Many Aspects Of RNA Metabolism Research Articles

NMR characterization of RNA binding property of the DEAD-box RNA helicase DDX3X and its implications for helicase activity

The DEAD-box RNA helicase (DDX) plays a central role in many aspects of RNA metabolism by remodeling the defined structure of RNA molecules. While a number of structural studies have revealed the atomistic details of the interaction between DDX and RNA ligands, the molecular mechanism of how this molecule unwinds a structured RNA into an unstructured single-stranded RNA (ssRNA) has largely remained elusive. This is due to challenges in structurally characterizing the unwinding intermediate state and the lack of thermodynamic details underlying this process. In this study, we use solution nuclear magnetic resonance (NMR) spectroscopy to characterize the interaction of human DDX3X, a member of the DDX family, with various RNA ligands. Our results show that the inherent binding affinity of DDX3X for ssRNA is significantly higher than that for structured RNA elements. This preferential binding, accompanied by the formation of a domain-closed conformation in complex with ssRNA, effectively stabilizes the denatured ssRNA state and thus underlies the unwinding activity of DDX3X. Our results provide a thermodynamic and structural basis for the DDX function, whereby DDX can recognize and remodel a distinct set of structured RNAs to participate in a wide range of physiological processes.

TDP-43 chronic deficiency leads to dysregulation of transposable elements and gene expression by affecting R-loop and 5hmC crosstalk

TDP-43 is an RNA/DNA-binding protein that forms aggregates in various brain disorders. TDP-43 engages in many aspects of RNA metabolism, but its molecular roles in regulating genes and transposable elements (TEs) have not been extensively explored. Chronic TDP-43 knockdown impairs cell proliferation and cellular responses to DNA damage. At the molecular level, TDP-43 chronic deficiency affects gene expression either locally or distally by concomitantly altering the crosstalk between R-loops and 5-hydroxymethylcytosine (5hmC) in gene bodies and long-range enhancer/promoter interactions. Furthermore, TDP-43 knockdown induces substantial disease-relevant TE activation by influencing their R-loop and 5hmC homeostasis in a locus-specific manner. Together, our findings highlight the genomic roles of TDP-43 in modulating R-loop-5hmC coordination in coding genes, distal regulatory elements, and TEs, presenting a general and broad molecular mechanism underlying the contributions of proteinopathies to the etiology of neurodegenerative disorders.

Caenorhabditis elegansddx-15 helicase fails to complement loss of Prp43p in Saccharomyces cerevisiae.

Helicase proteins have important roles in many aspects of RNA metabolism in the cell. The function of these highly conserved proteins is commonly preserved between organisms, yet in a few cases these homologues are found to have slightly different biochemical functions. Prp43 is a protein with varied roles in yeast, but here we show that the C. elegans homologue of this protein is unable to rescue the loss of Prp43p. By employing a transcriptional repression experiment, the expression of DDX-15 protein in yeast is not enough to complement the loss of Prp43p, which is a yeast essential protein.

The polyA tail facilitates splicing of last introns with weak 3' splice sites via PABPN1.

The polyA tail of mRNAs is important for many aspects of RNA metabolism. However, whether and how it regulates pre-mRNA splicing is still unknown. Here, we report that the polyA tail acts as a splicing enhancer for the last intron via the nuclear polyA binding protein PABPN1 in HeLa cells. PABPN1-depletion induces the retention of a group of introns with a weaker 3' splice site, and they show a strong 3'-end bias and mainly locate in nuclear speckles. The polyA tail is essential for PABPN1-enhanced last intron splicing and functions in a length-dependent manner. Tethering PABPN1 to nonpolyadenylated transcripts also promotes splicing, suggesting a direct role for PABPN1 in splicing regulation. Using TurboID-MS, we construct the PABPN1 interactome, including many spliceosomal and RNA-binding proteins. Specifically, PABPN1 can recruit RBM26&27 to promote splicing by interacting with the coiled-coil and RRM domain of RBM27. PABPN1-regulated terminal intron splicing is conserved in mice. Together, our study establishes a novel mode of post-transcriptional splicing regulation via the polyA tail and PABPN1.

A systems-level mass spectrometry-based technique for accurate and sensitive quantification of the RNA cap epitranscriptome.

Chemical modifications of transcripts with a 5' cap occur in all organisms and function in many aspects of RNA metabolism. To facilitate analysis of RNA caps, we developed a systems-level mass spectrometry-based technique, CapQuant, for accurate and sensitive quantification of the cap epitranscriptome. The protocol includes the addition of stable isotope-labeled cap nucleotides (CNs) to RNA, enzymatic hydrolysis of endogenous RNA to release CNs, and off-line enrichment of CNs by ion-pairing high-pressure liquid chromatography, followed by a 17 min chromatography-coupled tandem quadrupole mass spectrometry run for the identification and quantification of individual CNs. The total time required for the protocol can be up to 7 d. In this approach, 26 CNs can be quantified in eukaryotic poly(A)-tailed RNA, bacterial total RNA and viral RNA. This protocol can be modified to analyze other types of RNA and RNA from in vitro sources. CapQuant stands out from other methods in terms of superior specificity, sensitivity and accuracy, and it is not limited to individual caps nor does it require radiolabeling. Thanks to its unique capability of accurately and sensitively quantifying RNA caps on a systems level, CapQuant can reveal both the RNA cap landscape and the transcription start site distribution of capped RNA in a broad range of settings.

FUS regulates a subset of snoRNA expression and modulates the level of rRNA modifications

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.

Altered m6A RNA methylation contributes to hippocampal memory deficits in Huntington’s disease mice

N6-methyladenosine (m6A) regulates many aspects of RNA metabolism and is involved in learning and memory processes. Yet, the impact of a dysregulation of post-transcriptional m6A editing on synaptic impairments in neurodegenerative disorders remains unknown. Here we investigated the m6A methylation pattern in the hippocampus of Huntington’s disease (HD) mice and the potential role of the m6A RNA modification in HD cognitive symptomatology. m6A modifications were evaluated in HD mice subjected to a hippocampal cognitive training task through m6A immunoprecipitation sequencing (MeRIP-seq) and the relative levels of m6A-modifying proteins (FTO and METTL14) by subcellular fractionation and Western blot analysis. Stereotaxic CA1 hippocampal delivery of AAV-shFTO was performed to investigate the effect of RNA m6A dysregulation in HD memory deficits. Our results reveal a m6A hypermethylation in relevant HD and synaptic related genes in the hippocampal transcriptome of Hdh+/Q111 mice. Conversely, m6A is aberrantly regulated in an experience-dependent manner in the HD hippocampus leading to demethylation of important components of synapse organization. Notably, the levels of RNA demethylase (FTO) and methyltransferase (METTL14) were modulated after training in the hippocampus of WT mice but not in Hdh+/Q111 mice. Finally, inhibition of FTO expression in the hippocampal CA1 region restored memory disturbances in symptomatic Hdh+/Q111 mice. Altogether, our results suggest that a differential RNA methylation landscape contributes to HD cognitive symptoms and uncover a role of m6A as a novel hallmark of HD.

Epitranscriptomic dynamics in brain development and disease.

Distinct cell types are generated at specific times during brain development and are regulated by epigenetic, transcriptional, and newly emerging epitranscriptomic mechanisms. RNA modifications are known to affect many aspects of RNA metabolism and have been implicated in the regulation of various biological processes and in disease. Recent studies imply that dysregulation of the epitranscriptome may be significantly associated with neuropsychiatric, neurodevelopmental, and neurodegenerative disorders. Here we review the current knowledge surrounding the role of the RNA modifications N6-methyladenosine, 5-methylcytidine, pseudouridine, A-to-I RNA editing, 2'O-methylation, and their associated machinery, in brain development and human diseases. We also highlight the need for the development of new technologies in the pursuit of directly mapping RNA modifications in both genome- and single-molecule-level approach.

The Role of DEAD-Box ATPases in Gene Expression and the Regulation of RNA-Protein Condensates.

DEAD-box ATPases constitute a very large protein family present in all cells, often in great abundance. From bacteria to humans, they play critical roles in many aspects of RNA metabolism, and due to their widespread importance in RNA biology, they have been characterized in great detail at both the structural and biochemical levels. DEAD-box proteins function as RNA-dependent ATPases that can unwind short duplexes of RNA, remodel ribonucleoprotein (RNP) complexes, or act as clamps to promote RNP assembly. Yet, it often remains enigmatic how individual DEAD-box proteins mechanistically contribute to specific RNA-processing steps. Here, we review the role of DEAD-box ATPases in the regulation of gene expression and propose that one common function of these enzymes is in the regulation of liquid-liquid phase separation of RNP condensates.

The expanding world of metabolic enzymes moonlighting as RNA binding proteins

RNA binding proteins play key roles in many aspects of RNA metabolism and function, including splicing, translation, localization, stability and degradation. Within the past few years, proteomics studies have identified dozens of enzymes in intermediary metabolism that bind to RNA. The wide occurrence and conservation of RNA binding ability across distant branches of the evolutionary tree suggest that these moonlighting enzymes are involved in connections between intermediary metabolism and gene expression that comprise far more extensive regulatory networks than previously thought.

Dysregulated mitochondrial and cytosolic tRNA m1A methylation in Alzheimer's disease.

RNA modifications affect many aspects of RNA metabolism and are involved in the regulation of many different biological processes. Mono-methylation of adenosine in the N1 position, N1-methyladensoine (m1A), is a reversible modification that is known to target rRNAs and tRNAs. m1A has been shown to increase tRNA structural stability and induce correct tRNA folding. Recent studies have begun to associate the dysregulation of epitranscriptomic control with age-related disorders such as Alzheimer's disease. Here, we applied the newly developed m1A-quant-seq approach to map the brain abundant m1A RNA modification in the cortex of an Alzheimer's disease mouse model, 5XFAD. We observed hypomethylation in both mitochondrial and cytosolic tRNAs in 5XFAD mice compared with wild type. Furthermore, the main enzymes responsible for the addition of m1A in mitochondrial (TRMT10C, HSD17B10) and cytosolic tRNAs (TRMT61A) displayed decreased expression in 5XFAD compared with wild-type mice. Knockdown of these enzymes results in a more severe phenotype in a Drosophila tau model, and differential m1A methylation is correlated with differences in mature mitochondrial tRNA expression. Collectively, this work suggests that hypo m1A modification in tRNAs may play a role in Alzheimer's disease pathogenesis.

RNA m6A Methylation Regulators Multi-Omics Analysis in Prostate Cancer.

RNA N6-methyladenosine (m6A) methylation is known to be the most popular RNA modification in animals. Many research reports have elaborated on the effects of m6A regulators in medical practice, such as diagnosis, prognosis, and treatment. M6A modification has evident impacts on many aspects of RNA metabolism, just like RNA splicing, processing, translation, and stability. M6A also has a magnificent role in numerous types of cancers. We analyzed the prostate cancer datasets, from The Cancer Genome Atlas (TCGA) database, for every recognized m6A regulator in their gene expression, DNA methylation status and copy number variations (CNVs). We also systematically analyzed the relationship between different m6A regulators and the prognosis of prostate cancer. The results illustrated considerable differences in the expression of various m6A regulators between the prostate and normal cancer samples. At the same time, there were evident differences in the expression of various m6A regulators in prostate cancers with different Gleason scores. Subsequently, we determined CBLL1, FTO, YTHDC1, HNRNPA2B1 as crucial m6A regulators of prostate cancer. Premised on the expression of CBLL1, we also identified potential therapeutic agents for prostate cancer, and knockdown of FTO prominently inhibited prostate cells migration and invasion in vitro experiment.

The Amyloid Region of Hfq Riboregulator Promotes DsrA:rpoS RNAs Annealing.

Simple SummaryRNA:amyloid protein interactions have been observed in the past few years. Nevertheless, the molecular basis and physiological implications of these interactions are still poorly understood. Here we focus on a bacterial amyloid protein, Hfq. This protein is a pleiotropic bacterial regulator that mediates many aspects of RNA metabolism. The protein notably mediates mRNA stability and translation efficiency by using stress-related small noncoding regulatory RNAs. This regulation contributes to bacterial adaptation to stresses. Our results show that the amyloid region of Hfq significantly influences the efficiency of annealing between DsrA small noncoding RNA to its target mRNA. This unexpected result opens perspectives for a novel physiological role of amyloids, including those associated with neurodegenerative diseases.Hfq is a bacterial RNA chaperone which promotes the pairing of small noncoding RNAs to target mRNAs, allowing post-transcriptional regulation. This RNA annealing activity has been attributed for years to the N-terminal region of the protein that forms a toroidal structure with a typical Sm-fold. Nevertheless, many Hfqs, including that of Escherichia coli, have a C-terminal region with unclear functions. Here we use a biophysical approach, Synchrotron Radiation Circular Dichroism (SRCD), to probe the interaction of the E. coli Hfq C-terminal amyloid region with RNA and its effect on RNA annealing. This C-terminal region of Hfq, which has been described to be dispensable for sRNA:mRNA annealing, has an unexpected and significant effect on this activity. The functional consequences of this novel property of the amyloid region of Hfq in relation to physiological stress are discussed.

M6A-express: uncovering complex and condition-specific m6A regulation of gene expression.

N6-methyladenosine (m6A) is the most abundant form of mRNA modification and controls many aspects of RNA metabolism including gene expression. However, the mechanisms by which m6A regulates cell- and condition-specific gene expression are still poorly understood, partly due to a lack of tools capable of identifying m6A sites that regulate gene expression under different conditions. Here we develop m6A-express, the first algorithm for predicting condition-specific m6A regulation of gene expression (m6A-reg-exp) from limited methylated RNA immunoprecipitation sequencing (MeRIP-seq) data. Comprehensive evaluations of m6A-express using simulated and real data demonstrated its high prediction specificity and sensitivity. When only a few MeRIP-seq samples may be available for the cellular or treatment conditions, m6A-express is particularly more robust than the log-linear model. Using m6A-express, we reported that m6A writers, METTL3 and METTL14, competitively regulate the transcriptional processes by mediating m6A-reg-exp of different genes in Hela cells. In contrast, METTL3 induces different m6A-reg-exp of a distinct group of genes in HepG2 cells to regulate protein functions and stress-related processes. We further uncovered unique m6A-reg-exp patterns in human brain and intestine tissues, which are enriched in organ-specific processes. This study demonstrates the effectiveness of m6A-express in predicting condition-specific m6A-reg-exp and highlights the complex, condition-specific nature of m6A-regulation of gene expression.

The expanding world of metabolic enzymes moonlighting as RNA binding proteins.

RNA binding proteins play key roles in many aspects of RNA metabolism and function, including splicing, transport, translation, localization, stability and degradation. Within the past few years, proteomics studies have identified dozens of enzymes in intermediary metabolism that bind to RNA. The wide occurrence and conservation of RNA binding ability across distant branches of the evolutionary tree suggest that these moonlighting enzymes are involved in connections between intermediary metabolism and gene expression that comprise far more extensive regulatory networks than previously thought. There are many outstanding questions about the molecular structures and mechanisms involved, the effects of these interactions on enzyme and RNA functions, and the factors that regulate the interactions. The effects on RNA function are likely to be wider than regulation of translation, and some enzyme-RNA interactions have been found to regulate the enzyme's catalytic activity. Several enzyme-RNA interactions have been shown to be affected by cellular factors that change under different intracellular and environmental conditions, including concentrations of substrates and cofactors. Understanding the molecular mechanisms involved in the interactions between the enzymes and RNA, the factors involved in regulation, and the effects of the enzyme-RNA interactions on both the enzyme and RNA functions will lead to a better understanding of the role of the many newly identified enzyme-RNA interactions in connecting intermediary metabolism and gene expression.

N6-methyladenosine dynamics in neurodevelopment and aging, and its potential role in Alzheimer\u2019s disease

BackgroundN6-methyladenosine (m6A) modification is known to impact many aspects of RNA metabolism, including mRNA stability and translation, and is highly prevalent in the brain.ResultsWe show that m6A modification displays temporal and spatial dynamics during neurodevelopment and aging. Genes that are temporally differentially methylated are more prone to have mRNA expression changes and affect many pathways associated with nervous system development. Furthermore, m6A shows a distinct tissue-specific methylation profile, which is most pronounced in the hypothalamus. Tissue-specific methylation is associated with an increase in mRNA expression and is associated with tissue-specific developmental processes. During the aging process, we observe significantly more m6A sites as age increases, in both mouse and human. We show a high level of overlap between mouse and human; however, humans at both young and old ages consistently show more m6A sites compared to mice. Differential m6A sites are found to be enriched in alternative untranslated regions of genes that affect aging-related pathways. These m6A sites are associated with a strong negative effect on mRNA expression. We also show that many Alzheimer-related transcripts exhibit decreased m6A methylation in a mouse model of Alzheimer’s disease, which is correlated with reduced protein levels.ConclusionsOur results suggest that m6A exerts a critical function in both early and late brain development in a spatio-temporal fashion. Furthermore, m6A controls protein levels of key genes involved in Alzheimer’s disease-associated pathways, suggesting that m6A plays an important role in aging and neurodegenerative disease.

RMVar: an updated database of functional variants involved in RNA modifications.

Distinguishing the few disease-related variants from a massive number of passenger variants is a major challenge. Variants affecting RNA modifications that play critical roles in many aspects of RNA metabolism have recently been linked to many human diseases, such as cancers. Evaluating the effect of genetic variants on RNA modifications will provide a new perspective for understanding the pathogenic mechanism of human diseases. Previously, we developed a database called ‘m6AVar’ to host variants associated with m6A, one of the most prevalent RNA modifications in eukaryotes. To host all RNA modification (RM)-associated variants, here we present an updated version of m6AVar renamed RMVar (http://rmvar.renlab.org). In this update, RMVar contains 1 678 126 RM-associated variants for 9 kinds of RNA modifications, namely m6A, m6Am, m1A, pseudouridine, m5C, m5U, 2′-O-Me, A-to-I and m7G, at three confidence levels. Moreover, RBP binding regions, miRNA targets, splicing events and circRNAs were integrated to assist investigations of the effects of RM-associated variants on posttranscriptional regulation. In addition, disease-related information was integrated from ClinVar and other genome-wide association studies (GWAS) to investigate the relationship between RM-associated variants and diseases. We expect that RMVar may boost further functional studies on genetic variants affecting RNA modifications.

TDP-43 dysfunction restricts dendritic complexity by inhibiting CREB activation and altering gene expression

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two related neurodegenerative diseases that present with similar TDP-43 pathology in patient tissue. TDP-43 is an RNA-binding protein which forms aggregates in neurons of ALS and FTD patients as well as in a subset of patients diagnosed with other neurodegenerative diseases. Despite our understanding that TDP-43 is essential for many aspects of RNA metabolism, it remains obscure how TDP-43 dysfunction contributes to neurodegeneration. Interestingly, altered neuronal dendritic morphology is a common theme among several neurological disorders and is thought to precede neurodegeneration. We previously found that both TDP-43 overexpression (OE) and knockdown (KD) result in reduced dendritic branching of cortical neurons. In this study, we used TRIBE (targets of RNA-binding proteins identified by editing) as an approach to identify signaling pathways that regulate dendritic branching downstream of TDP-43. We found that TDP-43 RNA targets are enriched for pathways that signal to the CREB transcription factor. We further found that TDP-43 dysfunction inhibits CREB activation and CREB transcriptional output, and restoring CREB signaling rescues defects in dendritic branching. Finally, we demonstrate, using RNA sequencing, that TDP-43 OE and KD cause similar changes in the abundance of specific messenger RNAs, consistent with their ability to produce similar morphological defects. Our data therefore provide a mechanism by which TDP-43 dysfunction interferes with dendritic branching, and may define pathways for therapeutic intervention in neurodegenerative diseases.

Chaperone Mediated Autophagy Degrades TDP-43 Protein and Is Affected by TDP-43 Aggregation.

TAR DNA binding protein 43 kDa (TDP-43) is a ribonuclear protein regulating many aspects of RNA metabolism. Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD) are fatal neurodegenerative diseases with the presence of TDP-43 aggregates in neuronal cells. Chaperone Mediated Autophagy (CMA) is a lysosomal degradation pathway participating in the proteostasis of several cytosolic proteins including neurodegenerative associated proteins. In addition, protein oligomers or aggregates can affect the status of CMA. In this work, we studied the relationship between CMA and the physiological and pathological forms of TDP-43. First, we found that recombinant TDP-43 was specifically degraded by rat liver’s CMA+ lysosomes and that endogenous TDP-43 is localized in rat brain’s CMA+ lysosomes, indicating that TDP-43 can be a CMA substrate in vivo. Next, by using a previously reported TDP-43 aggregation model, we have shown that wild-type and an aggregate-prone form of TDP-43 are detected in CMA+ lysosomes isolated from cell cultures. In addition, their protein levels increased in cells displaying CMA down-regulation, indicating that these two TDP-43 forms are CMA substrates in vitro. Finally, we observed that the aggregate-prone form of TDP-43 is able to interact with Hsc70, to co-localize with Lamp2A, and to up-regulate the levels of these molecular components of CMA. The latter was followed by an up-regulation of the CMA activity and lysosomal damage. Altogether our data shows that: (i) TDP-43 is a CMA substrate; (ii) CMA can contribute to control the turnover of physiological and pathological forms of TDP-43; and (iii) TDP-43 aggregation can affect CMA performance. Overall, this work contributes to understanding how a dysregulation between CMA and TDP-43 would participate in neuropathological mechanisms associated with TDP-43 aggregation.

In Vivo Tethering System to Isolate RNA-Binding Proteins Regulating mRNA Decay in Leishmania.

RNA-binding proteins (RBPs) play key roles in many aspects of RNA metabolism. In Leishmania, a unicellular eukaryote that favors the posttranscriptional mode of regulation for controlling gene expression levels, the function of RBPs becomes even more critical. However, due largely to limited in vivo approaches available for identifying RBPs in these parasites, there have been no significant advances to our understanding of the role these proteins play in posttranscriptional control through binding to cis-acting elements in the 3' untranslated region (3'UTR) of mRNAs. Here we describe an optimized in vivo RNA tethering approach using the bacteriophage MS2 coat protein combined to immunoprecipitation and mass spectrometry analysis to identify RBPs specifically interacting with 3'UTR short interspersed degenerated retroposon elements (SIDERs). Members of the SIDER2 subfamily were shown previously to promote mRNA degradation through a novel mechanism of mRNA decay. Using this modified MS2 tethering approach, we have identifiedcandidate RBPs specifically interacting with SIDER2 elements and contributing to the decay mechanism.