scholarly journals Pseudouridine synthases modify human pre-mRNA co-transcriptionally and affect splicing

2020 ◽  
Author(s):  
Nicole M. Martinez ◽  
Amanda Su ◽  
Julia K. Nussbacher ◽  
Margaret C. Burns ◽  
Cassandra Schaening ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Genetic Manipulation ◽  
Rna Modification ◽  
Messenger Rnas ◽  
Specific Expression ◽  
Pseudouridine Synthases ◽  
In Cells

AbstractEukaryotic messenger RNAs are extensively decorated with modified nucleotides and the resulting epitranscriptome plays important regulatory roles in cells 1. Pseudouridine (Ψ) is a modified nucleotide that is prevalent in human mRNAs and can be dynamically regulated 2–5. However, it is unclear when in their life cycle RNAs become pseudouridylated and what the endogenous functions of mRNA pseudouridylation are. To determine if pseudouridine is added co-transcriptionally, we conducted pseudouridine profiling 2 on chromatin-associated RNA to reveal thousands of intronic pseudouridines in nascent pre-mRNA at locations that are significantly associated with alternatively spliced exons, enriched near splice sites, and overlap hundreds of binding sites for regulatory RNA binding proteins. Multiple distinct pseudouridine synthases with tissue-specific expression pseudouridylate pre-mRNA sites, and genetic manipulation of the predominant pre-mRNA modifying pseudouridine synthases PUS1, PUS7 and RPUSD4 induced widespread changes in alternative splicing in cells, supporting a role for pre-mRNA pseudouridylation in alternative splicing regulation. Consistently, we find that individual pseudouridines identified in cells are sufficient to directly affect splicing in vitro. Together with previously observed effects of artificial pseudouridylation on RNA-RNA6–8 and RNA-protein 9–11 interactions that are relevant for splicing, our results demonstrate widespread co-transcriptional pre-mRNA pseudouridylation and establish the enormous potential for this RNA modification to control human gene expression.

scholarly journals Loss of Pseudouridine Synthases in the RluA Family Causes Hypersensitive Nociception in Drosophila

2020 ◽  
Vol 10 (12) ◽  
pp. 4425-4438
Author(s):  
Wan Song ◽  
Susanne Ressl ◽  
W. Daniel Tracey
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Null Alleles ◽  
Rna Modification ◽  
Loss Of Function ◽  
Specific Expression ◽  
Nociceptive Neurons ◽  
Link Type ◽  
Pseudouridine Synthases ◽  
Link Loss

Nociceptive neurons of Drosophila melanogaster larvae are characterized by highly branched dendritic processes whose proper morphogenesis relies on a large number of RNA-binding proteins. Post-transcriptional regulation of RNA in these dendrites has been found to play an important role in their function. Here, we investigate the neuronal functions of two putative RNA modification genes, RluA-1 and RluA-2, which are predicted to encode pseudouridine synthases. RluA-1 is specifically expressed in larval sensory neurons while RluA-2 expression is ubiquitous. Nociceptor-specific RNAi knockdown of RluA-1 caused hypersensitive nociception phenotypes, which were recapitulated with genetic null alleles. These were rescued with genomic duplication and nociceptor-specific expression of UAS-RluA-1-cDNA. As with RluA-1, RluA-2 loss of function mutants also displayed hyperalgesia. Interestingly, nociceptor neuron dendrites showed a hyperbranched morphology in the RluA-1 mutants. The latter may be a cause or a consequence of heightened sensitivity in mutant nociception behaviors.

scholarly journals Loss of pseudouridine synthases in the RluA family causes hypersensitive nociception in Drosophila

2019 ◽  
Author(s):  
Wan Song ◽  
W. Daniel Tracey
Keyword(s):  
Sensory Neurons ◽  
Neuronal Excitability ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Null Alleles ◽  
Rna Modification ◽  
Loss Of Function ◽  
Specific Expression ◽  
Nociceptive Neurons ◽  
Pseudouridine Synthases

AbstractNociceptive neurons of Drosophila melanogaster larvae are characterized by highly branched dendritic processes whose proper morphogenesis relies on a large number of RNA-binding proteins. Post-transcriptional regulation of RNA in these dendrites has been found to play an important role in their function. Here, we investigate the neuronal functions of two putative RNA modification genes, RluA-1 and RluA-2, which are predicted to encode pseudouridine synthases. RluA-1 is specifically expressed in larval sensory neurons while RluA-2 expression is ubiquitous. Nociceptor-specific RNAi knockdown of RluA-1 caused hypersensitive nociception phenotypes, which were recapitulated with genetic null alleles. These were rescued with genomic duplication and nociceptor-specific expression of UAS-RluA-1-cDNA. As with RluA-1, RluA-2 loss of function mutants also displayed hyperalgesia. Interestingly, nociceptor neuron dendrites showed a hyperbranched morphology in the RluA-1 mutants. The latter may be a cause or a consequence of heightened sensitivity in mutant nociception behaviors.Author SummaryPseudouridine (Psi) is a C5-glycoside isomer of uridine and it is the most common posttranscriptional modification of RNAs, including noncoding tRNAs, rRNAs, snRNAs as well as mRNAs. Although first discovered in the 1950s, the biological functions of Psi in multicellular organisms are not well understood. Interestingly, a marker for sensory neurons in Drosophila encodes for a putative pseudouridine synthase called RluA-1. Here, we report our characterization of nociception phenotypes for larvae with RluA-1 loss of function along with that of a related gene RluA-2. Disrupting either or both RluA-1 and RluA-2 resulted in hypersensitive nociception. In addition, RluA-1 mutants have more highly branched nociceptor neurites that innervate the epidermis. Our studies suggest an important role for the RluA family in nociception. This may be through its action on RNAs that regulate neuronal excitability and/or dendrite morphogenesis.

scholarly journals Alteration of mRNA 5-Methylcytosine Modification in Neurons After OGD/R and Potential Roles in Cell Stress Response and Apoptosis

2021 ◽  
Vol 12 ◽  
Author(s):  
Huan Jian ◽  
Chi Zhang ◽  
ZhangYang Qi ◽  
Xueying Li ◽  
Yongfu Lou ◽  
...  
Keyword(s):  
Rna Binding ◽  
Ischemia Reperfusion Injury ◽  
Rna Binding Proteins ◽  
Ischemia Reperfusion ◽  
Mrna Translation ◽  
Cerebral Injury ◽  
Rna Modification ◽  
Hypermethylated Genes

Epigenetic modifications play an important role in central nervous system disorders. As a widespread posttranscriptional RNA modification, the role of the m5C modification in cerebral ischemia-reperfusion injury (IRI) remains poorly defined. Here, we successfully constructed a neuronal oxygen-glucose deprivation/reoxygenation (OGD/R) model and obtained an overview of the transcriptome-wide m5C profiles using RNA-BS-seq. We discovered that the distribution of neuronal m5C modifications was highly conserved, significantly enriched in CG-rich regions and concentrated in the mRNA translation initiation regions. After OGD/R, modification level of m5C increased, whereas the number of methylated mRNA genes decreased. The amount of overlap of m5C sites with the binding sites of most RNA-binding proteins increased significantly, except for that of the RBM3-binding protein. Moreover, hypermethylated genes in neurons were significantly enriched in pathological processes, and the hub hypermethylated genes RPL8 and RPS9 identified by the protein-protein interaction network were significantly related to cerebral injury. Furthermore, the upregulated transcripts with hypermethylated modification were enriched in the processes involved in response to stress and regulation of apoptosis, and these processes were not identified in hypomethylated transcripts. In final, we verified that OGD/R induced neuronal apoptosis in vitro using TUNEL and western blot assays. Our study identified novel m5C mRNAs associated with ischemia-reperfusion in neurons, providing valuable perspectives for future studies on the role of the RNA methylation in cerebral IRI.

scholarly journals Selective sorting of microRNAs into exosomes by phase-separated YBX1 condensates

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Xiao-Man Liu ◽  
Liang Ma ◽  
Randy Schekman
Keyword(s):  
Phase Separation ◽  
Rna Binding ◽  
Cultured Cells ◽  
Rna Binding Proteins ◽  
Point Mutations ◽  
Cell Communication ◽  
Mediate Cell ◽  
Local Enrichment ◽  
In Cells

Exosomes may mediate cell-to-cell communication by transporting various proteins and nucleic acids to neighboring cells. Some protein and RNA cargoes are significantly enriched in exosomes. How cells efficiently and selectively sort them into exosomes remains incompletely explored. Previously we reported that YBX1 is required in sorting of miR-223 into exosomes. Here we show that YBX1 undergoes liquid-liquid phase separation (LLPS) in vitro and in cells. YBX1 condensates selectively recruit miR-223 in vitro and into exosomes secreted by cultured cells. Point mutations that inhibit YBX1 phase separation impair the incorporation of YBX1 protein into biomolecular condensates formed in cells, and perturb miR-233 sorting into exosomes. We propose that phase separation-mediated local enrichment of cytosolic RNA binding proteins and their cognate RNAs enables their targeting and packaging by vesicles that bud into multivesicular bodies. This provides a possible mechanism for efficient and selective engulfment of cytosolic proteins and RNAs into intraluminal vesicles which are then secreted as exosomes from cells.

scholarly journals m6A methyltransferase RBM15 promotes cardiomyocytes survival under hypoxia by increasing Thbs4 RNA methylation and mediating PI3K/Akt signaling

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
H Cheng ◽  
X.Y Song ◽  
J.Q Xue ◽  
L Chen ◽  
R.D Xu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Basic Mechanism ◽  
Vital Role ◽  
Rna Modification ◽  
Binding Motif ◽  
Funding Source

Abstract Background mRNA modifications constitute ancient mechanisms in regulating gene expression after transcription. N6-methyladenosis (m6A), which is the most prevalent internal RNA modification, is not only installed by m6A methyltransferases, removed by demethylases, but also specifically bounded by RNA-binding proteins. As a significant component in the m6A methyltransferase complex, RNA binding motif protein 15 (RBM15) plays a vital role in m6A methylation. Nevertheless, its function and mechanism in myocardial infarction (MI) remain poorly defined. Purpose To investigate the role and mechanism of RBM15 in regulating its targets through m6A methylation in MI. The research results will not only add new content to the basic mechanism of myocardial protection but also provide new ideas and new targets for the prevention and treatment of MI. Methods Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to clarify the total m6A level, and Reverse transcription quantitative real-time PCR (RT-qPCR) and Western blot were used to determine the expression of RBM15 in normal and MI tissues. Then the effects of RBM15 on cardiomyocytes were clarified using apoptosis assay, and cell proliferation assay. Methylated RNA immunoprecipitation sequencing (MeRIP-seq), and transcriptomic RNA sequencing (RNA-seq) were used to perform the regulator mechanism of RBM15 on target gene Thbs4 in MI. Results In this research, we showed that total m6A methylation was increased in MI, and RBM15 was a main factor involved with this process. Silencing RBM15 remarkably decreased cell proliferation and increased apoptosis in vitro, and resulted in severe cardiac remodeling and further exacerbation of cardiac dysfunction in vivo, whereas its overexpression caused the opposite effects. Then, Thbs4 was identified as a direct downstream target of RBM15, and RBM15 induced m6A methylation on the 3'UTR of Thbs4 pre-mRNA. We also found that it showed faster Thbs4 mRNA decay and exhibited decreased mRNAs and levels of protein expression in RBM15-deficient cardiomyocytes under hypoxia. Furthermore, we confirmed that RBM15 contributed significantly to regulate the PI3k/Akt pathway. Conclusions Our work uncovers a complex RBM15-Thbs4-PI3K/Akt regulatory model based on m6A methylation and provides a new insight into the epi-transcriptomic dysregulation in MI development. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): National Natural Science Foundation of China

scholarly journals Selective sorting of microRNAs into exosomes by phase-separated YBX1 condensates

2021 ◽  
Author(s):  
Xiao-Man Liu ◽  
Liang Ma ◽  
Randy Schekman
Keyword(s):  
Phase Separation ◽  
Rna Binding ◽  
Cultured Cells ◽  
Rna Binding Proteins ◽  
Point Mutations ◽  
Cell Communication ◽  
Mediate Cell ◽  
Local Enrichment ◽  
In Cells

Exosomes may mediate cell-to-cell communication by transporting various proteins and nucleic acids to neighboring cells. Some protein and RNA cargoes are significantly enriched in exosomes. How cells efficiently and selectively sort them into exosomes remains incompletely explored. Previously we reported that YBX1 is required in sorting of miR-223 into exosomes. Here we show that YBX1 undergoes liquid-liquid phase separation (LLPS) in vitro and in cells. YBX1 condensates selectively recruit miR-223 in vitro and into exosomes secreted by cultured cells. Point mutations that inhibit YBX1 phase separation impair the incorporation of YBX1 protein into biomolecular condensates formed in cells, and perturb miR-233 sorting into exosomes. We propose that phase separation-mediated local enrichment of cytosolic RNA binding proteins and their cognate RNAs enables their targeting and packaging by vesicles that bud into multivesicular bodies. This provides a possible mechanism for efficient and selective engulfment of cytosolic proteins and RNAs into intraluminal vesicles which are then secreted as exosomes from cells.

scholarly journals Precise temporal regulation of alternative splicing during neural development

2018 ◽  
Author(s):  
Sebastien M. Weyn-Vanhentenryck ◽  
Huijuan Feng ◽  
Dmytro Ustianenko ◽  
Rachel Duffié ◽  
Qinghong Yan ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Neural Development ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Regulatory Mechanisms ◽  
Combinatorial Regulation ◽  
Temporal Regulation ◽  
Maturation Stages

AbstractAlternative splicing (AS) is a crucial step of gene expression that must be tightly controlled, but the precise timing of dynamic splicing switches during neural development and the underlying regulatory mechanisms are poorly understood. Here we systematically analyzed the temporal regulation of AS in a large number of transcriptome profiles of developing mouse cortices, in vivo purified neuronal subtypes, and neurons differentiated in vitro. Our analysis revealed early- and late-switch exons in genes with distinct functions, and these switches accurately define neuronal maturation stages. Integrative modeling suggests that these switches are under direct and combinatorial regulation by distinct sets of neuronal RNA-binding proteins including Nova, Rbfox, Mbnl and Ptbp. Surprisingly, various neuronal subtypes in the sensory systems lack Nova and/or Rbfox expression. These neurons retain the “immature” splicing program in early-switch exons, affecting numerous synaptic genes. These results provide new insights into the organization and regulation of the neurodevelopmental transcriptome.

scholarly journals MBNL1 and RBFOX1 co-regulate alternative splicing events transcriptome-wide through a conserved buffering mechanism

2021 ◽  
Author(s):  
J Andrew Berglund ◽  
Melissa Hale ◽  
Joseph Anthony Ellis ◽  
Ryan Meng ◽  
Sunny McDaniel ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Rna Binding ◽  
Mutational Analysis ◽  
Rna Binding Proteins ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Inducible Promoters ◽  
Vitro Binding ◽  
Dose Dependent

Alternative splicing (AS) is controlled by cis-regulatory elements recognized by networks of trans-acting factors. Here we investigate modes and mechanisms of AS co-regulation by MBNL1 and RBFOX1, two RNA binding proteins (RBPs) critical for developmental AS transitions. We generated two cell models that express each RBP under separate inducible promoters. Transcriptome-wide categorization of the impacts of RBFOX1 expression on MBNL1 splicing revealed a common co-regulatory mode through which RBFOX1 buffers MBNL1 dose-dependent splicing regulation by reducing the total range of exon inclusion or exclusion. Minigene mutational analysis and in vitro binding experiments suggest that this buffering mechanism occurs through a shared cis-regulatory element previously unidentified as critical for MBNL1-dependent activity. Overall, our studies define a conserved co-regulatory mechanism through which RBFOX1 and MBNL1 can fine-tune and provide redundancy for AS outcomes. These studies indicate overlapping use of RNA motifs with potential implications for when activity of RBPs is disrupted.

scholarly journals Bacterial 3′UTRs: A Useful Resource in Post-transcriptional Regulation

2021 ◽  
Vol 7 ◽  
Author(s):  
Pilar Menendez-Gil ◽  
Alejandro Toledo-Arana
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Untranslated Regions ◽  
Messenger Rnas ◽  
Specific Expression ◽  
Regulate Gene Expression ◽  
Transcriptional Regulatory ◽  
Transcriptional Regulatory Mechanisms ◽  
Species Specific ◽  
Post Transcriptional Regulation

Bacterial messenger RNAs (mRNAs) are composed of 5′ and 3′ untranslated regions (UTRs) that flank the coding sequences (CDSs). In eukaryotes, 3′UTRs play key roles in post-transcriptional regulatory mechanisms. Shortening or deregulation of these regions is associated with diseases such as cancer and metabolic disorders. Comparatively, little is known about the functions of 3′UTRs in bacteria. Over the past few years, 3′UTRs have emerged as important players in the regulation of relevant bacterial processes such as virulence, iron metabolism, and biofilm formation. This MiniReview is an update for the different 3′UTR-mediated mechanisms that regulate gene expression in bacteria. Some of these include 3′UTRs that interact with the 5′UTR of the same transcript to modulate translation, 3′UTRs that are targeted by specific ribonucleases, RNA-binding proteins and small RNAs (sRNAs), and 3′UTRs that act as reservoirs of trans-acting sRNAs, among others. In addition, recent findings regarding a differential evolution of bacterial 3′UTRs and its impact in the species-specific expression of orthologous genes are also discussed.

scholarly journals Small RNAs reveal two target sites of the RNA-maturation factor Mbb1 in the chloroplast of Chlamydomonas

2013 ◽  
Vol 42 (5) ◽  
pp. 3286-3297 ◽  
Author(s):  
Karen Loizeau ◽  
Yujiao Qu ◽  
Sébastien Depp ◽  
Vincent Fiechter ◽  
Hannes Ruwe ◽  
...  
Keyword(s):  
Small Rnas ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Chloroplast Transformation ◽  
Site Directed Mutagenesis ◽  
Tetratricopeptide Repeat ◽  
Messenger Rnas ◽  
Mrna Accumulation ◽  
Cis Acting

Abstract Many chloroplast transcripts are protected against exonucleolytic degradation by RNA-binding proteins. Such interactions can lead to the accumulation of short RNAs (sRNAs) that represent footprints of the protein partner. By mining existing data sets of Chlamydomonas reinhardtii small RNAs, we identify chloroplast sRNAs. Two of these correspond to the 5′-ends of the mature psbB and psbH messenger RNAs (mRNAs), which are both stabilized by the nucleus-encoded protein Mbb1, a member of the tetratricopeptide repeat family. Accordingly, we find that the two sRNAs are absent from the mbb1 mutant. Using chloroplast transformation and site-directed mutagenesis to survey the psbB 5′ UTR, we identify a cis-acting element that is essential for mRNA accumulation. This sequence is also found in the 5′ UTR of psbH, where it plays a role in RNA processing. The two sRNAs are centered on these cis-acting elements. Furthermore, RNA binding assays in vitro show that Mbb1 associates with the two elements specifically. Taken together, our data identify a conserved cis-acting element at the extremity of the psbH and psbB 5′ UTRs that plays a role in the processing and stability of the respective mRNAs through interactions with the tetratricopeptide repeat protein Mbb1 and leads to the accumulation of protected sRNAs.

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