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Site specific pseudouridine (Psi) on Leishmania rRNA regulates translation of subset of proteins

ABSTRACT: Leishmania are unicellular parasites that cycle between the insect and mammalian host and is the causative agent of Leishmaniasis. Previous studies from our lab suggested that individual rRNA modification such as pseudouridine (Psi) and 2-O-methylation are developmentally regulated during the complex life cycle of these parasites. Ablation of a single snoRNA guiding Psi in the inter-subunit bridge, helix69 of the ribosome affects its affinity to translate specific mRNA. Mutational analysis in the pseudouridylation pocket of this snoRNA supports the notion that the guided modification and not the chaperone activity, is essential for the observed phenotype. Structural studies using CryoEM suggested that a single Psi modification can affect the rRNA structure. We propose that alteration in rRNA modifications could generate ribosomes preferentially translating state-beneficial proteins.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Leishmania Major Strain Friedlin

SUBMITTER: Tirza Doniger

LAB HEAD: Prof. Shulamit Michaeli

PROVIDER: PXD034116 | Pride | 2024-05-09

REPOSITORIES: Pride

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Role of H/ACA snoRNA in translation

Project description:Trypanosomes are unicellular parasites that cycle between the insect and mammalian host and is the causative agent of African sleeping sickness and the wasting disease, nagana, in cattle. Previous studies from our lab suggested that individual rRNA modification such as pseudouridine (Y) and 2â€™-O-methylation are developmentally regulated during the complex life cycle of these parasites. In this study we identified that the Y composition of translating ribosomes are different from rRNA derived from total RNA. Ablation of single snoRNA guiding Y in one of the inter-subunit bridges of the ribosome affects its affinity to translate specific mRNA. Mutational analysis in the pseudouridylation pocket of this snoRNA supports for the notion that the guided modification and not the chaperonic activity, is essential for the observed phenotype. Structural probing of rRNA using DMS-seq suggested that a single modification can affect the rRNA structure locally as wells as globally. Quantitative profiling of the composition of mutant ribosomes suggested the absence of eS12 ribosomal protein in the mature ribosomes. Addback experiments validated the role of eS12 during the loss of a single Y in these mutant cells. Orthologous experiments in related leishmania spp. also verify importance of a single Y in the function of ribosome. Ongoing experiments intend to visualize such ribosomes lacking a single RNA modification and decipher their significance towards ribosome structure.

2024-01-26 | PXD030544 | Pride

A radiolabeling-free, qPCR-based method for locus-specific pseudouridine detection

Project description:Pseudouridine (Ψ) is the most abundant post-transcriptional RNA modification. Various methods have been developed to achieve locus-specific Ψ detection; however, the existing methods often involve radiolabeling of RNA, require advanced experimental skills and can be time-consuming. Herein we report a radiolabeling-free，qPCR-based method to detect locus-specific Ψs in rRNA and mRNA. This method is based on Ψ chemical adduct (Ψ-CMC) induced mutation/deletion during reverse transcription (RT), leading to qPCR products of different melting temperatures. Utilizing high-throughput sequencing, we demonstrate that such misincorporation is a general feature of Ψ-CMC adduct during our improved RT conditions. We validated this method on known Ψ sites in rRNA and showed that the melting curves correlate with the modification level. Moreover, we successfully detected Ψs in mRNA and lncRNA of different abundance, and identified Ψ synthase that targets mRNA. Our facile method takes only 1.5 days to complete, and with slight adjustment it can be applied to detect other epitranscriptomic marks in the transcriptome.

2017-10-03 | GSE102476 | GEO

Heterozygous SNORA31 mutations in patients with herpes simplex encephalitis

Project description:Pseudo-seq was used to measure differences in the extent of pseudouridine (Ψ) modification in rRNA from human cell lines with heterozygous, and homozygous disruptions of SNORA31.

2019-10-14 | GSE102078 | GEO

Chemical Pull-Down Reveals Comprehensive and Dynamic Pseudouridylation in Mammalian Transcriptome

Project description:Pseudouridine (Ψ) is the most abundant RNA modification, yet little is known about its content, dynamics and function in mRNA and ncRNA. Here, we perform quantitative MS analysis and develop CAP-seq for transcriptome-wide Ψ profiling. The unexpected high Ψ content (Ψ/U ratio: ~ 0.2% to 0.6%) indicates that pseudouridylation in mammalian mRNA is much more prevalent and comprehensive than previously believed. In concordance, CAP-seq identified 2,084 Ψ sites within 1,929 human transcripts. We prove four previously unknown Ψ sites in rRNA and EEF1A1 mRNA. Genetic and biochemical analysis uncover PUS1 as a major human mRNA Ψ synthase. In response to stimuli, Ψ level and sites are dynamically modulated in stimulus-specific manners. Comparisons between human and mouse pseudouridylation reveal conserved and unique sites across tissue and species. We observe stop codon pseudouridylation and readthrough events simultaneously for HSPB1 mRNA, indicating a role in nonsense suppression. Together, these approaches allow in-depth analysis of transcriptome-wide pseudouridylation events and our comprehensive study provides a resource for functional studies of Ψ-mediated epigenetic regulation. Here we report a transcriptome-wide profiling method that utilizes a chemically synthesized N3-CMC, which pre-enriches the Ψ-containing RNAs and blocks the reverse transcription.Mapping the Ψ sites in human transcriptome was performed using HEK293T and PUS1 dependent Ψ sites were identified by comparing PUS1 knock out cells with wildtype cells. Stress inducible or suppressed Ψ sites were identified by comparing stress treated cells with untreated cells. And mouse brain and liver were used to map Ψ sites in mouse transcriptome.

2015-06-05 | E-GEOD-63655 | biostudies-arrayexpress

Transcriptome-wide analysis of pseudouridinylation in Drosophila melanogaster

Project description:Pseudouridine (Psi) is one of the most frequent post-transcriptional modification of RNA. Enzymatic Psi modification occurs on rRNA, snRNA, snoRNA, tRNA, non-coding RNA and has recently been discovered on mRNA. Transcriptome-wide detection of Psi (Psi-seq) has yet to be performed for the widely studied model organism Drosophila melanogaster. Here, we optimized Psi-seq analysis for this species and have identified thousands of Psi modifications throughout the female fly head transcriptome. We find that Psi is widespread on both cellular and mitochondrial rRNAs. In addition, more than a thousand Psi sites were found on mRNAs. When pseudouridinylated, mRNAs frequently had many Psi sites. Many mRNA Psi sites are present in genes encoding for ribosomal proteins, and many are found in mitochondrial encoded RNAs, further implicating the importance of pseudouridinylation for ribosome and mitochondrial function. The 7SLRNA of the signal recognition particle is the non-coding RNA most enriched for Psi. The three mRNAs most enriched for Psi encode highly-expressed yolk proteins (YP1, YP2, YP3). By comparing the pseudouridine profiles in the RluA-2 mutant and the w1118 control genotype, we identified Psi sites that were missing in the mutant RNA as potential RluA-2 targets. Finally, differential gene expression analysis of the mutant transcriptome indicates a major impact of loss of RluA-2 on the ribosome and translational machinery.

2022-12-13 | GSE213312 | GEO

Chemical Pull-Down Reveals Comprehensive and Dynamic Pseudouridylation in Mammalian Transcriptome

2015-06-05 | GSE63655 | GEO

Pseudouridylation in Naf1+/- mice

Project description:Pseudo-seq was used to measure differences in the extent of pseudouridine (Ψ) modification in rRNA from mice haploinsufficient for the H/ACA biogenesis factor Naf1 compared to wild- type. We measured telomerase RNA levels as well as a sample of box H/ACA RNAs and found they were all decreased in Naf1+/- mice. Nevertheless, Naf1+/- mice had minimally decreased levels of pseudouridylation at the rRNA sites queried. This was not associated with any phenotypic abnormalities in vivo in these mice which we examined for hematopoeitic, liver, testes and brain defects.

2016-08-07 | GSE78063 | GEO

Quantification of mock microbial communities with metagenomes, 16S rRNA gene amplicons and metaproteomics

Project description:In this study we developed metaproteomics based methods for quantifying taxonomic composition of microbiomes (microbial communities). We also compared metaproteomics based quantification to other quantification methods, namely metagenomics and 16S rRNA gene amplicon sequencing. The metagenomic and 16S rRNA data can be found in the European Nucleotide Archive (Study number: PRJEB19901). For the method development and comparison of the methods we analyzed three types of mock communities with all three methods. The communities contain between 28 to 32 species and strains of bacteria, archaea, eukaryotes and bacteriophage. For each community type 4 biological replicate communities were generated. All four replicates were analyzed by 16S rRNA sequencing and metaproteomics. Three replicates of each community type were analyzed with metagenomics. The "C" type communities have same cell/phage particle number for all community members (C1 to C4). The "P" type communities have the same protein content for all community members (P1 to P4). The "U" (UNEVEN) type communities cover a large range of protein amounts and cell numbers (U1 to U4). We also generated proteomic data for four pure cultures to test the specificity of the protein inference method. This data is also included in this submission.

2017-05-12 | PXD006118 | Pride

ChIP-seq of H1 human embryonic stem cells (hESCs) to map the global binding profile of EZH2 to investigate the association of PRDM14 binding with the PRC2 in hESCs

Project description:An experiment was performed to map the global binding profile of EZH2 in human embryonic stem cells (hESCs). How the Polycomb Repressive Complex 2 (PRC2) is recruited to the DNA in hESC remains largely unknown. Previous studies on the transcription factor PRDM14 suggested that PRDM14 plays a repressive role in hESCs. Here, we mapped the global binding profile of EZH2 in hESC to investigate the association of PRDM14 binding with the PRC2 in hESCs. EZH2 binding is found to be enriched in PRDM14 binding sites. The PRC2 dependent repressive role of PRDM14 is further supported by the strong correlation of PRDM14 binding with the repressive histone modification H3K27me3.

2012-11-01 | E-MTAB-1305 | biostudies-arrayexpress

BID-seq for transcriptome-wide quantitative sequencing of mRNA pseudouridine at base resolution

Project description:Pseudouridine (Ψ) is an abundant RNA modification that is present in and impacts the functions of diverse non-coding RNA species, including rRNA, tRNA, snRNA, etc. Pseudouridine also exists in mammalian mRNA and likely exhibits functional roles; however, functional investigations of pseudouridine in mammalian mRNA have been hampered by the lack of a quantitative method that detects Ψ at base precision. We have recently developed Bisulfite-Induced Deletion sequencing (BID-seq), which provides the community with a quantitative method to map RNA Ψ distribution transcriptome-wide at single-base resolution. Here, we describe an optimized BID-seq protocol to generate highly reproducible results, displaying nearly zero background deletions at unmodified uridines after bisulfite treatment and uncovering 8,407 Ψ sites starting from as little as 10 ng mouse embryonic stem cell (mESC) polyA+ RNA, with the steps that are fast in both library preparation and data analysis. This optimized BID-seq workflow takes 5 days to complete and includes four main sections: RNA preparation, library construction, next-generation sequencing (NGS), and data analysis. The library construction can be completed by researchers who have basic knowledge and skills in molecular biology and genetics. In addition to the experimental protocol, we provide BID-pipe (https://github.com/y9c/pseudoU-BIDseq), a user-friendly data analysis pipeline for BID-seq, requiring only basic bioinformatic and computational skills to uncover Ψ signatures from NGS data.

2023-08-07 | GSE238245 | GEO

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