RESUMO
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 associated with parasite fitness gain, with genome instability causing highly reproducible, gene dosage-independent and -dependent changes. Reduction of flagellar transcripts and increase in coding and non-coding RNAs implicated in ribosomal biogenesis and protein translation were not correlated to dosage changes of the corresponding genes, revealing a gene dosage-independent, post-transcriptional mechanism of regulation. In contrast, abundance of gene products implicated in post-transcriptional regulation itself correlated to corresponding gene dosage changes. Thus, RNA abundance during parasite adaptation is controled by direct and indirect gene dosage changes. 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 modified 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.
Assuntos
Leishmania donovani , Leishmaniose Visceral , Regulação da Expressão Gênica , Instabilidade Genômica , Humanos , Leishmania donovani/genética , Leishmaniose Visceral/parasitologia , ProteômicaRESUMO
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.
Assuntos
Adaptação Fisiológica/genética , Epistasia Genética , Genoma de Protozoário , Instabilidade Genômica , Leishmania/genética , Dosagem de Genes , Aptidão Genética , Humanos , Leishmaniose/parasitologiaRESUMO
Trypanosoma brucei, the parasite that causes sleeping sickness, cycles between an insect and a mammalian host. However, the effect of RNA modifications such as pseudouridinylation on its ability to survive in these two different host environments is unclear. Here, two genome-wide approaches were applied for mapping pseudouridinylation sites (Ψs) on small nucleolar RNA (snoRNA), 7SL RNA, vault RNA, and tRNAs from T. brucei. We show using HydraPsiSeq and RiboMeth-seq that the Ψ on C/D snoRNA guiding 2'-O-methylation increased the efficiency of the guided modification on its target, rRNA. We found differential levels of Ψs on these noncoding RNAs in the two life stages (insect host and mammalian host) of the parasite. Furthermore, tRNA isoform abundance and Ψ modifications were characterized in these two life stages demonstrating stage-specific regulation. We conclude that the differential Ψ modifications identified here may contribute to modulating the function of noncoding RNAs involved in rRNA processing, rRNA modification, protein synthesis, and protein translocation during cycling of the parasite between its two hosts.
Assuntos
Interações Hospedeiro-Parasita , Estágios do Ciclo de Vida , Pseudouridina , Pequeno RNA não Traduzido , Trypanosoma brucei brucei , Animais , Interações Hospedeiro-Parasita/fisiologia , Estágios do Ciclo de Vida/fisiologia , Pseudouridina/genética , Pseudouridina/metabolismo , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/metabolismo , Pequeno RNA não Traduzido/genética , RNA de Transferência/genética , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/crescimento & desenvolvimento , Trypanosoma brucei brucei/metabolismoRESUMO
The parasite Trypanosoma brucei cycles between an insect and a mammalian host and is the causative agent of sleeping sickness. Here, we performed high-throughput mapping of pseudouridines (Ψs) on mRNA from two life stages of the parasite. The analysis revealed ~273 Ψs, including developmentally regulated Ψs that are guided by homologs of pseudouridine synthases (PUS1, 3, 5, and 7). Mutating the U that undergoes pseudouridylation in the 3' UTR of valyl-tRNA synthetase destabilized the mRNA level. To investigate the mechanism by which Ψ affects the stability of this mRNA, proteins that bind to the 3' UTR were identified, including the RNA binding protein RBSR1. The binding of RBSR1 protein to the 3' UTR was stronger when lacking Ψ compared to transcripts carrying the modification, suggesting that Ψ can inhibit the binding of proteins to their target and thus affect the stability of mRNAs. Consequently, Ψ modification on mRNA adds an additional level of regulation to the dominant post-transcriptional control in these parasites.
Assuntos
Transferases Intramoleculares/metabolismo , Pseudouridina/genética , Pseudouridina/metabolismo , RNA Mensageiro/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Regiões 3' não Traduzidas , Animais , Regulação da Expressão Gênica , Ensaios de Triagem em Larga Escala/métodos , Transferases Intramoleculares/genética , Ligação Proteica , Estabilidade de RNA , Proteínas de Ligação a RNA/metabolismoRESUMO
There is growing evidence that post-transcriptional RNA modifications are highly dynamic and can be used to improve crop production. Although more than 172 unique types of RNA modifications have been identified throughout the kingdom of life, we are yet to leverage upon the understanding to optimize RNA modifications in crops to improve productivity. The contributions of internal mRNA modifications such as N6-methyladenosine (m6 A) and 5-methylcytosine (m5 C) methylations to embryonic development, root development, leaf morphogenesis, flowering, fruit ripening and stress response are sufficiently known, but the roles of the two most abundant RNA modifications, pseudouridine (Ψ) and 2'-O-methylation (Nm), in the cell remain unclear due to insufficient advances in high-throughput technologies in plant development. Therefore, in this review, we discuss the latest methods and insights gained in mapping internal Ψ and Nm and their unique properties in plants and other organisms. In addition, we discuss the limitations that remain in high-throughput technologies for qualitative and quantitative mapping of these RNA modifications and highlight future challenges in regulating the plant epitranscriptome.
Assuntos
Pseudouridina , Transcriptoma , 5-Metilcitosina , Plantas/genética , Plantas/metabolismo , Pseudouridina/genética , Pseudouridina/metabolismo , RNA/metabolismo , Processamento Pós-Transcricional do RNA/genética , Transcriptoma/genéticaRESUMO
Cardiovascular disease is the leading cause of morbidity and mortality all over the world. Emerging evidence emphasize the importance of extracellular vesicles (EVs) in the cell to cell communication in the cardiovascular system which is majorly mediated through non-coding RNA cargo. Advancement in sequencing technologies revealed a major proportion of human genome is composed of non-coding RNAs viz., miRNAs, lncRNAs, tRNAs, snoRNAs, piRNAs and rRNAs. However, our understanding of the role of ncRNAs-containing EVs in cardiovascular health and disease is still in its infancy. This book chapter provides a comprehensive update on our understanding on the role of EVs derived ncRNAs in the cardiovascular pathophysiology and their therapeutic potential.
Assuntos
Sistema Cardiovascular , Vesículas Extracelulares , MicroRNAs , Humanos , MicroRNAs/genética , RNA não Traduzido/genéticaRESUMO
Post-transcriptional RNA modification has been observed in all kingdoms of life and more than a hundred different types of RNA modifications decorate the chemical and topological properties of these ribose nucleotides. These RNA modifications can potentially alter the RNA structure and also affect the binding affinity of proteins, thus regulating the mRNA stability as well as translation. Emerging evidence suggest that these modifications are not static, but are dynamic; vary upon different cues and are cell-type or tissue-specific. The cardiac transcriptome is not exceptional to such RNA modifications and is enriched with the abundant base methylation such as N6-methyladenosine (m6A) and also 2'-O-Methylation (Nm). In this review we will focus on the technologies available to map these modifications and as well as the contribution of these post-transcriptional modifications during various pathological conditions of the heart.
Assuntos
Doenças Cardiovasculares/patologia , Metilação de DNA , Epigênese Genética , Epigenoma , Regulação da Expressão Gênica , Coração/fisiopatologia , Processamento Pós-Transcricional do RNA , Animais , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/metabolismo , Humanos , TranscriptomaRESUMO
The parasite Trypanosoma brucei, the causative agent of sleeping sickness, cycles between an insect and a mammalian host. Here, we investigated the presence of pseudouridines (Ψs) on the spliceosomal small nuclear RNAs (snRNAs), which may enable growth at the very different temperatures characterizing the two hosts. To this end, we performed the first high-throughput mapping of spliceosomal snRNA Ψs by small RNA Ψ-seq. The analysis revealed 42 Ψs on T. brucei snRNAs, which is the highest number reported so far. We show that a trypanosome protein analogous to human protein WDR79, is essential for guiding Ψ on snRNAs but not on rRNAs. snoRNA species implicated in snRNA pseudouridylation were identified by a genome-wide approach based on ligation of RNAs following in vivo UV cross-linking. snRNA Ψs are guided by single hairpin snoRNAs, also implicated in rRNA modification. Depletion of such guiding snoRNA by RNAi compromised the guided modification on snRNA and reduced parasite growth at elevated temperatures. We further demonstrate that Ψ strengthens U4/U6 RNA-RNA and U2B"/U2A' proteins-U2 snRNA interaction at elevated temperatures. The existence of single hairpin RNAs that modify both the spliceosome and ribosome RNAs is unique for these parasites, and may be related to their ability to cycle between their two hosts that differ in temperature.
Assuntos
Proteínas de Protozoários/metabolismo , Pseudouridina/metabolismo , RNA Nuclear Pequeno/metabolismo , RNA Nucleolar Pequeno/metabolismo , Spliceossomos/metabolismo , Trypanosoma brucei brucei/metabolismo , Animais , Sequência de Bases , Humanos , Ligação Proteica , Proteínas de Protozoários/genética , Pseudouridina/genética , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , RNA Nuclear Pequeno/genética , RNA Nucleolar Pequeno/genética , Ribonucleoproteínas Nucleares Pequenas/genética , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Spliceossomos/genética , Trypanosoma brucei brucei/genéticaRESUMO
The vault ribonucleoprotein (RNP), comprising vault RNA (vtRNA) and telomerase-associated protein 1 (TEP1), is found in many eukaryotes. However, previous studies of vtRNAs, for example in mammalian cells, have failed to reach a definitive conclusion about their function. vtRNAs are related to Y RNAs, which are complexed with Ro protein and influence Ro's function in noncoding RNA (ncRNA) quality control and processing. In Trypanosoma brucei, the small noncoding TBsRNA-10 was first described in a survey of the ncRNA repertoire in this organism. Here, we report that TBsRNA-10 in T. brucei is a vtRNA, based on its association with TEP1 and sequence similarity to those of other known and predicted vtRNAs. We observed that like vtRNAs in other species, TBsRNA-10 is transcribed by RNA polymerase III, which in trypanosomes also generates the spliceosomal U-rich small nuclear RNAs. In T. brucei, spliced leader (SL)-mediated trans-splicing of pre-mRNAs is an obligatory step in gene expression, and we found here that T. brucei's vtRNA is highly enriched in a non-nucleolar locus in the cell nucleus implicated in SL RNP biogenesis. Using a newly developed permeabilized cell system for the bloodstream form of T. brucei, we show that down-regulated vtRNA levels impair trans-spliced mRNA production, consistent with a role of vtRNA in trypanosome mRNA metabolism. Our results suggest a common theme for the functions of vtRNAs and Y RNAs. We conclude that by complexing with their protein-binding partners TEP1 and Ro, respectively, these two RNA species modulate the metabolism of various RNA classes.
Assuntos
Proteínas de Protozoários/genética , RNA de Protozoário/genética , Trans-Splicing/genética , Trypanosoma brucei brucei/genética , Partículas de Ribonucleoproteínas em Forma de Abóbada/genética , Pareamento de Bases/genética , Sequência de Bases , Nucléolo Celular/metabolismo , Sequência Conservada/genética , DNA Polimerase III/metabolismo , Proteínas de Protozoários/metabolismo , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Protozoário/química , Transcrição GênicaRESUMO
The parasite Trypanosoma brucei cycles between insect and mammalian hosts, and is the causative agent of sleeping sickness. Here, we performed genome-wide mapping of 2'-O-methylations (Nms) on trypanosome rRNA using three high-throughput sequencing methods; RibOxi-seq, RiboMeth-seq and 2'-OMe-seq. This is the first study using three genome-wide mapping approaches on rRNA from the same species showing the discrepancy among the methods. RibOxi-seq detects all the sites, but RiboMeth-seq is the only method to evaluate the level of a single Nm site. The sequencing revealed at least ninety-nine Nms guided by eighty-five snoRNAs among these thirty-eight Nms are trypanosome specific sites. We present the sequence and target of the C/D snoRNAs guiding on rRNA. This is the highest number of Nms detected to date on rRNA of a single cell parasite. Based on RiboMeth-seq, several Nm sites were found to be differentially regulated at the two stages of the parasite life cycle, the insect procyclic form (PCF) versus the bloodstream form (BSF) in the mammalian host.
Assuntos
RNA de Protozoário , RNA Ribossômico , RNA Nucleolar Pequeno/genética , Trypanosoma brucei brucei/genética , Biologia Computacional/métodos , Conectoma , Perfilação da Expressão Gênica , Conformação de Ácido Nucleico , TranscriptomaRESUMO
BACKGROUND: The discovery of PIWI-interacting RNAs (piRNAs) has fundamentally changed our understanding of post transcriptional regulation of transposons and other genes. Unlike miRNA and siRNA, the piRNAs are the most abundant but least studied RNA species in mammals. Although the expression of PIWI proteins and piRNAs has long been regarded as germline specific, increasing evidences suggest the expression of piRNAs in somatic cells. METHODS: In this study, the small RNA sequencing executed during induction of cardiac hypertrophy in both in vivo and in vitro conditions were annotated for the expression of piRNAs. The expression of piRNAs was validated by qPCR and RNA immunoprecipitation. In addition, the presence of piRNAs in circulation of myocardial infarction patients was studied by qPCR. RESULTS: We identified an abundant and altered expression of piRNAs during cardiac hypertrophy. The differentially expressed piRNAs was validated by qPCR and RNA immunoprecipitation. The significantly and differentially expressed piRNAs were predicted to target different retrotransposons and mRNAs in the rat genome. The detection of specific piRNA in serum of myocardial infarction patients suggests the potential of piRNA for diagnosis. CONCLUSION: Overall this study is the first to provide a whole-genome analysis of the large repertoire of piRNAs in the cardiac system and this would pave a new path to understanding the molecular aetiology of piRNA and retrotransposons in the physiology and pathology of the cardiac system.
Assuntos
Cardiomegalia/metabolismo , Regulação da Expressão Gênica , RNA Interferente Pequeno/biossíntese , Animais , Estudo de Associação Genômica Ampla , RatosRESUMO
N 6-methyladenosine (m6A) in eukaryotes is the most common and widespread internal modification in mRNA. The modification regulates mRNA stability, translation efficiency, and splicing, thereby fine-tuning gene regulation. In plants, m6A is dynamic and critical for various growth stages, embryonic development, morphogenesis, flowering, stress response, crop yield, and biomass. Although recent high-throughput sequencing approaches have enabled the rapid identification of m6A modification sites, the site-specific mechanism of this modification remains unclear in trees. In this review, we discuss the functional significance of m6A in trees under different stress conditions and discuss recent advancements in the quantification of m6A. Quantitative and functional insights into the dynamic aspect of m6A modification could assist researchers in engineering tree crops for better productivity and resistance to various stress conditions.
RESUMO
As cardiovascular diseases continue to be the leading cause of death worldwide, groundbreaking research is being conducted to mitigate their effects. This review looks into the potential of small nucleolar RNAs (snoRNAs) and the opportunity to use these molecular agents as therapeutic biomarkers for cardiovascular issues specific to the heart. Through an investigation of snoRNA biogenesis, functionality, and roles in cardiovascular diseases, this review relates our past and present knowledge of snoRNAs to the current scientific literature. Considering the initial discovery of snoRNAs and the studies thereafter analyzing the roles of snoRNAs in disease, we look forward to uncovering many other noncanonical functions that could lead researchers closer to finding preventive and curative solutions for cardiovascular diseases.
RESUMO
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 â¼2.4-3 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.
Assuntos
Pseudouridina , RNA de Transferência , Ribossomos , Pseudouridina/metabolismo , Ribossomos/metabolismo , RNA de Transferência/metabolismo , RNA de Transferência/genética , Leishmania/metabolismo , Leishmania/genética , Microscopia Crioeletrônica , RNA Ribossômico/metabolismo , RNA Ribossômico/química , RNA Ribossômico/genética , Conformação de Ácido Nucleico , Modelos MolecularesRESUMO
Trypanosomes are protozoan parasites that cycle between insect and mammalian hosts and are the causative agent of sleeping sickness. Here, we describe the changes of pseudouridine (Ψ) modification on rRNA in the two life stages of the parasite using four different genome-wide approaches. CRISPR-Cas9 knock-outs of all four snoRNAs guiding Ψ on helix 69 (H69) of the large rRNA subunit were lethal. A single knock-out of a snoRNA guiding Ψ530 on H69 altered the composition of the 80S monosome. These changes specifically affected the translation of only a subset of proteins. This study correlates a single site Ψ modification with changes in ribosomal protein stoichiometry, supported by a high-resolution cryo-EM structure. We propose that alteration in rRNA modifications could generate ribosomes preferentially translating state-beneficial proteins.
Assuntos
Parasitos , Trypanosoma brucei brucei , Animais , Parasitos/genética , Trypanosoma brucei brucei/metabolismo , Pseudouridina/metabolismo , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Ribossomos/metabolismo , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/metabolismo , Mamíferos/genéticaRESUMO
The nucleolus is a subnuclear 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 the lytic reactivation of KSHV. This was manifested by the redistribution of key nucleolar proteins, including the rRNA transcription factor UBF. Distinct delocalization patterns were evident; certain nucleolar proteins remained together whereas others dissociated, implying that nucleolar proteins undergo nonrandom programmed dispersion. Significantly, the redistribution of UBF was 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. 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 (Ψ) and 2'-O-methylation (Nm) were documented across the rRNA; however, none were localized to the functional domain. Taken together, our results suggest that despite dramatic changes in the nucleolar organization, rRNA transcription and processing persist during lytic reactivation of KSHV. Whether the observed nucleolar alterations favor productive infection or signify cellular anti-viral responses remains to be determined.
Assuntos
Herpesvirus Humano 8 , Sarcoma de Kaposi , Replicação do DNA , DNA Viral , Regulação Viral da Expressão Gênica , Herpesvirus Humano 8/genética , Humanos , Hibridização in Situ Fluorescente , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Precursores de RNA , Replicação ViralRESUMO
The parasite Trypanosoma brucei causes African sleeping sickness that is fatal to patients if untreated. Parasite differentiation from a replicative slender form into a quiescent stumpy form promotes host survival and parasite transmission. Long noncoding RNAs (lncRNAs) are known to regulate cell differentiation in other eukaryotes. To determine whether lncRNAs are also involved in parasite differentiation, we used RNA sequencing to survey the T. brucei genome, identifying 1428 previously uncharacterized lncRNA genes. We find that grumpy lncRNA is a key regulator that promotes parasite differentiation into the quiescent stumpy form. This function is promoted by a small nucleolar RNA encoded within the grumpy lncRNA. snoGRUMPY binds to messenger RNAs of at least two stumpy regulatory genes, promoting their expression. grumpy overexpression reduces parasitemia in infected mice. Our analyses suggest that T. brucei lncRNAs modulate parasite-host interactions and provide a mechanism by which grumpy regulates cell differentiation in trypanosomes.
RESUMO
During spaceflight, astronauts are exposed to various physiological and psychological stressors that have been associated with adverse health effects. Therefore, there is an unmet need to develop novel diagnostic tools to predict early alterations in astronauts' health. Small nucleolar RNA (snoRNA) is a type of short non-coding RNA (60-300 nucleotides) known to guide 2'-O-methylation (Nm) or pseudouridine (ψ) of ribosomal RNA (rRNA), small nuclear RNA (snRNA), or messenger RNA (mRNA). Emerging evidence suggests that dysregulated snoRNAs may be key players in regulating fundamental cellular mechanisms and in the pathogenesis of cancer, heart, and neurological disease. Therefore, we sought to determine whether the spaceflight-induced snoRNA changes in astronaut's peripheral blood (PB) plasma extracellular vesicles (PB-EV) and peripheral blood mononuclear cells (PBMCs). Using unbiased small RNA sequencing (sRNAseq), we evaluated changes in PB-EV snoRNA content isolated from astronauts (n = 5/group) who underwent median 12-day long Shuttle missions between 1998 and 2001. Using stringent cutoff (fold change > 2 or log2-fold change >1, FDR < 0.05), we detected 21 down-and 9-up-regulated snoRNAs in PB-EVs 3 days after return (R + 3) compared to 10 days before launch (L-10). qPCR validation revealed that SNORA74A was significantly down-regulated at R + 3 compared to L-10. We next determined snoRNA expression levels in astronauts' PBMCs at R + 3 and L-10 (n = 6/group). qPCR analysis further confirmed a significant increase in SNORA19 and SNORA47 in astronauts' PBMCs at R + 3 compared to L-10. Notably, many downregulated snoRNA-guided rRNA modifications, including four Nms and five ψs. Our findings revealed that spaceflight induced changes in PB-EV and PBMCs snoRNA expression, thus suggesting snoRNAs may serve as potential novel biomarkers for monitoring astronauts' health.
RESUMO
In the parasite Trypanosoma brucei, the causative agent of human African sleeping sickness, all mRNAs are trans-spliced to generate a common 5' exon derived from the spliced leader (SL) RNA. Perturbations of protein translocation across the endoplasmic reticulum (ER) induce the spliced leader RNA silencing (SLS) pathway. SLS activation is mediated by a serine-threonine kinase, PK3, which translocates from the cytosolic face of the ER to the nucleus, where it phosphorylates the TATA-binding protein TRF4, leading to the shutoff of SL RNA transcription, followed by induction of programmed cell death. Here, we demonstrate that SLS is also induced by depletion of the essential ER-resident chaperones BiP and calreticulin, ER oxidoreductin 1 (ERO1), and the Golgi complex-localized quiescin sulfhydryl oxidase (QSOX). Most strikingly, silencing of Rhomboid-like 1 (TIMRHOM1), involved in mitochondrial protein import, also induces SLS. The PK3 kinase, which integrates SLS signals, is modified by phosphorylation on multiple sites. To determine which of the phosphorylation events activate PK3, several individual mutations or their combination were generated. These mutations failed to completely eliminate the phosphorylation or translocation of the kinase to the nucleus. The structures of PK3 kinase and its ATP binding domain were therefore modeled. A conserved phenylalanine at position 771 was proposed to interact with ATP, and the PK3F771L mutation completely eliminated phosphorylation under SLS, suggesting that the activation involves most if not all of the phosphorylation sites. The study suggests that the SLS occurs broadly in response to failures in protein sorting, folding, or modification across multiple compartments. IMPORTANCE In this study, we found that SLS is induced by depletion of the essential ER-resident chaperones BiP and calreticulin, ER oxidoreductin 1 (ERO1), and the Golgi complex-localized quiescin sulfhydryl oxidase (QSOX). Most strikingly, silencing of Rhomboid-like 1 (TIMRHOM1), involved in mitochondrial protein import, also induces SLS. We also report on the autophosphorylation of PK3 during SLS induction. This study has implications for our understanding of how trypanosomes keep the homeostasis between the ER and the mitochondria and suggests that PK3 may participate in the connection between these two organelles. The pathway, when induced, leads to the suicide of these parasites, and its induction offers a potential novel drug target against these parasites.
Assuntos
Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Protozoários/metabolismo , RNA de Protozoário/genética , RNA Líder para Processamento/genética , Trypanosoma brucei brucei/genética , Tripanossomíase Africana/parasitologia , Núcleo Celular/genética , Núcleo Celular/metabolismo , Retículo Endoplasmático/genética , Complexo de Golgi/genética , Humanos , Proteínas Mitocondriais/genética , Proteínas Serina-Treonina Quinases/genética , Transporte Proteico , Proteínas de Protozoários/genética , Interferência de RNA , Splicing de RNA , RNA de Protozoário/metabolismo , RNA Líder para Processamento/metabolismo , Proteína de Ligação a TATA-Box/genética , Proteína de Ligação a TATA-Box/metabolismo , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/metabolismoRESUMO
The parasite Trypanosoma brucei is the causative agent of sleeping sickness and cycles between insect and mammalian hosts. The parasite appears to lack conventional transcriptional regulation of protein coding genes, and mRNAs are processed from polycistronic transcripts by the concerted action of trans-splicing and polyadenylation. Regulation of mRNA function is mediated mainly by RNA binding proteins affecting mRNA stability and translation. In this study, we describe the identification of 62 non-coding (nc) RNAs that are developmentally regulated and/or respond to stress. We characterized two novel anti-sense RNA regulators (TBsRNA-33 and 37) that originate from the rRNA loci, associate with ribosomes and polyribosomes, and interact in vivo with distinct mRNA species to regulate translation. Thus, this study suggests for the first-time anti-sense RNA regulators as an additional layer for controlling gene expression in these parasites.