This review provides a thorough survey of long noncoding RNAs that bear the RNA modification N6-methyladenosine (m6A) and current work to understand the resulting mechanistic and biological consequences. We give an overview of lncRNA and m6A biology first, describing the writers, erasers, and readers of m6A and their targeting of lncRNAs. Next, we give an in-depth review of the field of nuclear lncRNAs that regulate chromatin and their regulation via m6A. We then describe the growing appreciation of liquid–liquid phase separation properties in lncRNA and m6A biology. Finally, we cover examples of cytoplasmic lncRNAs regulated by m6A. Overall, this review aims to emphasize how epitranscriptomics influences noncoding RNA mechanisms to provide additional layers of regulation, integrated into downstream biological processes.

Regulation of gene expression is central to the development of immune cells and their ability to respond to infection. As part of a clinical evaluation, we identified two sisters with recurrent infections, hypogammaglobulinemia, and memory B cell deficiency, diagnosed as common variable immunodeficiency. Whole exome sequencing identified a heterozygous variant (Leu50Ser, L50S) in a conserved region of EZH2, the catalytic subunit of the epigenetic gene repressor Polycomb Repressive Complex 2 (PRC2). EZH2-catalyzed histone H3 lysine 27 methylation (H3K27me) in bulk was not overall significantly disrupted by this variant, in patient samples or cell lines expressing EZH2-L50S. EZH2-L50S protein is expressed similar to wild-type and can form PRC2. However, we find that specific genomic regions that normally have high wild-type levels of H3K27me3 are deficient in the L50S context, particularly around gene promoters. EZH2-L50S is still recruited to these sites, but is not as active. Using recombinant purified PRC2, we determine that L50S affects methylation of nucleosomes and disrupts allosteric stimulation that normally amplifies H3K27me3, consistent with the location of L50 in the allosteric regulatory region of PRC2. Thus, variation of EZH2 L50, occurring at low frequency in the population may interfere with normal B cell gene expression patterns, contributing to immunodeficiency. This study has implications for genetic variation in PRC2 in the general population.

The Saccharomyces cerevisiae silent information regulator (SIR) complex performs all core heterochromatin functions. It first drives histone deacetylation in an iterative, spreading manner, then stably incorporates with nucleosomes to compact and epigenetically repress the chromatin. How this switch between dynamic spreading and stable compaction occurs has remained poorly understood, partly due to limited structural data on the intact complex. Using crosslinking mass spectrometry, we identified an uncharacterized intersubunit interaction connecting these two states: the Sir2 deacetylase interacts with the scaffolding subunit Sir4 through its coiled-coil domain, which also contacts the Sir3 compaction subunit. This interaction hub contains conserved Sir2 residues that can adopt multiple conformations, including orientation toward the active site, alongside co-evolved residues that enable species-specific Sir4 interactions. Mutations disrupting this hub disrupt heterochromatic repression in vivo and affect the deacetylation activity, directly linking catalysis to compaction. Our findings reveal how a multifunctional complex stages a stepwise transition to achieve epigenetic gene repression.

Organisms with smaller genomes often perform multiple functions using one multi-subunit protein complex. The S. cerevisiae Silent Information Regulator complex (SIRc) carries out all of the core functions of heterochromatin. SIR complexes first drive the initiation and spreading of histone deacetylation in an iterative manner. Subsequently, the same complexes are incorporated stably with nucleosomes, driving compaction and repression of the underlying chromatin domain. These two distinct functions of SIRc have each been characterized in much detail, but the mechanism by which the dynamic spreading state switches to stable compaction is not well-understood. This incomplete knowledge of potential intra-complex communication is partly due to a lack of structural information of the complex as a whole; only structures of fragments have been determined to date. Using cross-linking mass spectrometry in solution, we identified a novel inter-subunit interaction that physically connects the two states of SIRc. The Sir2 deacetylase makes direct interactions with the scaffolding subunit Sir4 through its coiled-coil domain, which also interacts with the Sir3 compaction/repression subunit. Within the hub of interactions are conserved residues in Sir2 that can sense deacetylation state, as well as amino acids that likely diverged and co-evolved to interact with Sir4, promoting species-specific functions. Mutation of this interaction hub disrupts heterochromatic repression, potentially by disrupting a conserved mechanism that communicates completion of deacetylation to switch to compaction. Our work highlights how a single multi-functional chromatin regulatory complex can stage a step-wise mechanism that requires a major transition in activities to achieve epigenetic gene repression.

A key challenge in synthetic biology is achieving durable amplification of low-level inputs in gene regulation systems. Current RNA-based tools primarily operate post-transcriptionally and often yield limited, transient responses. An underexplored feature of lowly expressed long non-coding RNAs (lncRNAs) is their ability to induce outsized effects on chromatin regulation across large genomic regions. Mechanistic insights from basic research are bringing the field closer to designing lncRNAs for epigenetic engineering. We review foundational studies on ectopic expression to uncover lncRNA-mediated epigenetic mechanisms and state-of-the-art transgenic systems for studying lncRNA-driven epigenetic regulation. We present perspectives on strategies for testing the composability of modular lncRNA elements to build rationally designed systems with programmable chromatin-modifying functions and potential biomedical applications such as gene dosage correction. Deepening mechanistic insights into lncRNA function, combined with the development of lncRNA-based technologies for genome regulation, will pave the way for significant advances in cell state control.

Organisms with smaller genomes often perform multiple functions using one multi-subunit protein complex. The S. cerevisiae Silent Information Regulator complex (SIRc) carries out all of the core functions of heterochromatin. SIR complexes first drive the initiation and spreading of histone deacetylation in an iterative manner. Subsequently, the same complexes are incorporated stably with nucleosomes, driving compaction and repression of the underlying chromatin domain. These two distinct functions of SIRc have each been characterized in much detail, but the mechanism by which the dynamic spreading state switches to stable compaction is not well-understood. This incomplete knowledge of intra-complex communication is partly due to a lack of structural information of the complex as a whole; only structures of fragments have been determined to date. Using cross-linking mass spectrometry in solution, we identified a novel inter-subunit interaction that physically connects the two states of SIRc. The Sir2 deacetylase makes direct interactions with the scaffolding subunit Sir4 through its coiled-coil domain, which also interacts with the Sir3 compaction/repression subunit. Within the hub of interactions are conserved residues in Sir2 that can sense deacetylation state, as well as amino acids that likely diverged and co-evolved to interact with Sir4, promoting species-specific functions. Mutation of this interaction hub disrupts heterochromatic repression, potentially by disrupting a conserved mechanism that communicates completion of deacetylation to switch to compaction. Our work highlights how a single multi-functional chromatin regulatory complex can stage a step-wise mechanism that requires a major transition in activities to achieve epigenetic gene repression. ### Competing Interest Statement The authors have declared no competing interest.

N6-methyladenosine (m6A) modification of RNA regulates normal and cancer biology, but knowledge of its function on long noncoding RNAs (lncRNAs) remains limited. Here, we reveal that m6A regulates the breast cancer-associated human lncRNA HOTAIR. Mapping m6A in breast cancer cell lines, we identify multiple m6A sites on HOTAIR, with 1 single consistently methylated site (A783) that is critical for HOTAIR-driven proliferation and invasion of triple-negative breast cancer (TNBC) cells. Methylated A783 interacts with the m6A "reader" YTHDC1, promoting chromatin association of HOTAIR, proliferation and invasion of TNBC cells, and gene repression. A783U mutant HOTAIR induces a unique antitumor gene expression profile and displays loss-of-function and antimorph behaviors by impairing and, in some cases, causing opposite gene expression changes induced by wild-type (WT) HOTAIR. Our work demonstrates how modification of 1 base in an lncRNA can elicit a distinct gene regulation mechanism and drive cancer-associated phenotypes.

RNA-binding proteins (RBPs), essential for skeletal muscle regeneration, cause muscle degeneration and neuromuscular disease when mutated. Why mutations in these ubiquitously expressed RBPs orchestrate complex tissue regeneration and direct cell fate decisions in skeletal muscle remains poorly understood. Single-cell RNA-sequencing of regenerating Mus musculus skeletal muscle reveals that RBP expression, including the expression of many neuromuscular disease-associated RBPs, is temporally regulated in skeletal muscle stem cells and correlates with specific stages of myogenic differentiation. By combining machine learning with RBP engagement scoring, we discovered that the neuromuscular disease-associated RBP Hnrnpa2b1 is a differentiation-specifying regulator of myogenesis that controls myogenic cell fate transitions during terminal differentiation in mice. The timing of RBP expression specifies cell fate transitions by providing post-transcriptional regulation of messenger RNAs that coordinate stem cell fate decisions during tissue regeneration.

Human Polycomb Repressive Complex 2 (PRC2) catalysis of histone H3 lysine 27 methylation at certain loci depends on long noncoding RNAs (lncRNAs). Yet, in apparent contradiction, RNA is a potent catalytic inhibitor of PRC2. Here, we show that intermolecular RNA-RNA interactions between the lncRNA HOTAIR and its targets can relieve RNA inhibition of PRC2. RNA bridging is promoted by heterogeneous nuclear ribonucleoprotein B1, which uses multiple protein domains to bind HOTAIR regions via multivalent protein-RNA interactions. Chemical probing demonstrates that establishing RNA-RNA interactions changes HOTAIR structure. Genome-wide HOTAIR/PRC2 activity occurs at genes whose transcripts can make favorable RNA-RNA interactions with HOTAIR. We demonstrate that RNA-RNA matches of HOTAIR with target gene RNAs can relieve the inhibitory effect of a single lncRNA for PRC2 activity after B1 dissociation. Our work highlights an intrinsic switch that allows PRC2 activity in specific RNA contexts, which could explain how many lncRNAs work with PRC2.

Methylation at the N 6 position of adenosine (m 6 A) is one of the most abundant RNA modifications found in eukaryotes; however, accurate detection of specific m 6 A nucleotides within transcripts has been historically challenging due to m 6 A and unmodified adenosine having virtually indistinguishable chemical properties. While previous strategies such as methyl-RNA immunoprecipitation and sequencing (MeRIP-seq) have relied on m 6 A-specific antibodies to isolate RNA fragments containing the modification, these methods do not allow for precise identification of individual m 6 A residues. More recently, modified cross-linking and immunoprecipitation (CLIP)-based approaches that rely on inducing specific mutations during reverse transcription via UV cross-linking of the anti-m 6 A antibody to methylated RNA have been used to overcome this limitation. However, the most utilized version of this approach, miCLIP, can be technically challenging to use for achieving high-complexity libraries. Here we present an improved methodology that yields high library complexity and allows for the straightforward identification of individual m 6 A residues with reliable confidence metrics. Based on enhanced CLIP (eCLIP), our m 6 A-eCLIP (meCLIP) approach couples the improvements of eCLIP with the inclusion of an input sample and an easy-to-use computational pipeline to allow for precise calling of m 6 A sites at true single-nucleotide resolution. As the effort to accurately identify m 6 As in an efficient and straightforward way intensifies, this method is a valuable tool for investigators interested in unraveling the m 6 A epitranscriptome.

28 Human Polycomb Repressive Complex 2 (PRC2) catalysis of histone H3 lysine 27 methylation at certain 29 loci depends on long noncoding RNAs (lncRNAs). Yet, in apparent contradiction, RNA is a potent catalytic 30 inhibitor of PRC2. Here we show that intermolecular RNA-RNA interactions between the lncRNA HOTAIR 31 and its targets can relieve RNA inhibition of PRC2. RNA bridging is promoted by heterogenous nuclear 32 ribonucleoprotein B1, which uses multiple protein domains to bind HOTAIR regions via multi-valent 33 protein-RNA interactions. Chemical probing demonstrates that establishing RNA-RNA interactions 34 changes HOTAIR structure. Genome-wide HOTAIR/PRC2 activity occurs at genes whose transcripts can 35 make favorable RNA-RNA interactions with HOTAIR. We demonstrate that RNA-RNA matches of 36 HOTAIR with target gene RNAs can relieve the inhibitory effect of a single lncRNA for PRC2 activity after 37 B1 dissociation. Our work highlights an intrinsic switch that allows PRC2 activity in specific RNA contexts, 38 which could explain how many lncRNAs work with PRC2.

Beyond being the product of gene expression, RNA can also influence the regulation of chromatin. The majority of the human genome has the capacity to be transcribed and the majority of the non-protein-coding transcripts made by RNA Polymerase II are enriched in the nucleus. Many chromatin regulators can bind to these ncRNAs in the nucleus; in some cases, there are clear examples of direct RNA-mediated chromatin regulation mechanisms stemming from these interactions, while others have yet to be determined. Recent studies have highlighted examples of chromatin regulation via RNA matchmaking, a term we use broadly here to describe intermolecular base-pairing interactions between one RNA molecule and an RNA or DNA match. This review provides examples of RNA matchmaking that regulates chromatin processes and summarizes the technical approaches used to capture these events.

ABSTRACTHuman Polycomb Repressive Complex 2 (PRC2) catalysis of histone H3 lysine 27 methylation at certain loci depends on long noncoding RNAs (lncRNAs). Yet, in apparent contradiction, RNA is a potent catalytic inhibitor of PRC2. Here we show that intermolecular RNA-RNA interactions between the lncRNA HOTAIR and its target genes can relieve RNA inhibition of PRC2. RNA matchmaking is promoted by heterogenous nuclear ribonucleoprotein (hnRNP) B1, which uses multiple protein domains to bind regions of HOTAIR via multi-valent protein-RNA interactions. Chemical probing demonstrates that RNA matchmaking changes HOTAIR RNA structure. Genome-wide HOTAIR/PRC2 activity occurs at genes whose transcripts can make favorable RNA-RNA interactions with HOTAIR. We demonstrate that RNA-RNA matches of HOTAIR with target gene RNAs can relieve the inhibitory effect of a single lncRNA for PRC2 activity. Our work highlights an intrinsic switch that allows PRC2 activity in specific RNA contexts, which could explain how many lncRNAs work with PRC2.

Over the past decade, long noncoding RNAs (lncRNAs) have been identified as significant players in gene regulation. They are often differentially expressed and widely-associated with a majority of cancer types. The aberrant expression of these transcripts has been linked to tumorigenesis, metastasis, cancer stage progression and patient survival. Despite their apparent link to cancer, it has been challenging to gain a mechanistic understanding of how they contribute to cancer, partially due the difficulty in discriminating functional RNAs from other noncoding transcription events. However, there are several well-studied lncRNAs where specific mechanisms have been more clearly defined, leading to new discoveries into how these RNAs function. One major observation that has come to light is the context-dependence of lncRNA mechanisms, where they often have unique function in specific cell types and environment. Here, we review the molecular mechanisms of lncRNAs with a focus on cancer pathways, illustrating a few informative examples. Together, this type of detailed insight will lead to a greater understanding of the potential for the application of lncRNAs as targets of cancer therapies and diagnostics.

A dominant histopathological feature in neuromuscular diseases, including amyotrophic lateral sclerosis and inclusion body myopathy, is cytoplasmic aggregation of the RNA-binding protein TDP-43. Although rare mutations in TARDBP-the gene that encodes TDP-43-that lead to protein misfolding often cause protein aggregation, most patients do not have any mutations in TARDBP. Therefore, aggregates of wild-type TDP-43 arise in most patients by an unknown mechanism. Here we show that TDP-43 is an essential protein for normal skeletal muscle formation that unexpectedly forms cytoplasmic, amyloid-like oligomeric assemblies, which we call myo-granules, during regeneration of skeletal muscle in mice and humans. Myo-granules bind to mRNAs that encode sarcomeric proteins and are cleared as myofibres mature. Although myo-granules occur during normal skeletal-muscle regeneration, myo-granules can seed TDP-43 amyloid fibrils in vitro and are increased in a mouse model of inclusion body myopathy. Therefore, increased assembly or decreased clearance of functionally normal myo-granules could be the source of cytoplasmic TDP-43 aggregates that commonly occur in neuromuscular disease.

DNA packaged into chromatin is the core structure of the human genome. Nearly all eukaryotic genome regulation must interface with this genomic structure, and modification of the chromatin can influence molecular mechanisms that regulate the underlying DNA. Many processes are governed by regulated stepwise assembly mechanisms that build complex machinery on chromatin to license a specific activity such as transcription. Transcriptional activators drive the initial steps of gene expression, regulated in part by chromatin. Here we describe tools to study the stepwise assembly of protein complexes on chromatin in a highly controlled manner using reconstituted human chromatin platforms and quantitative proteomic profiling. We profile the early steps in transcriptional activation and highlight the potential for understanding the multiple ways chromatin can influence transcriptional regulation. We also describe modifications of this approach to study the activity of a long noncoding RNA to act as a dynamic scaffold for proteins to be recruited to chromatin. This approach has the potential to provide a more comprehensive understanding of important macromolecular complex assembly that occurs on the human genome. The reconstituted nature of the chromatin substrate offers a tunable system that can be trapped at specific substeps to understand how chromatin interfaces with genome regulation machinery.

Heterochromatin formation in budding yeast is regulated by the silent information regulator (SIR) complex. The SIR complex comprises the NAD-dependent deacetylase Sir2, the scaffolding protein Sir4, and the nucleosome-binding protein Sir3. Transcriptionally active regions present a challenge to SIR complex-mediated de novo heterochromatic silencing due to the presence of antagonistic histone post-translational modifications, including acetylation and methylation. Methylation of histone H3K4 and H3K79 is dependent on monoubiquitination of histone H2B (H2B-Ub). The SIR complex cannot erase H2B-Ub or histone methylation on its own. The deubiquitinase (DUB) Ubp10 is thought to promote heterochromatic silencing by maintaining low H2B-Ub at sub-telomeres. Here, we biochemically characterized the interactions between Ubp10 and the SIR complex machinery. Wedemonstrate that a direct interaction between Ubp10 and the Sir2/4 sub-complex facilitates Ubp10 recruitment to chromatin via a co-assembly mechanism. Using hydrolyzable H2B-Ub analogs, we show that Ubp10 activity is lower on nucleosomes compared with H2B-Ub in solution. We find that Sir2/4 stimulates Ubp10 DUB activity on nucleosomes, likely through a combination of targeting and allosteric regulation. This coupling mechanism between the silencing machinery and its DUB partner allows erasure of active PTMs and the de novo transition of a transcriptionally active DNA region to a silent chromatin state.

Mono-ubiquitinated histone H2B (H2B-Ub) is important for chromatin regulation of transcription, chromatin assembly, and also influences heterochromatin. In this review, we discuss the effects of H2B-Ub from nucleosome to higher-order chromatin structure. We then assess what is currently known of the role of H2B-Ub in heterochromatic silencing in budding and fission yeasts ( S. cerevisiae and S. pombe ), which have distinct silencing mechanisms. In budding yeast, the SIR complex initiates heterochromatin assembly with the aid of a H2B-Ub deubiquitinase, Ubp10. In fission yeast, the RNAi-dependent pathway initiates heterochromatin in the context of low H2B-Ub. We examine how the different silencing machineries overcome the challenge of H2B-Ub chromatin and highlight the importance of using these microorganisms to further our understanding of H2B-Ub in heterochromatic silencing pathways.