Cohen-Chalamish, S. | Search bioRxiv

bioRxiv2021

Post-transcriptional regulation of Leishmania fitness gain

The protozoan parasite Leishmania donovani causes fatal human visceral leishmaniasis in absence of treatment. Genome instability has been recognized as a driver in Leishmania fitness gain in response to environmental change or chemotherapy. How genome instability generates beneficial phenotypes despite potential deleterious gene dosage effects is unknown. Here we address this important open question applying experimental evolution and integrative systems approaches on parasites adapting to in vitro culture. Phenotypic analyses of parasites from early and late stages of culture adaptation revealed an important fitness tradeoff, with selection for accelerated growth in promastigote culture (fitness gain) impairing infectivity (fitness costs). Comparative genomics, transcriptomics and proteomics analyses revealed a complex regulatory network driving parasite fitness, with genome instability causing highly reproducible, gene dosage-dependent changes in protein abundance linked to post-transcriptional regulation. These in turn were associated with a gene dosage-independent reduction in abundance of flagellar transcripts and a coordinated increase in abundance of coding and non-coding RNAs implicated in ribosomal biogenesis and protein translation. We correlated differential expression of small nucleolar RNAs (snoRNAs) with changes in rRNA modification, providing first evidence that Leishmania fitness gain in culture may be controlled by post-transcriptional and epitranscriptomic regulation. Our findings propose a novel model for Leishmania fitness gain in culture, where differential regulation of mRNA stability and the generation of fitness-adapted ribosomes may potentially filter deleterious from beneficial gene dosage effects and provide proteomic robustness to genetically heterogenous, adapting parasite populations. This model challenges the current, genome-centric approach to Leishmania epidemiology and identifies the Leishmania transcriptome and non-coding small RNome as potential novel sources for the discovery of biomarkers that may be associated with parasite phenotypic adaptation in clinical settings.

microbiology↗

bioRxiv2020

Lytic Reactivation of the Kaposis sarcoma-associated herpesvirus (KSHV) is Accompanied by Major Nucleolar Alterations

The nucleolus is a sub-nuclear compartment whose primary function is the biogenesis of ribosomal subunits. Certain viral infections affect the morphology and composition of the nucleolar compartment and influence ribosomal RNA (rRNA) transcription and maturation. However, no description of nucleolar morphology and function during infection with Kaposis sarcoma-associated herpesvirus (KSHV) is available to date. Using immunofluorescence microscopy, we documented extensive destruction of the nuclear and nucleolar architecture during lytic reactivation of KSHV. This was manifested by redistribution of key nucleolar proteins, including the rRNA transcription factor, UBF, the essential pre-rRNA processing factor Fibrillarin, and the nucleolar multifunctional phosphoproteins Nucleophosmin (NPM1) and Nucleolin. Distinct delocalization patterns were evident; certain nucleolar proteins remained together whereas others dissociated, implying that nucleolar proteins undergo nonrandom programmed dispersion. Of note, neither Fibrillarin nor UBF colocalized with promyelocytic leukemia (PML) nuclear bodies or with the viral protein LANA-1, and their redistribution was not dependent on viral DNA replication or late viral gene expression. No significant changes in pre-rRNA levels and no accumulation of pre-rRNA intermediates were found by RT-qPCR and Northern blot analysis, respectively. Furthermore, fluorescent in situ hybridization (FISH), combined with immunofluorescence, revealed an overlap between Fibrillarin and internal transcribed spacer 1 (ITS1), which represents the primary product of the pre-rRNA, suggesting that the processing of rRNA proceeds during lytic reactivation. Finally, small changes in the levels of pseudouridylation were documented across the rRNA. Taken together, our results suggest that rRNA transcription and processing persist during lytic reactivation of KSHV, yet they may become uncoupled. Whether the observed nucleolar alterations favor productive infection or signify cellular anti-viral responses remains to be determined. Author SummaryWe describe the extensive destruction of the nuclear and nucleolar architecture during lytic reactivation of KSHV. Distinct delocalization patterns are illustrated: certain nucleolar proteins remained associated with each other whereas others dissociated, implying that nucleolar proteins undergo nonrandom programmed dispersion. Of note, no significant changes in pre-rRNA levels and no accumulation of pre-rRNA intermediates were found, suggesting that pre-RNA transcription and processing continue and could be uncoupled during lytic reactivation. Small changes in the levels of pseudouridylation were documented across the rRNA. Previous studies showed that the different forms of KSHV infection are controlled through cellular and viral functions, which reprogram host epigenetic, transcriptomic, post-transcriptomic and proteomic landscapes. The ability of KSHV to affect the nucleolus and rRNA modifications constitutes a novel interaction network between viral and cellular components. The study of rRNA modifications is still in its infancy; however, the notion of altering cell fate by regulating rRNA modifications has recently begun to emerge, and its significance in viral infection is intriguing.

microbiology↗