RNA Interactions Research Articles

Synthetic antibodies for accelerated RNA crystallography.

RNA structure is crucial to a wide range of cellular processes. The intimate relationship between macromolecular structure and function necessitates the determination of high-resolution structures of functional RNA molecules. X-ray crystallography is the predominant technique used for macromolecular structure determination; however, solving RNA structures has been more challenging than their protein counterparts, as reflected in their poor representation in the Protein Data Bank (<1%). Antibody-assisted RNA crystallography is a relatively new technique that promises to accelerate RNA structure determination by employing synthetic antibodies (Fabs) as crystallization chaperones that are specifically raised against target RNAs. Antibody chaperones facilitate the formation of ordered crystal lattices by minimizing RNA flexibility and replacing unfavorable RNA-RNA contacts with contacts between chaperone molecules. Atomic coordinates of these antibody fragments can also be used as search models to obtain phase information during structure determination. Antibody-assisted RNA crystallography has enabled the structure determination of 15 unique RNA targets, including 11 in the last 6 years. In this review, I cover the historical development of antibody fragments as crystallization chaperones and their application to diverse RNA targets. I discuss how the first structures of antibody-RNA complexes informed the design of second-generation antibodies and led to the development of portable crystallization modules that have greatly reduced the uncertainties associated with RNA crystallography. Finally, I outline unexplored avenues that can increase the impact of this technology in structural biology research and discuss potential applications of antibodies as affinity reagents for interrogating RNA biology outside of their use in crystallography. This article is categorized under: RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry RNA Interactions with Proteins and Other Molecules > Protein-RNA Recognition RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes.

Logical regulation of endogenous gene expression using programmable, multi-input processing CRISPR guide RNAs.

The CRISPR-Cas system provides a versatile RNA-guided approach for a broad range of applications. Thanks to advances in RNA synthetic biology, the engineering of guide RNAs (gRNAs) has enabled the conditional control of the CRISPR-Cas system. However, achieving precise regulation of the CRISPR-Cas system for efficient modulation of internal metabolic processes remains challenging. In this work, we developed a robust dCas9 regulator with engineered conditional gRNAs to enable tight control of endogenous genes. Our conditional gRNAs in Escherichia coli can control gene expression upon specific interaction with trigger RNAs with a dynamic range as high as 130-fold, evaluating up to a three-input logic A OR (B AND C). The conditional gRNA-mediated targeting of endogenous metabolic genes, lacZ, malTand poxB, caused differential regulation of growth in Escherichia coli via metabolic flux control. Further, conditional gRNAs could regulate essential cytoskeleton genes, ftsZ and mreB, to control cell filamentation and division. Finally, three types of two-input logic gates could be applied for the conditional control of ftsZ regulation, resulting in morphological changes. The successful operation and application of conditional gRNAs based on programmable RNA interactions suggests that our system could be compatible with other Cas-effectors and implemented in other host organisms.

Circ_0000758 Facilitates Bladder Cancer Cell Growth, Migration and Angiogenesis Via Severing as miR-1236-3p Sponge.

Circular RNA (circRNA) has been reported to regulate the development of bladder cancer (BCa). However, the role of circ_0000758 in BCa progression is unknown. Circ_0000758 and miR-1236-3p expression, as well as ZEB2 mRNA expression were determined by qRT-PCR. BCa cell biological functions were determined by MTT assay, EdU assay, flow cytometry, wound healing assay and tube formation assay. Protein expression was detected by western blot analysis. RNA pull-down assay and dual-luciferase reporter assay were used to confirm RNA interaction. Xenograft mice models were constructed to assess the effect of circ_0000758 on BCa tumor growth. Circ_0000758 had increased expression in BCa tissues and cells. Circ_0000758 silencing could inhibit BCa cell growth, migration, angiogenesis in vitro, and tumor growth in vivo. Circ_0000758 served as a molecular sponge for miR-1236-3p, and miR-1236-3p inhibitor reversed circ_0000758 knockdown-mediated the inhibition effect on BCa cell progression. ZEB2 was targeted by miR-1236-3p, and its overexpression also revoked the suppressive effect of miR-1236-3p on BCa cell growth, migration, and angiogenesis. Besides, circ_0000758 knockdown also restrained BCa tumor growth. Circ_0000758 might promote BCa cell growth, migration, and angiogenesis by regulating the miR-1236-3p/ZEB2 axis.

Arid5a uses disordered extensions of its core ARID domain for distinct DNA- and RNA-recognition and gene regulation

AT-rich interacting domain (ARID)-containing proteins, Arids, are a heterogeneous DNA-binding protein family involved in transcription regulation and chromatin processing. For the member Arid5a, no exact DNA-binding preference has been experimentally defined so far. Additionally, the protein binds to mRNA motifs for transcript stabilization, supposedly through the DNA-binding ARID domain. To date, however, no unbiased RNA motif definition and clear dissection of nucleic acid-binding through the ARID domain have been undertaken. Using NMR-centered biochemistry, we here define the Arid5a DNA preference. Further, high-throughput in vitro binding (RBNS) reveals a consensus RNA-binding motif engaged by the core ARID domain. Finally, transcriptome-wide binding (iCLIP2) reveals that Arid5a has a weak preference for (A)U-rich regions in pre-mRNA transcripts of factors related to RNA processing. We find that the intrinsically disordered regions (IDR) flanking the ARID domain modulate the specificity and affinity of DNA-binding, while they appear crucial for RNA interactions. Ultimately, our data suggest that Arid5a uses its extended ARID domain for bi-functional gene regulation and that the involvement of IDR extensions is a more general feature of Arids in interacting with different nucleic acids at the chromatin-mRNA interface.

Exploring the Regulatory Landscape of Dementia: Insights from Non-Coding RNAs.

Dementia, a multifaceted neurological syndrome characterized by cognitive decline, poses significant challenges to daily functioning. The main causes of dementia, including Alzheimer's disease (AD), frontotemporal dementia (FTD), Lewy body dementia (LBD), and vascular dementia (VD), have different symptoms and etiologies. Genetic regulators, specifically non-coding RNAs (ncRNAs) such as microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs), are known to play important roles in dementia pathogenesis. MiRNAs, small non-coding RNAs, regulate gene expression by binding to the 3' untranslated regions of target messenger RNAs (mRNAs), while lncRNAs and circRNAs act as molecular sponges for miRNAs, thereby regulating gene expression. The emerging concept of competing endogenous RNA (ceRNA) interactions, involving lncRNAs and circRNAs as competitors for miRNA binding, has gained attention as potential biomarkers and therapeutic targets in dementia-related disorders. This review explores the regulatory roles of ncRNAs, particularly miRNAs, and the intricate dynamics of ceRNA interactions, providing insights into dementia pathogenesis and potential therapeutic avenues.

Hsa_circ_0004872 mitigates proliferation, metastasis and immune escape of meningioma cells by suppressing PD-L1

Meningioma is a prevalent intracranial malignancy known for its aggressive growth. Circular RNAs (circRNAs) play a crucial role in the development of various cancers. However, their involvement in meningioma remains understudied. This study aimed to investigate the function and underlying mechanism of hsa_circ_0004872 in meningioma. The molecular expression of hsa_circ_0004872, PD-L1 and EIF4A3 was identified by RT-qPCR and/or western blot assays. Cell viability, migration, and invasion were assessed through CCK-8 and Transwell assays, respectively. Cytotoxicity was determined using an LDH assay, and cell apoptosis was monitored by flow cytometry. The RNA and protein interactions were assessed through RNA-protein immunoprecipitation (RIP) and RNA pull down analyses. Our findings revealed that hsa_circ_0004872 expression was significantly downregulated in both meningioma tissue samples and cells. Overexpression of hsa_circ_0004872 inhibited the proliferation, metastasis, and immune escape of meningioma cells, as well as enhanced the cytotoxicity of CD8+ T cells by suppressing PD-L1. Furthermore, hsa_circ_0004872 directly interacted with EIF4A3, leading to the degradation of PD-L1 mRNA. Finally, inhibiting EIF4A3 improved the proliferation, metastasis, and immune escape of meningioma cells, as well as the cytotoxicity of CD8+ T cells. Our study demonstrated that hsa_circ_0004872 mitigated the proliferation, metastasis,and immune escape of meningioma cells by targeting the EIF4A3/PD-L1 axis. These findings suggested that hsa_circ_0004872 and EIF4A3 might serve as promising biological markers and therapeutic targets for meningioma treatment.

Long non-coding RNA TUG1 is down-regulated in Friedreich’s ataxia

Abstract Friedreich's Ataxia (FRDA) is a neurodegenerative disorder caused by reduced frataxin (FXN) levels. It leads to motor and sensory impairments and has a median life expectancy of around 35 years. As the most common inherited form of ataxia, FRDA lacks reliable, non-invasive biomarkers, prolonging and inflating the cost of clinical trials. This study proposes TUG1, a long non-coding RNA, as a promising blood-based biomarker for FRDA, which is known to regulate various cellular processes. In a previous study using a frataxin knockdown mouse model, we observed several hallmark FRDA symptoms. Building on this, we hypothesized that a dual-source approach—comparing the data from peripheral blood samples from FRDA patients with tissue samples from affected areas in FRDA knockdown mice, tissues usually unattainable from patients—would effectively identify robust biomarkers. A comprehensive reanalysis was conducted on gene expression data from 183 age- and sex-matched peripheral blood samples of FRDA patients, carriers, and controls, and 192 tissue datasets from FRDA knockdown mice. Blood and tissue samples underwent RNA isolation and quantitative reverse transcription PCR, and frataxin knockdown was confirmed through ELISA. Tug1 RNA interaction was explored via RNA pull-down assays. Validation was performed in serum samples on an independent set of 45 controls and 45 FRDA patients, and in blood samples from 66 heterozygous carriers and 72 FRDA patients. Tug1 and Slc40a1 emerged as potential blood-based biomarkers, confirmed in the FRDA knockdown mouse model (One-way ANOVA, p ≤ 0.05). Tug1 was consistently downregulated after Fxn knockdown and correlated strongly with Fxn levels (R2 = 0.71 during depletion, R2 = 0.74 during rescue). Slc40a1 showed a similar but tissue-specific pattern. Further validation of Tug1's downstream targets strengthened its biomarker candidacy. In additional human samples, TUG1 levels were significantly downregulated in both whole blood and serum of FRDA patients compared to controls (Wilcoxon signed-rank test, p &amp;lt; 0.05). Regression analyses revealed a negative correlation between TUG1 fold-change and disease onset (p &amp;lt; 0.0037), and positive correlations with disease duration and Functional Disability Stage score (p &amp;lt; 0.04). This suggests that elevated TUG1 levels correlate with earlier onset and more severe cases. This study identifies TUG1 as a potential blood-based biomarker for FRDA, showing consistent expression variance in human and mouse tissues related to disease severity and key FRDA pathways. It correlates with frataxin levels, indicating its promise as an early, non-invasive marker. TUG1 holds potential for FRDA monitoring and therapeutic development, meriting additional research.

NaProGraph: Network Analyzer for Interactions between Nucleic Acids and Proteins

Background: Interactions of RNA and DNA with proteins are crucial for elucidating intracellular processes in living organisms, diagnosing disorders, designing aptamer drugs, and other applications. Therefore, investigating the relationships between these macromolecules is essential to life science research. Methods: This study proposes an online network provider tool (NaProGraph) that offers an intuitive and user-friendly interface for studying interactions between nucleic acids (NA) and proteins. NaPro- Graph utilizes a comprehensive and curated dataset encompassing nearly all interacting macromolecules in the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB). Results: Researchers can employ this online tool to focus on a specific portion of the PDB, investigate its associated relationships, and visualize and extract pertinent information. This tool provides insights into the frequency of atoms and residues between proteins and nucleic acids (NAs) and the similarity of the macromolecules' primary structures. Conclusion: Furthermore, the functional similarity of proteins can be inferred using protein families and clans from Pfam.

Revealing the hidden RBP-RNA interactions with RNA modification enzyme-based strategies.

RNA-binding proteins (RBPs) are powerful and versatile regulators in living creatures, playing fundamental roles in organismal development, metabolism, and various diseases by the regulation of gene expression at multiple levels. The requirements of deep research on RBP function have promoted the rapid development of RBP-RNA interplay detection methods. Recently, the detection method of fusing RNA modification enzymes (RME) with RBP of interest has become a hot topic. Here, we reviewed RNA modification enzymes in adenosine deaminases that act onRNA (ADAR), terminal nucleotidyl transferase (TENT), and activation-induced cytosine deaminase/ApoB mRNA editing enzyme catalytic polypeptide-like (AID/APOBEC) protein family, regarding the biological function, biochemical activity, and substrate specificity originated from enzyme selves, their domains and partner proteins. In addition, we discussed the RME activity screening system, and the RME mutations with engineered enzyme activity. Furthermore, we provided a systematic overview of the basic principles, advantages, disadvantages, and applications of the RME-based and cross-linking and immunopurification (CLIP)-based RBP target profiling strategies, including targets of RNA-binding proteins identified by editing (TRIBE), RNA tagging, surveying targets by APOBEC-mediated profiling (STAMP), CLIP-seq, and their derivative technology. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Recognition RNA Processing > RNA Editing and Modification.

LINC00152: Potential driver oncogene in pan-cancer.

Long noncoding RNAs (lncRNA) are a class of non-coding RNAs greater than 200 bp in length with limited peptide-coding function. The transcription of LINC00152 is derived from chromosome 2p11.2. Many studies prove that LINC00152 influences the progression of various tumors via promoting the tumor cells malignant phenotype, chemoresistance, and immune escape. LINC00152 is regulated by multiple transcription factors and DNA hypomethylation. In addition, LINC00152 participates in the regulation of complex molecular signaling networks through epigenetic regulation, protein interactions, and competitive endogenous RNA (ceRNA). Here, we provide a systematic review of the upstream regulatory factors of LINC00152 expression level in different types of tumors. In addition, we revisit the main functions and mechanisms of LINC00152 as driver oncogene and biomarker in pan-cancer. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Methods > RNA Analyses in Cells RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes.

Large-scale analysis of small molecule-RNA interactions using multiplexed RNA structure libraries

The large-scale analysis of small-molecule binding to diverse RNA structures is key to understanding the required interaction properties and selectivity for developing RNA-binding molecules toward RNA-targeted therapies. Here, we report a new system for performing the large-scale analysis of small molecule–RNA interactions using a multiplexed pull-down assay with RNA structure libraries. The system profiled the RNA-binding landscapes of G-clamp and thiazole orange derivatives, which recognizes an unpaired guanine base and are good probes for fluorescent indicator displacement (FID) assays, respectively. We discuss the binding preferences of these molecules based on their large-scale affinity profiles. In addition, we selected combinations of fluorescent indicators and different ranks of RNA based on the information and screened for RNA-binding molecules using FID. RNAs with high- and intermediate-rank RNA provided reliable results. Our system provides fundamental information about small molecule–RNA interactions and facilitates the discovery of novel RNA-binding molecules.

Mapping RNA-protein interactions with subcellular resolution using colocalization CLIP.

RNA-binding proteins (RBPs) are essential for RNA metabolism and profoundly impact health and disease. The subcellular organization of RBP interaction networks with target RNAs remains largely unexplored. Here, we develop colocalization CLIP (coCLIP), a method that combines cross-linking and immunoprecipitation (CLIP) with proximity labeling, to explore in-depth the subcellular RNA interactions of the RBP human antigen R (HuR). Using this method, we uncover HuR's dynamic and location-specific interactions with RNA, revealing alterations in sequence preferences and interactions in the nucleus, cytosol, or stress granule (SG) compartments. We uncover HuR's unique binding preferences within SGs during arsenite stress, illuminating intricate interactions that conventional methodologies cannot capture. Overall, coCLIP provides a powerful method for revealing RBP-RNA interactions based on localization and lays the foundation for an advanced understanding of RBP models that incorporate subcellular location as a critical determinant of their functions.

Comprehensive analysis of lncRNA-miRNA-mRNA ceRNA network in ischemic stroke

ObjectiveCompetitive endogenous RNA (ceRNA) networks have uncovered a novel mode of RNA interaction, and are implicated in various biological processes and the pathogenesis of IS. This study aimed to explore the potential mechanisms underlying the ceRNA network in IS. MethodsFour public datasets containing lncRNA and mRNA (GSE22255 and GSE16561) and miRNA (GSE55937 and GSE43618) expression profiles from the GEO database were systematically analyzed to explore the role of RNAs in ischemic stroke (IS). Differentially expressed mRNAs (DEmRNAs), lncRNAs (DElncRNAs), and miRNAs (DEmiRNAs) between IS and normal control samples were identified. LncRNA-miRNA and miRNA-mRNA interactions were predicted, and the competing endogenous RNA (ceRNA) regulatory network was constructed using the Cytoscape software. The correlation between the RNAs in the ceRNA network and the clinical features of the samples was evaluated. Finally, principal component analysis was performed on the RNAs that constitute the ceRNA regulatory network, and their differential expression and principal component relationships among different types of samples were observed. ResultsA total of 224 DEmRNAs, 7 DEmiRNAs, and four DElncRNAs related to IS in four datasets were identified. Then, through target gene prediction, a lncRNA–miRNA–mRNA ceRNA network that contained 3 DElncRNAs, 2 DEmiRNAs, and 24 DEmRNAs was constructed. Correlations of the clinical characteristics showed that PART1 and SERPINH1 were related to clinical diseases, WNK1 was related to lifestyle, and seven RNAs were related to age. PCA results indicate that three principal components of PC1, PC2, and PC3 can clearly distinguish between control and IS samples. ConclusionOverall, we constructed a ceRNA network in IS, which could offer insights into the molecular mechanism and potential prognostic biomarkers for further research.

Development of Allosteric Ribozymes for ATP and l-Histidine Based on the R3C Ligase Ribozyme.

During the evolution of the RNA, short RNAs are thought to have joined together to form long RNAs, enhancing their function as ribozymes. Previously, the artificial R3C ligase ribozyme (73 nucleotides) was successfully reduced to 46 nucleotides; however, its activity decreased significantly. Therefore, we aimed to develop allosteric ribozymes, whose activities could be regulated by effector compounds, based on the reduced R3C ligase ribozyme (R3C-A). Among the variants prepared by fusing an ATP-binding aptamer RNA with R3C-A, one mutant showed increased ligation activity in an ATP-dependent manner. Melting temperature measurements of the two RNA mutants suggested that the region around the aptamer site was stabilized by the addition of ATP. This resulted in a suitable conformation for the reaction at the ligation site. Another ribozyme was prepared by fusing R3C-A with a l-histidine-binding aptamer RNA, and the ligase activity increased with increasing l-histidine concentrations. Both ATP and l-histidine play prominent roles in current molecular biology and the interaction of RNAs and these molecules could be a key step in the evolution of the world of RNAs. Our results suggest promise in the development of general allosteric ribozymes that are independent of the type of effector molecule and provide important clues to the evolution of the RNA world.

Decoding dynamic miRNA:ceRNA interactions unveils therapeutic insights and targets across predominant cancer landscapes.

Competing endogenous RNAs play key roles in cellular molecular mechanisms through cross-talk in post-transcriptional interactions. Studies on ceRNA cross-talk, which is particularly dependent on the abundance of free transcripts, generally involve large- and small-scale studies involving the integration of transcriptomic data from tissues and correlation analyses. This abundance-dependent nature of ceRNA interactions suggests that tissue- and condition-specific ceRNA dynamics may fluctuate. However, there are no comprehensive studies investigating the ceRNA interactions in normal tissue, ceRNAs that are lost and/or appear in cancerous tissues or their interactions. In this study, we comprehensively analyzed the tumor-specific ceRNA fluctuations observed in the three highest-incidence cancers, LUAD, PRAD, and BRCA, compared to healthy lung, prostate, and breast tissues, respectively. Our observations pertaining to tumor-specific competing endogenous RNA (ceRNA) interactions revealed that, in the cases of lung adenocarcinoma (LUAD), prostate adenocarcinoma (PRAD), and breast invasive carcinoma (BRCA), 3,204, 1,233, and 406 ceRNAs, respectively, engage in post-transcriptional intercommunication within tumor tissues, in contrast to their absence in corresponding healthy samples. We also found that 90 ceRNAs are shared by the three cancer types and that these ceRNAs participate in ceRNA interactions in tumor tissues compared to those in normal tissues. Among the 90 ceRNAs that directly interact with miRNAs, we uncovered a core network of 165 miRNAs and 63 ceRNAs that should be considered in RNA-targeted and RNA-mediated approaches in future studies and could be used in these three aggressive cancer types. More specifically, in this core interaction network, ceRNAs such as GALNT7, KLF9, and DAB2 and miRNAs like miR-106a/b-5p, miR-20a-5p, and miR-519d-3p may have potential as common targets in the three critical cancers. In contrast to conventional methods that construct ceRNA networks using differentially expressed genes compared to normal tissues, our proposed approach identifies ceRNA players by considering their context within the ceRNA:miRNA interactions. Our results have the potential to reveal distinct and common ceRNA interactions in cancer types and to pinpoint critical RNAs, thereby paving the way for RNA-based strategies in the battle against cancer.

RNAscape: geometric mapping and customizable visualization of RNA structure.

Analyzing and visualizing the tertiary structure and complex interactions of RNA is essential for being able to mechanistically decipher their molecular functions in vivo. Secondary structure visualization software can portray many aspects of RNA; however, these layouts are often unable to preserve topological correspondence since they do not consider tertiary interactions between different regions of an RNA molecule. Likewise, quaternary interactions between two or more interacting RNA molecules are not considered in secondary structure visualization tools. The RNAscape webserver produces visualizations that can preserve topological correspondence while remaining both visually intuitive and structurally insightful. RNAscape achieves this by designing a mathematical structural mapping algorithm which prioritizes the helical segments, reflecting their tertiary organization. Non-helical segments are mapped in a way that minimizes structural clutter. RNAscape runs a plotting script that is designed to generate publication-quality images. RNAscape natively supports non-standard nucleotides, multiple base-pairing annotation stylesand requires no programming experience. RNAscape can also be used to analyze RNA/DNA hybrid structures and DNA topologies, including G-quadruplexes. Users can upload their own three-dimensional structures or enter a Protein Data Bank (PDB) ID of an existing structure. The RNAscape webserver allows users to customize visualizations through various settings as desired. URL: https://rnascape.usc.edu/.

Integrative analyses of whole-transcriptome sequencing reveals CeRNA regulatory network in pulmonary hypertension treated with FGF21

Noncoding RNAs have been shown to play essential roles in hypoxic pulmonary hypertension (HPH). Our preliminary data showed that HPH is attenuated by fibroblast growth factor 21 (FGF21) administration. Therefore, we further investigated the whole transcriptome RNA expression patterns and interactions in a mice HPH model treated with FGF21. By whole-transcriptome sequencing, differentially expressed mRNAs, miRNAs, lncRNAs, and circRNAs were successfully identified in normoxia (Nx) vs. hypoxia (Hx) and Hx vs. hypoxia + FGF21 (Hx + F21). Differentially expressed mRNAs, miRNAs, lncRNAs, and circRNAs regulated by hypoxia and FGF21 were selected through intersection analysis. Based on prediction databases and sequencing data, differentially co-expressed mRNAs, miRNAs, lncRNAs, and circRNAs were further screened, followed by functional enrichment analysis. MAPK signaling pathway and epigenetic modification were enriched and may play fundamental roles in the therapeutic effects of FGF21. The ceRNA regulatory network of lncRNA–miRNA–mRNA and circRNA–miRNA–mRNA was constructed with miR-7a-5p, miR-449c-5p, miR-676-3p and miR-674-3p as the core. In addition, quantitative real-time PCR experiments were employed to verify the whole-transcriptome sequencing data. The results of luciferase reporter assays highlighted the relationship between miR-449c-5p and XR_878320.1, miR-449c-5p and Stab2, miR-449c-5p and circ_mtcp1, which suggesting that miR-449c-5p may be a key regulator of FGF21 in the treatment of PH. Taken together, this study provides potential biomarkers, pathways, and ceRNA regulatory networks in HPH treated with FGF21 and will provide an experimental basis for the clinical application of FGF21 in PH.

Loss-of-function mutation in PRMT9 causes abnormal synapse development by dysregulation of RNA alternative splicing

Protein arginine methyltransferase 9 (PRMT9) is a recently identified member of the PRMT family, yet its biological function remains largely unknown. Here, by characterizing an intellectual disability associated PRMT9 mutation (G189R) and establishing a Prmt9 conditional knockout (cKO) mouse model, we uncover an important function of PRMT9 in neuronal development. The G189R mutation abolishes PRMT9 methyltransferase activity and reduces its protein stability. Knockout of Prmt9 in hippocampal neurons causes alternative splicing of ~1900 genes, which likely accounts for the aberrant synapse development and impaired learning and memory in the Prmt9 cKO mice. Mechanistically, we discover a methylation-sensitive protein–RNA interaction between the arginine 508 (R508) of the splicing factor 3B subunit 2 (SF3B2), the site that is exclusively methylated by PRMT9, and the pre-mRNA anchoring site, a cis-regulatory element that is critical for RNA splicing. Additionally, using human and mouse cell lines, as well as an SF3B2 arginine methylation-deficient mouse model, we provide strong evidence that SF3B2 is the primary methylation substrate of PRMT9, thus highlighting the conserved function of the PRMT9/SF3B2 axis in regulating pre-mRNA splicing.

Single-cell multiplex chromatin and RNA interactions in ageing human brain

Dynamically organized chromatin complexes often involve multiplex chromatin interactions and sometimes chromatin-associated RNA1–3. Chromatin complex compositions change during cellular differentiation and ageing, and are expected to be highly heterogeneous among terminally differentiated single cells4–7. Here we introduce the multinucleic acid interaction mapping in single cells (MUSIC) technique for concurrent profiling of multiplex chromatin interactions, gene expression and RNA–chromatin associations within individual nuclei. When applied to 14 human frontal cortex samples from older donors, MUSIC delineated diverse cortical cell types and states. We observed that nuclei exhibiting fewer short-range chromatin interactions were correlated with both an ‘older’ transcriptomic signature and Alzheimer’s disease pathology. Furthermore, the cell type exhibiting chromatin contacts between cis expression quantitative trait loci and a promoter tends to be that in which these cis expression quantitative trait loci specifically affect the expression of their target gene. In addition, female cortical cells exhibit highly heterogeneous interactions between XIST non-coding RNA and chromosome X, along with diverse spatial organizations of the X chromosomes. MUSIC presents a potent tool for exploration of chromatin architecture and transcription at cellular resolution in complex tissues.

DUSP3 modulates IRES-dependent translation of mRNAs through dephosphorylation of the HNRNPC protein in cells under genotoxic stimulus.

The dual-specificity phosphatase 3 (DUSP3) regulates cell cycle progression, proliferation, senescence, and DNA repair pathways under genotoxic stress. This phosphatase interacts with HNRNPC protein suggesting an involvement in the regulation of HNRNPC-ribonucleoprotein complex stability. In this work, we investigate the impact of DUSP3 depletion on functions of HNRNPC aiming to suggest new roles for this enzyme. The DUSP3 knockdown results in the tyrosine hyperphosphorylation state of HNRNPC increasing its RNA binding ability. HNRNPC is present in the cytoplasm where it interacts with IRES trans-acting factors (ITAF) complex, which recruits the 40S ribosome on mRNA during protein synthesis, thus facilitating the translation of mRNAs containing IRES sequence in response to specific stimuli. In accordance with that, we found that DUSP3 is present in the 40S, monosomes and polysomes interacting with HNRNPC, just like other previously identified DUSP3 substrates/interacting partners such as PABP and NCL proteins. By downregulating DUSP3, Tyr-phosphorylated HNRNPC preferentially binds to IRES-containing mRNAs within ITAF complexes preferentially in synchronized or stressed cells, as evidenced by the higher levels of proteins such as c-MYC and XIAP, but not their mRNAs such as measured by qPCR. Under DUSP3 absence, this increased phosphorylated-HNRNPC/RNA interaction reduces HNRNPC-p53 binding in presence of RNAs releasing p53 for specialized cellular responses. Similarly, to HNRNPC, PABP physically interacts with DUSP3 in an RNA-dependent manner. Overall, DUSP3 can modulate cellular responses to genotoxic stimuli at the translational level by maintaining the stability of HNRNPC-ITAF complexes and regulating the intensity and specificity of RNA interactions with RRM-domain proteins.