Chromatin-associated RNA Research Articles

RNA m5C oxidation by TET2 regulates chromatin state and leukaemogenesis

Mutation of tet methylcytosine dioxygenase 2 (encoded by TET2) drives myeloid malignancy initiation and progression1–3. TET2 deficiency is known to cause a globally opened chromatin state and activation of genes contributing to aberrant haematopoietic stem cell self-renewal4,5. However, the open chromatin observed in TET2-deficient mouse embryonic stem cells, leukaemic cells and haematopoietic stem and progenitor cells5 is inconsistent with the designated role of DNA 5-methylcytosine oxidation of TET2. Here we show that chromatin-associated retrotransposon RNA 5-methylcytosine (m5C) can be recognized by the methyl-CpG-binding-domain protein MBD6, which guides deubiquitination of nearby monoubiquitinated Lys119 of histone H2A (H2AK119ub) to promote an open chromatin state. TET2 oxidizes m5C and antagonizes this MBD6-dependent H2AK119ub deubiquitination. TET2 depletion thereby leads to globally decreased H2AK119ub, more open chromatin and increased transcription in stem cells. TET2-mutant human leukaemia becomes dependent on this gene activation pathway, with MBD6 depletion selectively blocking proliferation of TET2-mutant leukaemic cells and largely reversing the haematopoiesis defects caused by Tet2 loss in mouse models. Together, our findings reveal a chromatin regulation pathway by TET2 through retrotransposon RNA m5C oxidation and identify the downstream MBD6 protein as a feasible target for developing therapies specific against TET2 mutant malignancies.

NSUN5/TET2-directed chromatin-associated RNA modification of 5-methylcytosine to 5-hydroxymethylcytosine governs glioma immune evasion

Malignant glioma exhibits immune evasion characterized by highly expressing the immune checkpoint CD47. RNA 5-methylcytosine(m5C) modification plays a pivotal role in tumor pathogenesis. However, the mechanism underlying m5C-modified RNA metabolism remains unclear, as does the contribution of m5C-modified RNA to the glioma immune microenvironment. In this study, we demonstrate that the canonical 28SrRNA methyltransferase NSUN5 down-regulates β-catenin by promoting the degradation of its mRNA, leading to enhanced phagocytosis of tumor-associated macrophages (TAMs). Specifically, the NSUN5-induced suppression of β-catenin relies on its methyltransferase activity mediated by cysteine 359 (C359) and is not influenced by its localization in the nucleolus. Intriguingly, NSUN5 directly interacts with and deposits m5C on CTNNB1 caRNA (chromatin-associated RNA). NSUN5-induced recruitment of TET2 to chromatin is independent of its methyltransferase activity. The m5C modification on caRNA is subsequently oxidized into 5-hydroxymethylcytosine (5hmC) by TET2, which is dependent on its binding affinity for Fe2+ and α-KG. Furthermore, NSUN5 enhances the chromatin recruitment of RBFOX2 which acts as a 5hmC-specific reader to recognize and facilitate the degradation of 5hmC caRNA. Notably, hmeRIP-seq analysis reveals numerous mRNA substrates of NSUN5 that potentially undergo this mode of metabolism. In addition, NSUN5 is epigenetically suppressed by DNA methylation and is negatively correlated with IDH1-R132H mutation in glioma patients. Importantly, pharmacological blockage of DNA methylation or IDH1-R132H mutant and CD47/SIRPα signaling synergistically enhances TAM-based phagocytosis and glioma elimination in vivo. Our findings unveil a general mechanism by which NSUN5/TET2/RBFOX2 signaling regulates RNA metabolism and highlight NSUN5 targeting as a potential strategy for glioma immune therapy.

MYH1G-AS is a chromatin-associated lncRNA that regulates skeletal muscle development in chicken

BackgroundSkeletal muscle development is pivotal for animal growth and health. Recently, long noncoding RNAs (lncRNAs) were found to interact with chromatin through diverse roles. However, little is known about how lncRNAs act as chromatin-associated RNAs to regulate skeletal muscle development. Here, we aim to investigate the regulation of chromatin-associated RNA (MYH1G-AS) during skeletal muscle development.MethodsWe provided comprehensive insight into the RNA profile and chromatin accessibility of different myofibers, combining RNA sequencing (RNA-seq) with an assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). The dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay were used to analyze the transcriptional regulation mechanism of MYH1G-AS. ALKBH5-mediated MYH1G-AS N6-methyladenosine (m6A) demethylation was assessed by a single-base elongation and ligation-based qPCR amplification method (SELECT) assay. Functions of MYH1G-AS were investigated through a primary myoblast and lentivirus/cholesterol-modified antisense oligonucleotide (ASO)-mediated animal model. To validate the interaction of MYH1G-AS with fibroblast growth factor 18 (FGF18) protein, RNA pull down and an RNA immunoprecipitation (RIP) assay were performed. Specifically, the interaction between FGF18 and SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5 (SMARCA5) protein was analyzed by coimmunoprecipitation (Co-IP) and a yeast two-hybrid assay.ResultsA total of 45 differentially expressed (DE) lncRNAs, with DE ATAC-seq peaks in their promoter region, were classified as open chromatin-associated lncRNAs. A skeletal muscle-specific lncRNA (MSTRG.15576.9; MYH1G-AS), which is one of the open chromatin-associated lncRNA, was identified. MYH1G-AS transcription is coordinately regulated by transcription factors (TF) SMAD3 and SP2. Moreover, SP2 represses ALKBH5 transcription to weaken ALKBH5-mediated m6A demethylation of MYH1G-AS, thus destroying MYH1G-AS RNA stability. MYH1G-AS accelerates myoblast proliferation but restrains myoblast differentiation. Moreover, MYH1G-AS drives a switch from slow-twitch to fast-twitch fibers and causes muscle atrophy. Mechanistically, MYH1G-AS inhibits FGF18 protein stabilization to reduce the interaction of FGF18 to SMARCA5, thus repressing chromatin accessibility of the SMAD4 promoter to activate the SMAD4-dependent pathway.ConclusionsOur results reveal a new pattern of the regulation of lncRNA expression at diverse levels and help expound the regulation of m6A methylation on chromatin status.Graphical

Base-Resolution Sequencing Methods for Whole-Transcriptome Quantification of mRNA Modifications.

RNA molecules are not merely a combination of four bases of A, C, G, and U. Chemical modifications occur in almost all RNA species and play diverse roles in gene expression regulation. The abundant cellular RNAs, such as ribosomal RNA (rRNA) and transfer RNA (tRNA), are known to have the highest density of RNA modifications, which exert critical functions in rRNA and tRNA biogenesis, stability, and subsequent translation. In recent years, modifications on low-abundance RNA species in mammalian cells, such as messenger RNA (mRNA), regulatory noncoding RNA (ncRNA), and chromatin-associated RNA (caRNA), have been shown to contain multiple different chemical modifications with functional significance. As the most abundant mRNA modification in mammals, N6-methyladenosine (m6A) affects nearly every stage of mRNA processing and metabolism, with the antibody-based m6A-MeRIP-seq (methylated RNA immunoprecipitation sequencing) followed by high-throughput sequencing widely employed in mapping m6A distribution transcriptome-wide in diverse biological systems. In addition to m6A, other chemical modifications such as pseudouridine (Ψ), 2'-O-methylation (Nm), 5-methylcytidine (m5C), internal N7-methylguanosine (m7G), N1-methyladenosine (m1A), N4-acetylcytidine (ac4C), etc. also exist in polyA-tailed RNA in mammalian cells, requiring effective mapping approaches for whole-transcriptome profiling of these non-m6A mRNA modifications. Like m6A, the antibody-based enrichment followed by sequencing has been the primary method to study distributions of these modifications. Methods to more quantitatively map these modifications would dramatically improve our understanding of distributions and modification density of these chemical marks on RNA, thereby bettering informing functional implications. In this Account, aimed at both single-base resolution and modification fraction quantification, we summarize our recent advances in developing a series of chemistry- or biochemistry-based methods to quantitatively map RNA modifications, including m6A, Ψ, m5C, m1A, 2'-O-methylation (Nm), and internal m7G, in mammalian mRNA at base resolution. These new methods, including m6A-SAC-seq, eTAM-seq, BID-seq, UBS-seq, DAMM-seq, m1A-quant-seq, Nm-Mut-seq, and m7G-quant-seq, promise to conduct base-resolution mapping of most major mRNA modifications with low RNA input and uncover dynamic changes in modification stoichiometry during biological and physiological processes, facilitating future investigations on these RNA modifications in regulating cellular gene expression and as potential biomarkers for clinical diagnosis and prognosis. These quantitative sequencing methods allow the mapping of most mRNA modifications with limited input sample requirements. The same modifications on diverse RNA species, such as caRNA, ncRNA, nuclear nascent RNA, mitochondrial RNA, cell-free RNA (cfRNA), etc., could be sequenced using the same methods.

Global mapping of RNA-chromatin contacts reveals a proximity-dominated connectivity model for ncRNA-gene interactions

Non-coding RNAs (ncRNAs) are transcribed throughout the genome and provide regulatory inputs to gene expression through their interaction with chromatin. Yet, the genomic targets and functions of most ncRNAs are unknown. Here we use chromatin-associated RNA sequencing (ChAR-seq) to map the global network of ncRNA interactions with chromatin in human embryonic stem cells and the dynamic changes in interactions during differentiation into definitive endoderm. We uncover general principles governing the organization of the RNA-chromatin interactome, demonstrating that nearly all ncRNAs exclusively interact with genes in close three-dimensional proximity to their locus and provide a model predicting the interactome. We uncover RNAs that interact with many loci across the genome and unveil thousands of unannotated RNAs that dynamically interact with chromatin. By relating the dynamics of the interactome to changes in gene expression, we demonstrate that activation or repression of individual genes is unlikely to be controlled by a single ncRNA.

Genome-wide analysis of RNA-chromatin interactions in lizards as a mean for functional lncRNA identification

BackgroundLong non-coding RNAs (lncRNAs) are defined as transcribed molecules longer than 200 nucleotides with little to no protein-coding potential. LncRNAs can regulate gene expression of nearby genes (cis-acting) or genes located on other chromosomes (trans-acting). Several methodologies have been developed to capture lncRNAs associated with chromatin at a genome-wide level. Analysis of RNA-DNA contacts can be combined with epigenetic and RNA-seq data to define potential lncRNAs involved in the regulation of gene expression.ResultsWe performed Chromatin Associated RNA sequencing (ChAR-seq) in Anolis carolinensis to obtain the genome-wide map of the associations that RNA molecules have with chromatin. We analyzed the frequency of DNA contacts for different classes of RNAs and were able to define cis- and trans-acting lncRNAs. We integrated the ChAR-seq map of RNA-DNA contacts with epigenetic data for the acetylation of lysine 16 on histone H4 (H4K16ac), a mark connected to actively transcribed chromatin in lizards. We successfully identified three trans-acting lncRNAs significantly associated with the H4K16ac signal, which are likely involved in the regulation of gene expression in A. carolinensis.ConclusionsWe show that the ChAR-seq method is a powerful tool to explore the RNA-DNA map of interactions. Moreover, in combination with epigenetic data, ChAR-seq can be applied in non-model species to establish potential roles for predicted lncRNAs that lack functional annotations.

Abstract 251: Regulation Of Endotheliopathy By Chromatin-associated Long Non-coding RNA

Lining the critical interface between circulating blood and vascular wall, endothelial cells (ECs) play vital functions in health and disease. The optimal gene expression in ECs is essential to maintain endothelial homeostasis, and its dysregulation can lead to EC dysfunction or endotheliopathy, a common mechanism underlying many metabolic and cardiovascular diseases. Through chromatin-RNA contact profiling, we have identified several chromatin-associated long non-coding RNA (lncRNAs) that play essential roles in the regulation of endothelial function, including endothelial nitric oxide production, angiogenesis, endothelial-mesenchymal transition, and inflammatory activation. These include nuclear genome-transcribed lncRNAs, e.g. lncRNA-enhancing endothelial nitric oxide synthase (LEENE) and LINC00607 that promotes expression of fibronectin and plasminogen activator inhibitor-1. Our recent study has also revealed that mitochondria transcribed RNAs (mtRNA) are pervasively attached to nuclear chromatin and such mtRNA-association with chromatin is increased by pro-inflammatory, diabetes-mimicking stimuli. Suppression of a mitochondrial lncRNA in ECs attenuates nascent RNA transcription from the nuclear genome, including that of critical genes promoting pro-inflammatory activation. Finally, we show increased nuclear localization of multiple mtRNAs in the ECs of human diabetic donors. Together, these data highlight an emerging role of chromatin-associated lncRNAs in regulating endothelial biology.

Regulation of Endotheliopathy by Chromatin-associated Long Noncoding RNA

Regulation of Endotheliopathy by Chromatin-associated Long Noncoding RNA

New horizons of regulatory RNA

Genetic information flows from DNA to protein through RNA in the central dogma. Different RNA species are known to accomplish essential tasks of protein encoding (mRNAs), amino acid loading (tRNAs), and translation machinery assembly (rRNAs). However, on top of these well-known roles, RNAs are central to various cellular regulatory pathways. Here we summarize newly emerging regulatory functions of RNA, specifically focusing on regulations through RNA modifications, RNP granules, and chromatin-associated regulatory RNA. In addition to being an essential building block of the central dogma, RNA can be critical to the regulation of many cellular processes.

Abstract IA008: Chromatin regulation by reversible chromatin-associated RNA methylation

Abstract Over 150 types of post-transcriptional RNA modifications have been identified in all kingdoms of life. We have previously discovered two RNA demethylases, FTO and ALKBH5, which catalyze oxidative demethylation of the most prevalent modifications of mammalian messenger RNA (mRNA) and other nuclear RNA, N6-methyladenosine (m6A). We have also characterized reader proteins that preferentially recognize methylated transcripts to facilitate either decay or translation of these target RNAs. Most recently we have discovered m6A methylation of chromatin-associated regulatory RNAs (carRNAs), which modulates chromatin state and transcription in mouse embryonic stem cells (mESCs). These findings suggest that reversible RNA modification could impact chromatin regulation analogous to the reversible DNA and histone chemical modifications. I will present recent mechanistic investigation of chromatin and transcription regulation through m6A methylation and demethylation, and impacts on mammalian development and cancer progression. Citation Format: Chuan He. Chromatin regulation by reversible chromatin-associated RNA methylation. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr IA008.

Small Interfering RNAs Targeting a Chromatin-Associated RNA Induce Its Transcriptional Silencing in Human Cells.

Transcriptional gene silencing by small interfering RNAs (siRNAs) has been widely described in various species, including plants and yeast. In mammals, its extent remains somewhat debated. Previous studies showed that siRNAs targeting gene promoters could induce the silencing of the targeted promoter, although the involvement of off-target mechanisms was also suggested. Here, by using nascent RNA capture and RNA polymerase II chromatin immunoprecipitation, we show that siRNAs targeting a chromatin-associated noncoding RNA induced its transcriptional silencing. Deletion of the sequence targeted by one of these siRNAs on the two alleles by genome editing further showed that this silencing was due to base-pairing of the siRNA to the target. Moreover, by using cells with heterozygous deletion of the target sequence, we showed that only the wild-type allele, but not the deleted allele, was silenced by the siRNA, indicating that transcriptional silencing occurred only in cis. Finally, we demonstrated that both Ago1 and Ago2 are involved in this transcriptional silencing. Altogether, our data demonstrate that siRNAs targeting a chromatin-associated RNA at a distance from its promoter induce its transcriptional silencing. Our results thus extend the possible repertoire of endogenous or exogenous interfering RNAs.

Non-coding RNAs associated with Prader-Willi syndrome regulate transcription of neurodevelopmental genes in human induced pluripotent stem cells.

Mutations and aberrant gene expression during cellular differentiation lead to neurodevelopmental disorders, such as Prader-Willi syndrome (PWS), which results from the deletion of an imprinted locus on paternally inherited chromosome 15. We analyzed chromatin-associated RNA in human induced pluripotent cells (iPSCs) upon depletion of hybrid small nucleolar long non-coding RNAs (sno-lncRNAs) and 5' snoRNA capped and polyadenylated long non-coding RNAs (SPA-lncRNAs) transcribed from the locus deleted in PWS. We found that rapid ablation of these lncRNAs affects transcription of specific gene classes. Downregulated genes contribute to neurodevelopment and neuronal maintenance, while upregulated genes are predominantly involved in the negative regulation of cellular metabolism and apoptotic processes. Our data reveal the importance of SPA-lncRNAs and sno-lncRNAs in controlling gene expression in iPSCs and provide a platform for synthetic experimental approaches in PWS studies. We conclude that ncRNAs transcribed from the PWS locus are critical regulators of a transcriptional signature, which is important for neuronal differentiation and development.

RBM45 is an m6A-binding protein that affects neuronal differentiation and the splicing of a subset of mRNAs.

SUMMARYN6-methyladenosine (m6A) is deposited co-transcriptionally on thousands of cellular mRNAs and plays important roles in mRNA processing and cellular function. m6A is particularly abundant within the brain and is critical for neurodevelopment. However, the mechanisms through which m6A contributes to brain development are incompletely understood. RBM45 acts as an m6A-binding protein that is highly expressed during neurodevelopment. We find that RBM45 binds to thousands of cellular RNAs, predominantly within intronic regions. Rbm45 depletion disrupts the constitutive splicing of a subset of target pre-mRNAs, leading to altered mRNA and protein levels through both m6A-dependent and m6A-independent mechanisms. Finally, we find that RBM45 is necessary for neuroblastoma cell differentiation and that its depletion impacts the expression of genes involved in several neurodevelopmental signaling pathways. Altogether, our findings show a role for RBM45 in controlling mRNA processing and neuronal differentiation, mediated in part by the recognition of methylated RNA.

P487: SMALL NON-CODING RNAS ASSOCIATE WITH CHROMATIN TO MAINTEIN PROPOGATION OF ACUTE MEYLOID LEUKEMIA

Background: Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by the clonal expansion of myeloid progenitors without terminal differentiation. The interplay between genome and epigenome alterations drives aberrant gene network linking to leukemia transformation. Altered transcription may occur at three-dimensional chromatin level and be orchestrated by a combination of multiple regulatory factors. Although some RNA molecules, especially non-coding RNAs, were reported to modulate transcription in leukemia cells, the regulatory role of genome-wide RNAs on transcription through chromatin association remains largely unexplored. Aims: To identify novel RNA molecules as chromatin-association factors critical for leukemogenesis through modulating transcription Methods: We performed in situ Mapping of RNA-Genome Interactome (iMARGI) in a human acute leukemia cell line – MV4-11 cells. Chromatin association of candidate RNAs was verified using Chromatin Isolation by RNA Purification (ChIRP) by means of biotinylated oligonucleotides for target capture. A series of ChIP-seq assays were performed in multiple AML cell lines to profile chromatin occupancy by Fibrillarin (FBL), a catalytic component of C/D box ribonucleoproteins (snoRNPs), as well as to observe the remodelling of chromatin states marked by H3K27ac and H3K9me3 upon shRNA-mediated knockdown of FBL. Loss-of-function experiments using antisense oligonucleotides were performed on candidate chromatin-associated RNAs in AML cell lines, AML primary samples, and CD34+ healthy bone marrow cells. Results: In total, about 52.1 million RNA-chromatin interactions were identified out of deep sequencing of four iMARGI libraries. Whereas the vast majority (50.5 million, 97%) of RNA-chromatin interactome were intra-chromosome, a small fraction (1.6 million, 3%) occurred inter chromosome. Though protein-coding mRNAs predominate the chromatin-associated RNA species, other non-coding RNAs were also captured. Intriguingly, different from protein-coding mRNAs and long intergenic non-coding RNAs (lincRNAs) which mainly form intra-chromosome interactions, two small non-coding RNA classes, small nucleolar RNAs (snoRNAs) and small nuclear RNAs (snRNAs), were found with prevalent frequency (62% and 92%, respectively) of inter-chromosome associations. SNORD3A (U3) and SNORD118 (U8) were C/D box snoRNAs ranked top by the number of their associated interactions in MV4-11 cells as well as in other cell types. Chromatin association of U3 and U8 was verified using ChIRP, with the observation of unique chromatin binding for both U3 and U8, and of specific binding motifs of transcription factors at their binding sites. ChIP-seq revealed that the snoRNP FBL occupied only a small set of genes (n=118), and shRNA-mediated knockdown of FBL had a minimal impact on H3K27ac and H3K9me3, suggesting that snoRNA-chromatin interactions may function independently on FBL-associated snoRNPs. ASO-mediated loss of U3 or U8 significantly impairs leukemia cells proliferation and colony forming capacity in multiple AML cell lines and primary AML blast samples, whereas only mildly affects CD34+ healthy cells. Summary/Conclusion: We identified chromatin-associated snoRNAs U3 and U8 which were essential for leukemia maintenance and may serve as therapeutic targets for leukemia eradication.

MIR205HG/LEADR Long Noncoding RNA Binds to Primed Proximal Regulatory Regions in Prostate Basal Cells Through a Triplex- and Alu-Mediated Mechanism.

Aside serving as host gene for miR-205, MIR205HG transcribes for a chromatin-associated long noncoding RNA (lncRNA) able to restrain the differentiation of prostate basal cells, thus being reannotated as LEADR (Long Epithelial Alu-interacting Differentiation-related RNA). We previously showed the presence of Alu sequences in the promoters of genes modulated upon MIR205HG/LEADR manipulation. Notably, an Alu element also spans the first and second exons of MIR205HG/LEADR, suggesting its possible involvement in target selection/binding. Here, we performed ChIRP-seq to map MIR205HG/LEADR chromatin occupancy at genome-wide level in prostate basal cells. Our results confirmed preferential binding to regions proximal to gene transcription start site (TSS). Moreover, enrichment of triplex-forming sequences was found upstream of MIR205HG/LEADR-bound genes, peaking at −1,500/−500 bp from TSS. Triplexes formed with one or two putative DNA binding sites within MIR205HG/LEADR sequence, located just upstream of the Alu element. Notably, triplex-forming regions of bound genes were themselves enriched in Alu elements. These data suggest, from one side, that triplex formation may be the prevalent mechanism by which MIR205HG/LEADR selects and physically interacts with target DNA, from the other that direct or protein-mediated Alu (RNA)/Alu (DNA) interaction may represent a further functional requirement. We also found that triplex-forming regions were enriched in specific histone modifications, including H3K4me1 in the absence of H3K27ac, H3K4me3 and H3K27me3, indicating that in prostate basal cells MIR205HG/LEADR may preferentially bind to primed proximal regulatory elements. This may underscore the need for basal cells to keep MIR205HG/LEADR target genes repressed but, at the same time, responsive to differentiation cues.

Pseudouridine synthases modify human pre-mRNA co-transcriptionally and affect pre-mRNA processing

Pseudouridine is a modified nucleotide that is prevalent in human mRNAs and is dynamically regulated. Here, we investigate when in their life cycle mRNAs become pseudouridylated to illuminate the potential regulatory functions of endogenous mRNA pseudouridylation. Using single-nucleotide resolution pseudouridine profiling on chromatin-associated RNA from human cells, we identified pseudouridines in nascent pre-mRNA at locations associated with alternatively spliced regions, enriched near splice sites, and overlapping hundreds of binding sites for RNA-binding proteins. Invitro splicing assays establish a direct effect of individual endogenous pre-mRNA pseudouridines on splicing efficiency. We validate hundreds of pre-mRNA sites as direct targets of distinct pseudouridine synthases and show that PUS1, PUS7, and RPUSD4-three pre-mRNA-modifying pseudouridine synthases with tissue-specific expression-control widespread changes in alternative pre-mRNA splicing and 3' end processing. Our results establish a vast potential for cotranscriptional pre-mRNA pseudouridylation to regulate human gene expression via alternative pre-mRNA processing.

Type I and II PRMTs inversely regulate post-transcriptional intron detention through Sm and CHTOP methylation.

Protein arginine methyltransferases (PRMTs) are required for the regulation of RNA processing factors. Type I PRMT enzymes catalyze mono- and asymmetric dimethylation; Type II enzymes catalyze mono- and symmetric dimethylation. To understand the specific mechanisms of PRMT activity in splicing regulation, we inhibited Type I and II PRMTs and probed their transcriptomic consequences. Using the newly developed Splicing Kinetics and Transcript Elongation Rates by Sequencing (SKaTER-seq) method, analysis of co-transcriptional splicing demonstrated that PRMT inhibition resulted in altered splicing rates. Surprisingly, co-transcriptional splicing kinetics did not correlate with final changes in splicing of polyadenylated RNA. This was particularly true for retained introns (RI). By using actinomycin D to inhibit ongoing transcription, we determined that PRMTs post-transcriptionally regulate RI. Subsequent proteomic analysis of both PRMT-inhibited chromatin and chromatin-associated polyadenylated RNA identified altered binding of many proteins, including the Type I substrate, CHTOP, and the Type II substrate, SmB. Targeted mutagenesis of all methylarginine sites in SmD3, SmB, and SmD1 recapitulated splicing changes seen with Type II PRMT inhibition, without disrupting snRNP assembly. Similarly, mutagenesis of all methylarginine sites in CHTOP recapitulated the splicing changes seen with Type I PRMT inhibition. Examination of subcellular fractions further revealed that RI were enriched in the nucleoplasm and chromatin. Taken together, these data demonstrate that, through Sm and CHTOP arginine methylation, PRMTs regulate the post-transcriptional processing of nuclear, detained introns.

Dissecting the plant chromatin interactome using mass spectrometry

The protein interactome mediates crucial functions in transcription, chromatin remodeling, and higher-order structural organization. Elucidating the proteins that interact with chromatin-associated RNA or proteins is key to understanding fundamental epigenetic regulatory pathways. We discuss the opportunities and challenges of mass spectrometry (MS)-based proteomics for characterizing the plant chromatin interactome.

Long Non-Coding RNA CAR10 Facilitates Non-Small Cell Lung Cancer Cell Migration and Invasion by Modulating the miR-892a/GJB2 Pathway.

IntroductionNon-coding RNAs, including long non-coding (lnc)RNAs and microRNAs (miRs), play crucial roles in numerous malignant tumors, including non-small cell lung cancer (NSCLC).MethodsThe expression levels of chromatin-associated RNA Intergenic 10 (CAR10), gap junction protein beta 2 (GJB2) and miR-892a in NSCLC were evaluated by reanalyzing three Gene Expression Omnibus (GEO) datasets, and performing reverse transcription-quantitative PCR, immunohistochemistry staining and Western blot analysis, accordingly. Functionally, Transwell and Matrigel assays were performed to measure changes in the migration and invasion abilities of the A549 and H1299 cell lines. The targeted binding effects between CAR10 and miR-892a, as well as between miR-892a and GJB2 were confirmed by conducting dual-luciferase reporter and RNA pull-down assays, respectively.ResultsThe present study demonstrated that CAR10 was upregulated in patients with NSCLC, which was also associated with a poor prognosis. Functionally, CAR10 was confirmed to be oncogenic and promoted NSCLC cell migration and invasion, using overexpression and knockdown Transwell assays. Furthermore, GJB2 expression was revealed to be upregulated and was positively correlated with CAR10 expression in NSCLC. A further mechanistic study revealed that GJB2 was a downstream target of CAR10, which induced the migration and invasive potential of the A549 and H1299 cell lines. More specifically, miR-892a was found to serve as a bridge between CAR10 and GJB2, via similar miRNA response elements. The RNA pull-down and luciferase assays indicated that miR-892a directly binds both CAR10 and GJB2.ConclusionCAR10 promoted NSCLC cell migration and invasion by upregulating GJB2 and sponging miR-892a. These findings illustrated that the CAR10/miR-892a/GJB2 axis may be a novel molecular target for the treatment of NSCLC.

Revealing nascent RNA processing dynamics with nano-COP.

During maturation, eukaryotic precursor RNAs undergo processing events including intron splicing, 3'-end cleavage, and polyadenylation. Here we describe nanopore analysis of co-transcriptional processing (nano-COP), a method for probing the timing and patterns of RNA processing. An extension of native elongating transcript sequencing, which quantifies transcription genome-wide through short-read sequencing of nascent RNA 3' ends, nano-COP uses long-read nascent RNA sequencing to observe global patterns of RNA processing. First, nascent RNA is stringently purified through a combination of 4-thiouridine metabolic labeling and cellular fractionation. In contrast to cDNA or short-read-based approaches relying on reverse transcription or amplification, the sample is sequenced directly through nanopores to reveal the native context of nascent RNA. nano-COP identifies both active transcription sites and splice isoforms of single RNA molecules during synthesis, providing insight into patterns of intron removal and the physical coupling between transcription and splicing. The nano-COP protocol yields data within 3 d.