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Dihydrouridine (D), a prevalent and evolutionarily conserved base in the transcriptome, primarily resides in tRNAs and, to a lesser extent, in mRNAs. Notably, this modification is found at position 2449 in the Escherichia coli 23S rRNA, strategically positioned near the ribosome's peptidyl transferase site. Despite the prior identification, in E. coli genome, of three dihydrouridine synthases (DUS), a set of NADPH and FMN-dependent enzymes known for introducing D in tRNAs and mRNAs, characterization of the enzyme responsible for D2449 deposition has remained elusive. This study introduces a rapid method for detecting D in rRNA, involving reverse transcriptase-blockage at the rhodamine-labeled D2449 site, followed by PCR amplification (RhoRT-PCR). Through analysis of rRNA from diverse E. coli strains, harboring chromosomal or single-gene deletions, we pinpoint the yhiN gene as the ribosomal dihydrouridine synthase, now designated as RdsA. Biochemical characterizations uncovered RdsA as a unique class of flavoenzymes, dependent on FAD and NADH, with a complex structural topology. In vitro assays demonstrated that RdsA dihydrouridylates a short rRNA transcript mimicking the local structure of the peptidyl transferase site. This suggests an early introduction of this modification before ribosome assembly. Phylogenetic studies unveiled the widespread distribution of the yhiN gene in the bacterial kingdom, emphasizing the conservation of rRNA dihydrouridylation. In a broader context, these findings underscore nature's preference for utilizing reduced flavin in the reduction of uridines and their derivatives.
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Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , ARN Ribosómico 23S/metabolismo , ARN Ribosómico 23S/genética , ARN Ribosómico 23S/química , Uridina/análogos & derivados , Uridina/metabolismo , Uridina/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , ARN Bacteriano/metabolismo , ARN Bacteriano/genética , ARN Bacteriano/químicaRESUMEN
Alterations in cell fate are often attributed to (epigenetic) regulation of gene expression. An emerging paradigm focuses on specialized ribosomes within a cell. However, little evidence exists for the dynamic regulation of ribosome composition and function. Here, we stimulated a chondrocytic cell line with transforming growth factor beta (TGF-ß2) and mapped changes in ribosome function, composition and ribosomal RNA (rRNA) epitranscriptomics. 35S Met/Cys incorporation was used to evaluate ribosome activity. Dual luciferase reporter assays were used to assess ribosomal modus. Ribosomal RNA expression and processing were determined by RT-qPCR, while RiboMethSeq and HydraPsiSeq were used to determine rRNA modification profiles. Label-free protein quantification of total cell lysates, isolated ribosomes and secreted proteins was done by LC-MS/MS. A three-day TGF-ß2 stimulation induced total protein synthesis in SW1353 chondrocytic cells and human articular chondrocytes. Specifically, TGF-ß2 induced cap-mediated protein synthesis, while IRES-mediated translation was not (P53 IRES) or little affected (CrPv IGR and HCV IRES). Three rRNA post-transcriptional modifications (PTMs) were affected by TGF-ß2 stimulation (18S-Gm1447 downregulated, 18S-ψ1177 and 28S-ψ4598 upregulated). Proteomic analysis of isolated ribosomes revealed increased interaction with eIF2 and tRNA ligases and decreased association of eIF4A3 and heterogeneous nuclear ribonucleoprotein (HNRNP)s. In addition, thirteen core ribosomal proteins were more present in ribosomes from TGF-ß2 stimulated cells, albeit with a modest fold change. A prolonged stimulation of chondrocytic cells with TGF-ß2 induced ribosome activity and changed the mode of translation. These functional changes could be coupled to alterations in accessory proteins in the ribosomal proteome.
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Condrocitos , Biosíntesis de Proteínas , ARN Ribosómico , Ribosomas , Factor de Crecimiento Transformador beta2 , Condrocitos/metabolismo , Condrocitos/efectos de los fármacos , Ribosomas/metabolismo , Humanos , ARN Ribosómico/metabolismo , ARN Ribosómico/genética , Factor de Crecimiento Transformador beta2/metabolismo , Factor de Crecimiento Transformador beta2/farmacología , Sitios Internos de Entrada al Ribosoma , Línea CelularRESUMEN
Dihydrouridine (D) is a common modified base found predominantly in transfer RNA (tRNA). Despite its prevalence, the mechanisms underlying dihydrouridine biosynthesis, particularly in prokaryotes, have remained elusive. Here, we conducted a comprehensive investigation into D biosynthesis in Bacillus subtilis through a combination of genetic, biochemical, and epitranscriptomic approaches. Our findings reveal that B. subtilis relies on two FMN-dependent Dus-like flavoprotein homologs, namely DusB1 and DusB2, to introduce all D residues into its tRNAs. Notably, DusB1 exhibits multisite enzyme activity, enabling D formation at positions 17, 20, 20a and 47, while DusB2 specifically catalyzes D biosynthesis at positions 20 and 20a, showcasing a functional redundancy among modification enzymes. Extensive tRNA-wide D-mapping demonstrates that this functional redundancy impacts the majority of tRNAs, with DusB2 displaying a higher dihydrouridylation efficiency compared to DusB1. Interestingly, we found that BsDusB2 can function like a BsDusB1 when overexpressed in vivo and under increasing enzyme concentration in vitro. Furthermore, we establish the importance of the D modification for B. subtilis growth at suboptimal temperatures. Our study expands the understanding of D modifications in prokaryotes, highlighting the significance of functional redundancy in this process and its impact on bacterial growth and adaptation.
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Bacillus subtilis , ARN de Transferencia , Uridina , Bacillus subtilis/enzimología , Bacillus subtilis/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , ARN Bacteriano/metabolismo , ARN Bacteriano/genética , ARN de Transferencia/metabolismo , ARN de Transferencia/genética , Uridina/metabolismo , Uridina/análogos & derivados , Expresión GénicaRESUMEN
Transfer RNA (tRNA) modifications play a crucial role in maintaining translational fidelity and efficiency, and they may function as regulatory elements in stress response and virulence. Despite their pivotal roles, a comprehensive mapping of tRNA modifications and their associated synthesis genes is still limited, with a predominant focus on free-living bacteria. In this study, we employed a multidisciplinary approach, incorporating comparative genomics, mass spectrometry, and next-generation sequencing, to predict the set of tRNA modification genes responsible for tRNA maturation in two intracellular pathogens-Bartonella henselae Houston I and Bartonella quintana Toulouse, which are causative agents of cat-scratch disease and trench fever, respectively. This analysis presented challenges, particularly because of host RNA contamination, which served as a potential source of error. However, our approach predicted 26 genes responsible for synthesizing 23 distinct tRNA modifications in B. henselae and 22 genes associated with 23 modifications in B. quintana. Notably, akin to other intracellular and symbiotic bacteria, both Bartonella species have undergone substantial reductions in tRNA modification genes, mostly by simplifying the hypermodifications present at positions 34 and 37. Bartonella quintana exhibited the additional loss of four modifications and these were linked to examples of gene decay, providing snapshots of reductive evolution.
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Respectful maternity care (RMC) for women living with HIV (WLHIV) improves birth outcomes and may influence women's long-term commitment to HIV care. In this study, we evaluated the MAMA training, a team-based simulation training for labor and delivery (L&D) providers to improve RMC and reduce stigma in caring for WLHIV. The study was conducted in six clinical sites in the Kilimanjaro Region of Tanzania. 60 L&D providers participated in the MAMA training, which included a two-and-a-half-day workshop followed by a half-day on-site refresher. We assessed the impact of the MAMA training using a pre-post quasi-experimental design. To assess provider impacts, participants completed assessments at baseline and post-intervention periods, measuring RMC practices, HIV stigma, and self-efficacy to provide care. To evaluate patient impacts, we enrolled birthing women at the study facilities in the pre- (n = 229) and post- (n = 214) intervention periods and assessed self-reported RMC and perceptions of provider HIV stigma. We also collected facility-level data on the proportion of patients who gave birth by cesarean section, disaggregated by HIV status. The intervention had a positive impact on all provider outcomes; providers reported using more RMC practices, lower levels of HIV stigma, and greater self-efficacy to provide care for WLHIV. We did not observe differences in self-reported patient outcomes. In facility-level data, we observed a trend in reduction in cesarean section rates for WLHIV (33.0% vs. 24.1%, p = 0.14). The findings suggest that the MAMA training may improve providers' attitudes and practices in caring for WLHIV giving birth and should be considered for scale-up.
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Infecciones por VIH , Servicios de Salud Materna , Estigma Social , Humanos , Femenino , Tanzanía/epidemiología , Infecciones por VIH/psicología , Infecciones por VIH/terapia , Embarazo , Adulto , Aprendizaje Basado en Problemas , Personal de Salud/educación , Personal de Salud/psicología , Entrenamiento Simulado , Respeto , Actitud del Personal de Salud , Parto Obstétrico , Complicaciones Infecciosas del Embarazo/prevención & control , Trabajo de Parto/psicologíaRESUMEN
During their maturation, ribosomal RNAs (rRNAs) are decorated by hundreds of chemical modifications that participate in proper folding of rRNA secondary structures and therefore in ribosomal function. Along with pseudouridine, methylation of the 2'-hydroxyl ribose moiety (Nm) is the most abundant modification of rRNAs. The majority of Nm modifications in eukaryotes are placed by Fibrillarin, a conserved methyltransferase belonging to a ribonucleoprotein complex guided by C/D box small nucleolar RNAs (C/D box snoRNAs). These modifications impact interactions between rRNAs, tRNAs and mRNAs, and some are known to fine tune translation rates and efficiency. In this study, we built the first comprehensive map of Nm sites in Drosophila melanogaster rRNAs using two complementary approaches (RiboMethSeq and Nanopore direct RNA sequencing) and identified their corresponding C/D box snoRNAs by whole-transcriptome sequencing. We de novo identified 61 Nm sites, from which 55 are supported by both sequencing methods, we validated the expression of 106 C/D box snoRNAs and we predicted new or alternative rRNA Nm targets for 31 of them. Comparison of methylation level upon different stresses show only slight but specific variations, indicating that this modification is relatively stable in D. melanogaster. This study paves the way to investigate the impact of snoRNA-mediated 2'-O-methylation on translation and proteostasis in a whole organism.
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Drosophila melanogaster , ARN Nucleolar Pequeño , Animales , ARN Nucleolar Pequeño/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Secuencia de Bases , ARN Ribosómico/metabolismo , MetilaciónRESUMEN
Transfer RNA (tRNA) modifications play a crucial role in maintaining translational fidelity and efficiency, and they may function as regulatory elements in stress response and virulence. Despite their pivotal roles, a comprehensive mapping of tRNA modifications and their associated synthesis genes is still limited, with a predominant focus on free-living bacteria. In this study, we employed a multidisciplinary approach, incorporating comparative genomics, mass spectrometry, and next-generation sequencing, to predict the set of tRNA modification genes responsible for tRNA maturation in two intracellular pathogens- Bartonella henselae Houston I and Bartonella quintana Toulouse, which are causative agents of cat-scratch disease and trench fever, respectively. This analysis presented challenges, particularly because of host RNA contamination, which served as a potential source of error. However, our approach predicted 26 genes responsible for synthesizing 23 distinct tRNA modifications in B. henselae and 22 genes associated with 23 modifications in B. quintana . Notably, akin to other intracellular and symbiotic bacteria, both Bartonella species have undergone substantial reductions in tRNA modification genes, mostly by simplifying the hypermodifications present at positions 34 and 37. B. quintana exhibited the additional loss of four modifications and these were linked to examples of gene decay, providing snapshots of reductive evolution.
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Transfer RNAs (tRNAs) play a central role in protein translation. Studying them has been difficult in part because a simple method to simultaneously quantify their abundance and chemical modifications is lacking. Here we introduce Nano-tRNAseq, a nanopore-based approach to sequence native tRNA populations that provides quantitative estimates of both tRNA abundances and modification dynamics in a single experiment. We show that default nanopore sequencing settings discard the vast majority of tRNA reads, leading to poor sequencing yields and biased representations of tRNA abundances based on their transcript length. Re-processing of raw nanopore current intensity signals leads to a 12-fold increase in the number of recovered tRNA reads and enables recapitulation of accurate tRNA abundances. We then apply Nano-tRNAseq to Saccharomyces cerevisiae tRNA populations, revealing crosstalks and interdependencies between different tRNA modification types within the same molecule and changes in tRNA populations in response to oxidative stress.
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Secuenciación de Nanoporos , Nanoporos , ARN , ARN de Transferencia/química , Análisis de Secuencia de ARN/métodosRESUMEN
BACKGROUND: Respectful maternity care (RMC) is a rights-based approach to childbirth that centers the dignity, autonomy, and well-being of birthing women. This study aimed to examine factors associated with RMC among women giving birth in Tanzania and to examine whether HIV status was associated with self-reported RMC. METHODS: We enrolled 229 postpartum women in six clinics in the Kilimanjaro Region; of them, 103 were living with HIV. Participants completed a survey within 48 h after birth before being discharged. RMC was measured using a 30-item scale with three subscales (dignity and respect; supportive care; communication and autonomy), each standardized from 0 to 100. Univariable and multivariable regression models examined factors associated with RMC. RESULTS: The median score of the full RMC score was 74, differing slightly by subscale: 83 for dignity and respect, 76 for supportive care, and 67 for communication and autonomy. RMC did not differ by HIV status (median 67.0 vs. 67.0, p = 0.89). In multivariable linear regression, women who would not recommend the birth facility to their friends and who did not receive breastfeeding education had significantly lower RMC scores on the full RMC scale. In the dignity and respect subscale, variables associated with significantly lower RMC scores were not being able to read and write, delivering in a public facility, and delivering vaginally. CONCLUSIONS: Although self-reported RMC was generally high, we identified areas for improvement. Practitioners need ongoing training on RMC principles and the delivery of equitable care.
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Eukaryotic ribosomes are complex molecular nanomachines translating genetic information from mRNAs into proteins. There is natural heterogeneity in ribosome composition. The pseudouridylation (ψ) of ribosomal RNAs (rRNAs) is one of the key sources of ribosome heterogeneity. Nevertheless, the functional consequences of ψ-based ribosome heterogeneity and its relevance for human disease are yet to be understood. Using HydraPsiSeq and a chronic disease model of non-osteoarthritic primary human articular chondrocytes exposed to osteoarthritic synovial fluid, we demonstrated that the disease microenvironment is capable of instigating site-specific changes in rRNA ψ profiles. To investigate one of the identified differential rRNA ψ sites (28S-ψ4966), we generated SNORA22 and SNORA33 KO SW1353 cell pools using LentiCRISPRv2/Cas9 and evaluated the ribosome translational capacity by 35S-Met/Cys incorporation, assessed the mode of translation initiation and ribosomal fidelity using dual luciferase reporters, and assessed cellular and ribosomal proteomes by LC-MS/MS. We uncovered that the depletion of SNORA33, but not SNORA22, reduced 28S-ψ4966 levels. The resulting loss of 28S-ψ4966 affected ribosomal protein composition and function and led to specific changes in the cellular proteome. Overall, our pioneering findings demonstrate that cells dynamically respond to disease-relevant changes in their environment by altering their rRNA pseudouridylation profiles, with consequences for ribosome function and the cellular proteome relevant to human disease.
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Proteoma , Espectrometría de Masas en Tándem , Humanos , Cromatografía Liquida , Proteoma/genética , Ribosomas/genética , Procesamiento Postranscripcional del ARN , ARN Ribosómico/genéticaRESUMEN
Burnout, characterized by emotional exhaustion, depersonalization, and a diminished sense of accomplishment, is a serious problem among healthcare workers. Burnout negatively impacts provider well-being, patient outcomes, and healthcare systems globally, and is especially worrisome in settings with a shortage of healthcare workers and resources. The goal of this study is to explore the experience of burnout in a population of labor and delivery (L&D) providers in Tanzania. We examined burnout using three data sources. A structured assessment of burnout was collected at four time points from a sample of 60 L&D providers in six clinics. The same providers participated in an interactive group activity from which we drew observational data on burnout prevalence. Finally, we conducted in-depth interviews (IDIs) with a subset of 15 providers to further explore their experience of burnout. At baseline, prior to any introduction to the concept, 18% of respondents met criteria for burnout. Immediately after a discussion and activity on burnout, 62% of providers met criteria. One- and three- months later, 29% and 33% of providers met criteria, respectively. In IDIs, participants saw the lack of understanding of burnout as the cause for low baseline rates and attributed the subsequent decrease in burnout to newly acquired coping strategies. The activity helped providers realize they were not alone in their experience of burnout. High patient load, low staffing, limited resources, and low pay emerged as contributing factors. Burnout was prevalent among a sample of L&D providers in northern Tanzania. However, a lack of exposure to the concept of burnout leads to providers being unaware of the issue as a collective burden. Therefore, burnout remains rarely discussed and not addressed, thus continuing to impact provider and patient health. Previously validated burnout measures cannot adequately assess burnout without a discussion of the context.
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Newly growing evidence highlights the essential role that epitranscriptomic marks play in the development of many cancers; however, little is known about the role and implications of altered epitranscriptome deposition in prostate cancer. Here, we show that the transfer RNA N7-methylguanosine (m7G) transferase METTL1 is highly expressed in primary and advanced prostate tumours. Mechanistically, we find that METTL1 depletion causes the loss of m7G tRNA methylation and promotes the biogenesis of a novel class of small non-coding RNAs derived from 5'tRNA fragments. 5'tRNA-derived small RNAs steer translation control to favour the synthesis of key regulators of tumour growth suppression, interferon pathway, and immune effectors. Knockdown of Mettl1 in prostate cancer preclinical models increases intratumoural infiltration of pro-inflammatory immune cells and enhances responses to immunotherapy. Collectively, our findings reveal a therapeutically actionable role of METTL1-directed m7G tRNA methylation in cancer cell translation control and tumour biology.
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Carcinogénesis , Neoplasias de la Próstata , Masculino , Humanos , Carcinogénesis/genética , Transformación Celular Neoplásica , Neoplasias de la Próstata/genética , Transcripción Genética , Procesamiento Postranscripcional del ARN , Metiltransferasas/genéticaRESUMEN
In plant cells, a large pool of iron (Fe) is contained in the nucleolus, as well as in chloroplasts and mitochondria. A central determinant for intracellular distribution of Fe is nicotianamine (NA) generated by NICOTIANAMINE SYNTHASE (NAS). Here, we used Arabidopsis thaliana plants with disrupted NAS genes to study the accumulation of nucleolar iron and understand its role in nucleolar functions and more specifically in rRNA gene expression. We found that nas124 triple mutant plants, which contained lower quantities of the iron ligand NA, also contained less iron in the nucleolus. This was concurrent with the expression of normally silenced rRNA genes from nucleolar organizer regions 2 (NOR2). Notably, in nas234 triple mutant plants, which also contained lower quantities of NA, nucleolar iron and rDNA expression were not affected. In contrast, in both nas124 and nas234, specific RNA modifications were differentially regulated in a genotype dependent manner. Taken together, our results highlight the impact of specific NAS activities in RNA gene expression. We discuss the interplay between NA and nucleolar iron with rDNA functional organization and RNA methylation.
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Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , ADN Ribosómico/metabolismo , Metilación , Hierro/metabolismo , ARN Ribosómico/genética , ARN Ribosómico/metabolismoRESUMEN
BACKGROUND: The experience of HIV stigma during intrapartum care can impact women's trust in the health care system and undermine their long-term commitment to HIV care engagement. Delivery of respectful maternity care (RMC) to women living with HIV (WLHIV) can improve quality of life and clinical outcomes. The goal of this study is to conduct an evaluation of MAMA (Mradi wa Afya ya Mama Mzazi, Project to Support the Health of Women Giving Birth), a simulation team-training curriculum for labor and delivery providers that addresses providers' instrumental and attitudinal stigma toward WLHIV and promotes the delivery of evidence-based RMC for WLHIV. METHODS: The MAMA intervention will be evaluated among healthcare providers across six clinics in the Kilimanjaro Region of Tanzania. To evaluate the impact of MAMA, we will enroll WLHIV who give birth in the facilities before (n = 103 WLHIV) and after (n = 103 WLHIV) the intervention. We will examine differences in the primary outcome (perceptions of RMC) and secondary outcomes (postpartum HIV care engagement; perceptions of HIV stigma in the facility; internal HIV stigma; clinical outcomes and evidence-based practices) between women enrolled in the two time periods. Will also assess participating providers (n = 60) at baseline, immediate post, 1-month post training, and 2-month post training. We will examine longitudinal changes in the primary outcome (practices of RMC) and secondary outcomes (stigma toward WLHIV; self-efficacy in delivery intrapartum care). Quality assurance data will be collected to assess intervention feasibility and acceptability. DISCUSSION: The implementation findings will be used to finalize the intervention for a train-the-trainer model that is scalable, and the outcomes data will be used to power a multi-site study to detect significant differences in HIV care engagement. TRIAL REGISTRATION: The trial is registered at clinicaltrials.gov, NCT05271903.
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Infecciones por VIH , Servicios de Salud Materna , Femenino , Humanos , Embarazo , Parto , Aprendizaje Basado en Problemas , Calidad de Vida , TanzaníaRESUMEN
More than 170 posttranscriptional RNA modifications are so far known on both coding and noncoding RNA species. Within this group, pseudouridine (Ψ) and queuosine (Q) represent conserved RNA modifications with fundamental functional roles in regulating translation. Current detection methods of these modifications, which both are reverse transcription (RT)-silent, are mostly based on the chemical treatment of RNA prior to analysis. To overcome the drawbacks associated with indirect detection strategies, we have engineered an RT-active DNA polymerase variant called RT-KTq I614Y that produces error RT signatures specific for Ψ or Q without prior chemical treatment of the RNA samples. Combining this polymerase with next-generation sequencing techniques allows the direct identification of Ψ and Q sites of untreated RNA samples using a single enzymatic tool.
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Nucleósido Q , Seudouridina , ARN Mensajero/metabolismo , Seudouridina/metabolismo , ARN , ARN no Traducido , Procesamiento Postranscripcional del ARNRESUMEN
Background : The experience of HIV stigma during intrapartum care can impact women's trust in the health care system and undermine their long-term commitment to HIV care engagement. Delivery of respectful maternity care (RMC) to WLHIV can improve quality of life and clinical outcomes. The goal of this study is to conduct an evaluation of MAMA (Mradi wa Afya ya Mama Mzazi, Project to Support the Health of Women Giving Birth), a simulation team-training curriculum for labor and delivery providers that addresses providers' instrumental and attitudinal stigma toward WLHIV and promotes the delivery of evidence-based RMC for WLHIV. Methods : The MAMA intervention will be evaluated among healthcare providers across six clinics in the Kilimanjaro Region of Tanzania. To evaluate the impact of MAMA, we will enroll WLHIV who give birth in the facilities before (n=103 WLHIV) and after (n=103 WLHIV) the intervention. We will examine differences in the primary outcome (perceptions of RMC) and secondary outcomes (postpartum HIV care engagement; perceptions of HIV stigma in the facility; internal HIV stigma; clinical outcomes and evidence-based practices) between women enrolled in the two time periods. Will also assess participating providers (n=60) at baseline, immediate post, 1-month post training, and 2-month post training. We will examine longitudinal changes in the primary outcome (practices of RMC) and secondary outcomes (stigma toward WLHIV; self-efficacy in delivery intrapartum care). Quality assurance data will be collected to assess intervention feasibility and acceptability. Discussion : The implementation findings will be used to finalize the intervention for a train-the-trainer model that is scalable, and the outcomes data will be used to power a multi-site study to detect significant differences in HIV care engagement. Trial Registration : The trial is registered at clinicaltrials.gov, NCT05271903.
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Pseudouridine, a modified RNA residue formed by the isomerization of its parental U nucleotide, is prevalent in a majority of cellular RNAs; its presence was reported in tRNA, rRNA, and sn/snoRNA as well as in mRNA/lncRNA. Multiple analytical deep sequencing-based approaches have been proposed for pseudouridine detection and quantification, among which the most popular relies on the use of soluble carbodiimide (termed CMCT). Recently, we developed an alternative protocol for pseudouridine mapping and quantification. The principle is based on protection of pseudouridine against random RNA cleavage by hydrazine/aniline treatment (HydraPsiSeq protocol). This "negative" detection mode requires higher sequencing depth and provides a precise quantification of the pseudouridine content. All "wet-lab" technical details of the HydraPsiSeq protocol have been described in recent publications. Here, we describe all bioinformatics analysis steps required for data processing from raw reads to the pseudouridylation profile of known or unknown RNA.
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ARN Largo no Codificante , ARN , ARN/química , Seudouridina/genética , ARN de Transferencia/genética , ARN Mensajero/genética , ARN Ribosómico/genética , Procesamiento Postranscripcional del ARNRESUMEN
FUS is a multifunctional protein involved in many aspects of RNA metabolism, including transcription, splicing, translation, miRNA processing, and replication-dependent histone gene expression. In this work, we show that FUS depletion results in the differential expression of numerous small nucleolar RNAs (snoRNAs) that guide 2'-O methylation (2'-O-Me) and pseudouridylation of specific positions in ribosomal RNAs (rRNAs) and small nuclear RNAs (snRNAs). Using RiboMeth-seq and HydraPsiSeq for the profiling of 2'-O-Me and pseudouridylation status of rRNA species, we demonstrated considerable hypermodification at several sites in HEK293T and SH-SY5Y cells with FUS knockout (FUS KO) compared to wild-type cells. We observed a similar direction of changes in rRNA modification in differentiated SH-SY5Y cells with the FUS mutation (R495X) related to the severe disease phenotype of amyotrophic lateral sclerosis (ALS). Furthermore, the pattern of modification of some rRNA positions was correlated with the abundance of corresponding guide snoRNAs in FUS KO and FUS R495X cells. Our findings reveal a new role for FUS in modulating the modification pattern of rRNA molecules, that in turn might generate ribosome heterogeneity and constitute a fine-tuning mechanism for translation efficiency/fidelity. Therefore, we suggest that increased levels of 2'-O-Me and pseudouridylation at particular positions in rRNAs from cells with the ALS-linked FUS mutation may represent a possible new translation-related mechanism that underlies disease development and progression.
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Esclerosis Amiotrófica Lateral , Neuroblastoma , Humanos , ARN Nucleolar Pequeño/genética , Células HEK293 , ARN Ribosómico/genética , Proteína FUS de Unión a ARN/genéticaRESUMEN
Modification of tRNA is an integral part of the epitranscriptome with a particularly pronounced potential to generate diversity in RNA expression. Eukaryotic tRNA contains modifications in up to 20% of their nucleotides, but not all sites are always fully modified. Combinations and permutations of partially modified sites in tRNAs can generate a plethora of tRNA isoforms, termed modivariants. Here, we investigate the stoichiometry of incompletely modified sites in tRNAs from human cell lines for their information content. Using a panel of RNA modification mapping methods, we assess the stoichiometry of sites that contain the modifications 5-methylcytidine (m5C), 2'-O-ribose methylation (Nm), 3-methylcytidine (m3C), 7-methylguanosine (m7G), and Dihydrouridine (D). We discovered that up to 75% of sites can be incompletely modified and that the differential modification status of a cellular tRNA population holds information that allows to discriminate e.g. different cell lines. As a further aspect, we investigated potential causal connectivity between tRNA modification and its processing into tRNA fragments (tiRNAs and tRFs). Upon exposure of cultured living cells to cell-penetrating angiogenin, the modification patterns of the corresponding RNA populations was changed. Importantly, we also found that tsRNAs were significantly less modified than their parent tRNAs at numerous sites, suggesting that tsRNAs might derive chiefly from hypomodified tRNAs.
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FTSJ1 is a conserved human 2'-O-methyltransferase (Nm-MTase) that modifies several tRNAs at position 32 and the wobble position 34 in the anticodon loop. Its loss of function has been linked to X-linked intellectual disability (XLID), and more recently to cancers. However, the molecular mechanisms underlying these pathologies are currently unclear. Here, we report a novel FTSJ1 pathogenic variant from an X-linked intellectual disability patient. Using blood cells derived from this patient and other affected individuals carrying FTSJ1 mutations, we performed an unbiased and comprehensive RiboMethSeq analysis to map the ribose methylation on all human tRNAs and identify novel targets. In addition, we performed a transcriptome analysis in these cells and found that several genes previously associated with intellectual disability and cancers were deregulated. We also found changes in the miRNA population that suggest potential cross-regulation of some miRNAs with these key mRNA targets. Finally, we show that differentiation of FTSJ1-depleted human neural progenitor cells into neurons displays long and thin spine neurites compared with control cells. These defects are also observed in Drosophila and are associated with long-term memory deficits. Altogether, our study adds insight into FTSJ1 pathologies in humans and flies by the identification of novel FTSJ1 targets and the defect in neuron morphology.