A pseudouridylation switch in rRNA is implicated in ribosome function during the life cycle of Trypanosoma brucei

Chikne, Vaibhav; Doniger, Tirza; Rajan, K. Shanmugha; Bartok, Osnat; Eliaz, Dror; Cohen-Chalamish, Smadar; Tschudi, Christian; Unger, Ron; Hashem, Yaser; Kadener, Sebastián; Michaeli, Shulamit

doi:10.1038/srep25296

Cited by 43 publications

(88 citation statements)

References 38 publications

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“…Recent work has shown that pseudouridylation of a given position is not binary; instead a specific site can be more or less pseudouridylated in different conditions (Chikne et al 2016). If pseudouridylation does affect RNA turnover, the modest change we observed could be the result of only 25% of the pool of an RNA being pseudouridylated and showing differential stability, while the other 75% shows no change in stability, thereby dampening the signal.…”

Section: Discussionmentioning

confidence: 51%

“…The majority of indications that pseudouridylation plays an important biological role stems indirectly from the observation that Ψ's are modulated in response to different cellular conditions. Changes in the extent of rRNA pseudouridylation at specific sites changes with temperature and development, while entirely novel sites of pseudouridylation appear in snRNAs and mRNAs with stresses such as heat shock, starvation, oxidative stress, and immune stimulation (Wu et al 2011;Basak and Query 2014;Carlile et al 2014;Schwartz et al 2014;Li et al 2015;Chikne et al 2016).…”

Section: Introductionmentioning

confidence: 99%

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mRNA pseudouridylation affects RNA metabolism in the parasite Toxoplasma gondii

Nakamoto

Lovejoy

Cygan

et al. 2017

RNA

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RNA contains over 100 modified nucleotides that are created post-transcriptionally, among which pseudouridine (Ψ) is one of the most abundant. Although it was one of the first modifications discovered, the biological role of this modification is still not fully understood. Recently, we reported that a pseudouridine synthase (TgPUS1) is necessary for differentiation of the singlecelled eukaryotic parasite Toxoplasma gondii from active to chronic infection. To better understand the biological role of pseudouridylation, we report here gel-based and deep-sequencing methods to identify TgPUS1-dependent Ψ's in Toxoplasma RNA, and the use of TgPUS1 mutants to examine the effect of this modification on mRNAs. In addition to identifying conserved sites of pseudouridylation in Toxoplasma rRNA, tRNA, and snRNA, we also report extensive pseudouridylation of Toxoplasma mRNAs, with the Ψ's being relatively depleted in the 3 ′ ′ ′ ′ ′ -UTR but enriched at position 1 of codons. We show that many Ψ's in tRNA and mRNA are dependent on the action of TgPUS1 and that TgPUS1-dependent mRNA Ψ's are enriched in developmentally regulated transcripts. RNA-seq data obtained from wild-type and TgPUS1-mutant parasites shows that genes containing a TgPUS1-dependent Ψ are relatively more abundant in mutant parasites, while pulse/chase labeling of RNA with 4-thiouracil shows that mRNAs containing TgPUS1-dependent Ψ have a modest but statistically significant increase in half-life in the mutant parasites. These data are some of the first evidence suggesting that mRNA Ψ's play an important biological role.

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Section: Discussionmentioning

confidence: 51%

Section: Introductionmentioning

confidence: 99%

mRNA pseudouridylation affects RNA metabolism in the parasite Toxoplasma gondii

Nakamoto

Lovejoy

Cygan

et al. 2017

RNA

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“…Furthermore, studies have shown that active regulation of rRNA modification is a conserved behavior. For example, pseudouridylation of rRNA is up-regulated in the bloodstream form of the Trypanosoma brucei parasite (Chikne et al 2016), and rRNA methylation by NSUN5 regulates life span and stress resistance in flies, worms and yeast (Schosserer et al 2015). Similarly, a study in X. tropicalis showed that failure to ubiquitylate the ribosome biogenesis factors NOLC1 and TCOF1 destabilizes these proteins, and subsequently disrupts neural crest formation during cell differentiation (Werner et al 2015).…”

Section: Posttranscriptional Modification Of Rrnamentioning

confidence: 99%

Does functional specialization of ribosomes really exist?

Ferretti

Karbstein

2019

RNA

105

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It has recently become clear that ribosomes are much more heterogeneous than previously thought, with diversity arising from rRNA sequence and modifications, ribosomal protein (RP) content and posttranslational modifications (PTMs), as well as bound nonribosomal proteins. In some cases, the existence of these diverse ribosome populations has been verified by biochemical or structural methods. Furthermore, knockout or knockdown of RPs can diversify ribosome populations, while also affecting the translation of some mRNAs (but not others) with biological consequences. However, the effects on translation arising from depletion of diverse proteins can be highly similar, suggesting that there may be a more general defect in ribosome function or stability, perhaps arising from reduced ribosome numbers. Consistently, overall reduced ribosome numbers can differentially affect subclasses of mRNAs, necessitating controls for specificity. Moreover, in order to study the functional consequences of ribosome diversity, perturbations including affinity tags and knockouts are introduced, which can also affect the outcome of the experiment. Here we review the available literature to carefully evaluate whether the published data support functional diversification, defined as diverse ribosome populations differentially affecting translation of distinct mRNA (classes). Based on these observations and the commonly observed cellular responses to perturbations in the system, we suggest a set of important controls to validate functional diversity, which should include gainof-function assays and the demonstration of inducibility under physiological conditions.

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“…Mapping the Pseudouridine nucleotides by the CMC method. Pseudouridine sites were mapped as previously described [90]. RNA from BAC16-mCherry-ORF45-infected iSLK cells and SLK cells that Figure S1.…”

Section: Methodsmentioning

confidence: 99%

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

Atari

Rajan

Chikne

et al. 2020

Preprint

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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 Kaposi's 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 , 3 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, posttranscriptomic and proteomic landscapes. The ability of KSHV to affect the nucleolus and rRNA modifications constitutes a novel interaction network between viral and cellula...

show abstract

A pseudouridylation switch in rRNA is implicated in ribosome function during the life cycle of Trypanosoma brucei

Cited by 43 publications

References 38 publications

mRNA pseudouridylation affects RNA metabolism in the parasite Toxoplasma gondii

mRNA pseudouridylation affects RNA metabolism in the parasite Toxoplasma gondii

Does functional specialization of ribosomes really exist?

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

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