post replication stress recovery 'phenocopies' ATR kinase inhibition by failing to attenuate ATR signalling. Collectively, our observations suggest a novel role of ATR kinase in mediating its own signal attenuation via PPM1D recruitment to chromatin as an essential mechanism for restarting the stalled forks, cell-cycle re-entry and cellular recovery from replication stress.