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ISSN / EISSN : 1355-8382 / 1469-9001
Published by: Cold Spring Harbor Laboratory (10.1261)
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Published: 20 October 2021
Abstract:
RNP granules are ubiquitous features of eukaryotic cells. Several observations argue that the formation of at least some RNP granules can be considered analogous to the formation of unfolded protein aggregates. First, unfolded protein aggregates form from the exposure of promiscuous protein interaction surfaces, while some mRNP granules form, at least in part, by promiscuous intermolecular RNA-RNA interactions due to exposed RNA surfaces when mRNAs are not engaged with ribosomes. Second, analogous to the role of protein chaperones in preventing misfolded protein aggregation, cells contain abundant “RNA chaperones” to limit inappropriate RNA-RNA interactions and prevent mRNP granule formation. Third, analogous to the role of protein aggregates in diseases, situations where RNA aggregation exceeds the capacity of RNA chaperones to disaggregate RNAs may contribute to human disease. Understanding that RNP granules can be considered as promiscuous, reversible RNA aggregation events allows insight into their composition and how cells have evolved functions for RNP granules.
Pedro Prudencio, Rosina Savisaar, Kenny Rebelo, Rui Goncalo Martinho, Maria Carmo-Fonseca
Published: 19 October 2021
Abstract:
Widespread co-transcriptional splicing has been demonstrated from yeast to human. However, most studies to date addressing the kinetics of splicing relative to transcription used either Saccharomyces cerevisiae or metazoan cultured cell lines. Here, we adapted native elongating transcript sequencing technology (NET-seq) to measure co-transcriptional splicing dynamics during the early developmental stages of Drosophila melanogaster embryos. Our results reveal the position of RNA polymerase II (Pol II) when both canonical and recursive splicing occur. We found heterogeneity in splicing dynamics, with some RNAs spliced immediately after intron transcription, whereas for other transcripts no splicing was observed over the first 100 nucleotides of the downstream exon. Introns that show splicing completion before Pol II has reached the end of the downstream exon are necessarily intron-defined. We studied the splicing dynamics of both nascent pre-mRNAs transcribed in the early embryo, which have few and short introns, as well as pre-mRNAs transcribed later in embryonic development, which contain multiple long introns. As expected, we found a relationship between the proportion of spliced reads and intron size. However, intron definition was observed at all intron sizes. We further observed that genes transcribed in the early embryo tend to be isolated in the genome whereas genes transcribed later are often overlapped by a neighboring convergent gene. In isolated genes, transcription termination occurred soon after the polyadenylation site, while in overlapped genes Pol II persisted associated with the DNA template after cleavage and polyadenylation of the nascent transcript. Taken together, our data unravels novel dynamic features of Pol II transcription and splicing in the developing Drosophila embryo.
Alexandre Garus, Chantal Autexier
Published: 23 September 2021
Abstract:
Dyskerin and its homologues are ancient and conserved enzymes that catalyse the most common posttranscriptional modification found in cells, pseudouridylation. The resulting pseudouridines provide stability to RNA molecules and regulate ribosome biogenesis and splicing events. Dyskerin does not act independently – it is the core component of a protein heterotetramer, which associates with RNAs that contain the H/ACA motif. The variety of H/ACA RNAs that guide the function of this ribonucleoprotein (RNP) complex highlight the diversity of cellular processes in which dyskerin participates. When associated with small nucleolar (sno) RNAs, it regulates ribosomal (r) RNAs and ribosome biogenesis. By interacting with small Cajal Body (sca) RNAs, it targets small nuclear (sn) RNAs to regulate pre-mRNA splicing. As a component of the telomerase holoenzyme, dyskerin binds to the telomerase RNA to modulate telomere maintenance. In a disease context, dyskerin malfunction can result in multiple detrimental phenotypes. Mutations in DKC1, the gene that encodes dyskerin, cause the premature aging syndrome X-linked dyskeratosis congenita (X-DC), a still incurable disorder that typically leads to bone marrow failure. In this review, we present the classical and most recent findings on this essential protein, discussing the evolutionary, structural and functional aspects of dyskerin and the H/ACA RNP. The latest research underscores the role that dyskerin plays in the regulation of gene expression, translation efficiency and telomere maintenance, along with the impacts that defective dyskerin has on aging, cell proliferation, haematopoietic potential and cancer.
Elena Martin, , Malgorzata Rogalska, Jorge Herrero, Juan Valcarcel
Published: 20 September 2021
Abstract:
The regulation of pre-mRNA processing has important consequences for cell division and the control of cancer cell proliferation but the underlying molecular mechanisms remain poorly understood. We report that three splicing factors, SPF45, SR140 and CHERP form a tight physical and functionally coherent complex that regulates a variety of alternative splicing events, frequently by repressing short exons flanked by suboptimal 3' splice sites. These comprise alternative exons embedded in genes with important functions in cell cycle progression, including the G2/M key regulator FOXM1 and the spindle regulator SPDL1. Knockdown of either of the three factors leads to G2/M arrest and to enhanced apoptosis in HeLa cells. Promoting the changes in FOXM1 or SPDL1 splicing induced by SPF45/SR140/CHERP knockdown partially recapitulate the effects on cell growth, arguing that the complex orchestrates a program of alternative splicing necessary for efficient cell proliferation.
, Margaret Gadek, , Kevin Wilkins,
Published: 17 September 2021
Abstract:
DDX3 is a DEAD-box RNA helicase that regulates translation and is encoded by the X- and Y-linked paralogs DDX3X and DDX3Y. While DDX3X is ubiquitously expressed in human tissues and essential for viability, DDX3Y is male-specific and shows lower and more variable expression than DDX3X in somatic tissues. Heterozygous genetic lesions in DDX3X mediate a class of developmental disorders called DDX3X syndrome, while loss of DDX3Y is implicated in male infertility. One possible explanation for female-bias in DDX3X syndrome is that DDX3Y encodes a polypeptide with different biochemical activity. In this study, we use ribosome profiling and in vitro translation to demonstrate that the X- and Y-linked paralogs of DDX3 play functionally redundant roles in translation. We find that transcripts that are sensitive to DDX3X depletion or mutation are rescued by complementation with DDX3Y. Our data indicate that DDX3X and DDX3Y proteins can functionally complement each other in the context of mRNA translation in human cells. DDX3Y is not expressed in a large fraction of the central nervous system. These findings suggest that expression differences, not differences in paralog-dependent protein synthesis, underlie the sex-bias of DDX3X-associated diseases.
Published: 17 September 2021
Abstract:
BACKGROUND: Conversion of adenosine to inosine in RNA by ADAR enzymes occurs at thousands of sites in the human transcriptome, and is essential for healthy brain development. This editing process is dysregulated in many neuropsychiatric diseases, but has not yet been investigated at the level of individual neurons. METHODS: We quantified RNA editing sites in full-length capture nuclear transcriptomes of 3055 neurons from six cortical regions of a neurotypical post-mortem female donor. Putative editing sites were intersected with sites in bulk human tissue transcriptomes including healthy and neuropsychiatric brain tissue, and sites identified in single nuclei from unrelated brain donors. Differential editing between cell types and cortical regions, and individual sites and genes therein, was quantified using linear models. Associations between gene abundance and editing were also tested. RESULTS: We identified 41,930 RNA editing sites with robust read coverage in at least ten neuronal nuclei. Most sites were located within Alu repeats in introns or 3’ UTRs, and approximately 80% were catalogued in published RNA editing databases. We identified 9285 putative novel RNA editing sites, 29% of which were also detectable in neuronal transcriptomes from unrelated donors. Among the strongest correlates of global editing rates were snoRNAs from the SNORD115 and SNORD116 cluster (15q11), known to modulate serotonin receptor processing and to colocalize with ADAR2. Autism related genes were enriched with editing sites predicted to modify RNA structure. Inhibitory neurons showed higher overall transcriptome editing than excitatory neurons. Additionally, we identified 29 genes preferentially edited in excitatory neurons and 43 genes edited more heavily in inhibitory neurons including RBFOX1, its target genes, and small nucleolar RNA-associated genes in the autism-associated Prader-Willi locus 15q11. These results provide cell-type and spatial context for 1730 sites that are differentially edited in the brains of schizophrenic patients, and 910 sites in autistic patients. CONCLUSIONS: RNA editing, including thousands of previously unreported sites, is robustly detectable in single neuronal nuclei, where gene editing differences are stronger between cell subtypes than between cortical regions. Insufficient editing of autism-related genes in inhibitory neurons may manifest in the specific perturbation of these cells in autism.
Nicolas J Tourasse, Fabien Darfeuille
Published: 16 September 2021
Abstract:
Type I toxin-antitoxin (T1TA) systems constitute a large class of genetic modules with antisense RNA (asRNA)-mediated regulation of gene expression. They are widespread in bacteria and consist of an mRNA coding for a toxic protein and a noncoding asRNA that acts as an antitoxin preventing the synthesis of the toxin by directly basepairing to its cognate mRNA. The co- and post-transcriptional regulation of T1TA systems is intimately linked to RNA sequence and structure, therefore it is essential to have an accurate annotation of the mRNA and asRNA molecules to understand this regulation. However, most T1TA systems have been identified by means of bioinformatic analyses solely based on the toxin protein sequences, and there is no central repository of information on their specific RNA features. Here we present the first database dedicated to type I TA systems, named T1TAdb. It is an open-access web database (https://d-lab.arna.cnrs.fr/t1tadb) with a collection of ~1,900 loci in ~500 bacterial strains in which a toxin-coding sequence has been previously identified. RNA molecules were annotated with a bioinformatic procedure based on key determinants of the mRNA structure and the genetic organization of the T1TA loci. Besides RNA and protein secondary structure predictions, T1TAdb also identifies promoter, ribosome-binding, and mRNA-asRNA interaction sites. It also includes tools for comparative analysis, such as sequence similarity search and computation of structural multiple alignments, which are annotated with covariation information. To our knowledge, T1TAdb represents the largest collection of features, sequences, and structural annotations on this class of genetic modules.
Man Wu, Liang-Zhong Yang, Ling-Ling Chen
Published: 15 September 2021
Abstract:
Although long noncoding RNAs (lncRNAs) are generally expressed at low levels, emerging evidence has revealed that many play important roles in gene regulation by a variety of mechanisms as they engage with proteins. Given that the abundance of proteins often greatly exceeds that of their interacting lncRNAs, quantification of the relative abundance, or even the exact stoichiometry in some cases, within lncRNA-protein complexes is helpful for understanding of the mechanism(s) of action of lncRNAs. We discuss methods used to examine lncRNA and protein expression at the single cell, sub-cellular and sub-organelle levels, the average and local lncRNA concentration in cells, as well as how lncRNAs can modulate the functions of their interacting proteins even at a low stoichiometric concentration.
Dominik Burri,
Published: 14 September 2021
Abstract:
During pre-mRNA maturation 3p end processing can occur at different polyadenylation sites in the 3 prime untranslated region (3p UTR) to give rise to transcript isoforms that differ in the length of their 3p UTRs. Longer 3p UTRs contain additional cis-regulatory elements that impact the fate of the transcript and/or of the resulting protein. Extensive alternative polyadenylation (APA) has been observed in cancers, but the mechanisms and roles remain elusive. In particular, it is unclear whether the APA occurs in the malignant cells or in other cell types that infiltrate the tumor. To resolve this, we developed a computational method, called SCUREL, that quantifies changes in 3p UTR length between groups of cells, including cells of the same type originating from tumor and control tissue. We used this method to study APA in human lung adenocarcinoma (LUAD). SCUREL relies solely on annotated 3p UTRs and on control systems, such as T cell activation and spermatogenesis gives qualitatively similar results at much greater sensitivity compared to the previously published scAPA method. In the LUAD samples, we find a general trend towards 3p UTR shortening not only in cancer cells compared to the cell type of origin, but also when comparing other cell types from the tumor vs. the control tissue environment. However, we also find high variability in the individual targets between patients. The findings help to understand the extent and impact of APA in LUAD, which may support improvements in diagnosis and treatment.
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