The Story of RNA Unfolded: The Molecular Function of DEAD- and DExH-Box ATPases and Their Complex Relationship with Membraneless Organelles.

DEAD- and DExH-box ATPases (DDX/DHXs) are abundant and highly conserved cellular enzymes ubiquitously involved in RNA processing. By remodeling RNA-RNA and RNA-protein interactions, they often function as gatekeepers that control the progression of diverse RNA maturation steps. Intriguingly, most DDX/DHXs localize to membraneless organelles (MLOs) such as nucleoli, nuclear speckles, stress granules, or processing bodies. Recent findings suggest not only that localization to MLOs can promote interaction between DDX/DHXs and their targets but also that DDX/DHXs are key regulators of MLO formation and turnover through their condensation and ATPase activity.In this review, we describe the molecular function of DDX/DHXs in ribosome biogenesis, messenger RNA splicing, export, translation, and storage or decay as well as their association with prominent MLOs. We discuss how the enzymatic function of DDX/DHXs in RNA processing is linked to DDX/DHX condensation, the accumulation of ribonucleoprotein particles and MLO dynamics. Future research will reveal how these processes orchestrate the RNA life cycle in MLO space and DDX/DHX time.

Capturing totipotency in human cells through spliceosomal repression.

The cleavage of zygotes generates totipotent blastomeres. In human 8-cell blastomeres, zygotic genome activation (ZGA) occurs to initiate the ontogenesis program. However, capturing and maintaining totipotency in human cells pose significant challenges. Here, we realize culturing human totipotent blastomere-like cells (hTBLCs). We find that splicing inhibition can transiently reprogram human pluripotent stem cells into ZGA-like cells (ZLCs), which subsequently transition into stable hTBLCs after long-term passaging. Distinct from reported 8-cell-like cells (8CLCs), both ZLCs and hTBLCs widely silence pluripotent genes. Interestingly, ZLCs activate a particular group of ZGA-specific genes, and hTBLCs are enriched with pre-ZGA-specific genes. During spontaneous differentiation, hTBLCs re-enter the intermediate ZLC stage and further generate epiblast (EPI)-, primitive endoderm (PrE)-, and trophectoderm (TE)-like lineages, effectively recapitulating human pre-implantation development. Possessing both embryonic and extraembryonic developmental potency, hTBLCs can autonomously generate blastocyst-like structures in vitro without external cell signaling. In summary, our study provides key criteria and insights into human cell totipotency.

Sweet splicing.

Glucose is the main source of energy for cells. In this issue of Cell, a study now shows that glucose has additional non-energetic functions, acting as a biomolecular cue that regulates alternative splicing during epidermal differentiation. As keratinocytes differentiate, glucose associates with RNA-binding protein DDX21 and modulates its interaction properties, which modifies splicing decisions.

Circular RNAs: Characterization, cellular roles, and applications.

Most circular RNAs are produced from the back-splicing of exons of precursor mRNAs. Recent technological advances have in part overcome problems with their circular conformation and sequence overlap with linear cognate mRNAs, allowing a better understanding of their cellular roles. Depending on their localization and specific interactions with DNA, RNA, and proteins, circular RNAs can modulate transcription and splicing, regulate stability and translation of cytoplasmic mRNAs, interfere with signaling pathways, and serve as templates for translation in different biological and pathophysiological contexts. Emerging applications of RNA circles to interfere with cellular processes, modulate immune responses, and direct translation into proteins shed new light on biomedical research. In this review, we discuss approaches used in circular RNA studies and the current understanding of their regulatory roles and potential applications.

Counteracting chromatin effects of a splicing-correcting antisense oligonucleotide improves its therapeutic efficacy in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a motor-neuron disease caused by mutations of the SMN1 gene. The human paralog SMN2, whose exon 7 (E7) is predominantly skipped, cannot compensate for the lack of SMN1. Nusinersen is an antisense oligonucleotide (ASO) that upregulates E7 inclusion and SMN protein levels by displacing the splicing repressors hnRNPA1/A2 from their target site in intron 7. We show that by promoting transcriptional elongation, the histone deacetylase inhibitor VPA cooperates with a nusinersen-like ASO to promote E7 inclusion. Surprisingly, the ASO promotes the deployment of the silencing histone mark H3K9me2 on the SMN2 gene, creating a roadblock to RNA polymerase II elongation that inhibits E7 inclusion. By removing the roadblock, VPA counteracts the chromatin effects of the ASO, resulting in higher E7 inclusion without large pleiotropic effects. Combined administration of the nusinersen-like ASO and VPA in SMA mice strongly synergizes SMN expression, growth, survival, and neuromuscular function.

Splicing regulation through biomolecular condensates and membraneless organelles.

Biomolecular condensates, sometimes also known as membraneless organelles (MLOs), can form through weak multivalent intermolecular interactions of proteins and nucleic acids, a process often associated with liquid-liquid phase separation. Biomolecular condensates are emerging as sites and regulatory platforms of vital cellular functions, including transcription and RNA processing. In the first part of this Review, we comprehensively discuss how alternative splicing regulates the formation and properties of condensates, and conversely the roles of biomolecular condensates in splicing regulation. In the second part, we focus on the spatial connection between splicing regulation and nuclear MLOs such as transcriptional condensates, splicing condensates and nuclear speckles. We then discuss key studies showing how splicing regulation through biomolecular condensates is implicated in human pathologies such as neurodegenerative diseases, different types of cancer, developmental disorders and cardiomyopathies, and conclude with a discussion of outstanding questions pertaining to the roles of condensates and MLOs in splicing regulation and how to experimentally study them.

Co-transcriptional gene regulation in eukaryotes and prokaryotes.

Many steps of RNA processing occur during transcription by RNA polymerases. Co-transcriptional activities are deemed commonplace in prokaryotes, in which the lack of membrane barriers allows mixing of all gene expression steps, from transcription to translation. In the past decade, an extraordinary level of coordination between transcription and RNA processing has emerged in eukaryotes. In this Review, we discuss recent developments in our understanding of co-transcriptional gene regulation in both eukaryotes and prokaryotes, comparing methodologies and mechanisms, and highlight striking parallels in how RNA polymerases interact with the machineries that act on nascent RNA. The development of RNA sequencing and imaging techniques that detect transient transcription and RNA processing intermediates has facilitated discoveries of transcription coordination with splicing, 3'-end cleavage and dynamic RNA folding and revealed physical contacts between processing machineries and RNA polymerases. Such studies indicate that intron retention in a given nascent transcript can prevent 3'-end cleavage and cause transcriptional readthrough, which is a hallmark of eukaryotic cellular stress responses. We also discuss how coordination between nascent RNA biogenesis and transcription drives fundamental aspects of gene expression in both prokaryotes and eukaryotes.

Dynamic imaging of nascent RNA reveals general principles of transcription dynamics and stochastic splice site selection.

The activities of RNA polymerase and the spliceosome are responsible for the heterogeneity in the abundance and isoform composition of mRNA in human cells. However, the dynamics of these megadalton enzymatic complexes working in concert on endogenous genes have not been described. Here, we establish a quasi-genome-scale platform for observing synthesis and processing kinetics of single nascent RNA molecules in real time. We find that all observed genes show transcriptional bursting. We also observe large kinetic variation in intron removal for single introns in single cells, which is inconsistent with deterministic splice site selection. Transcriptome-wide footprinting of the U2AF complex, nascent RNA profiling, long-read sequencing, and lariat sequencing further reveal widespread stochastic recursive splicing within introns. We propose and validate a unified theoretical model to explain the general features of transcription and pervasive stochastic splice site selection.

Pharmacologic modulation of RNA splicing enhances anti-tumor immunity.

Although mutations in DNA are the best-studied source of neoantigens that determine response to immune checkpoint blockade, alterations in RNA splicing within cancer cells could similarly result in neoepitope production. However, the endogenous antigenicity and clinical potential of such splicing-derived epitopes have not been tested. Here, we demonstrate that pharmacologic modulation of splicing via specific drug classes generates bona fide neoantigens and elicits anti-tumor immunity, augmenting checkpoint immunotherapy. Splicing modulation inhibited tumor growth and enhanced checkpoint blockade in a manner dependent on host T cells and peptides presented on tumor MHC class I. Splicing modulation induced stereotyped splicing changes across tumor types, altering the MHC I-bound immunopeptidome to yield splicing-derived neoepitopes that trigger an anti-tumor T cell response in vivo. These data definitively identify splicing modulation as an untapped source of immunogenic peptides and provide a means to enhance response to checkpoint blockade that is readily translatable to the clinic.

Spliceosome-targeted therapies trigger an antiviral immune response in triple-negative breast cancer.

Many oncogenic insults deregulate RNA splicing, often leading to hypersensitivity of tumors to spliceosome-targeted therapies (STTs). However, the mechanisms by which STTs selectively kill cancers remain largely unknown. Herein, we discover that mis-spliced RNA itself is a molecular trigger for tumor killing through viral mimicry. In MYC-driven triple-negative breast cancer, STTs cause widespread cytoplasmic accumulation of mis-spliced mRNAs, many of which form double-stranded structures. Double-stranded RNA (dsRNA)-binding proteins recognize these endogenous dsRNAs, triggering antiviral signaling and extrinsic apoptosis. In immune-competent models of breast cancer, STTs cause tumor cell-intrinsic antiviral signaling, downstream adaptive immune signaling, and tumor cell death. Furthermore, RNA mis-splicing in human breast cancers correlates with innate and adaptive immune signatures, especially in MYC-amplified tumors that are typically immune cold. These findings indicate that dsRNA-sensing pathways respond to global aberrations of RNA splicing in cancer and provoke the hypothesis that STTs may provide unexplored strategies to activate anti-tumor immune pathways.

Splice site m6A methylation prevents binding of U2AF35 to inhibit RNA splicing.

The N6-methyladenosine (m6A) RNA modification is used widely to alter the fate of mRNAs. Here we demonstrate that the C. elegans writer METT-10 (the ortholog of mouse METTL16) deposits an m6A mark on the 3' splice site (AG) of the S-adenosylmethionine (SAM) synthetase pre-mRNA, which inhibits its proper splicing and protein production. The mechanism is triggered by a rich diet and acts as an m6A-mediated switch to stop SAM production and regulate its homeostasis. Although the mammalian SAM synthetase pre-mRNA is not regulated via this mechanism, we show that splicing inhibition by 3' splice site m6A is conserved in mammals. The modification functions by physically preventing the essential splicing factor U2AF35 from recognizing the 3' splice site. We propose that use of splice-site m6A is an ancient mechanism for splicing regulation.

ELAVL4, splicing, and glutamatergic dysfunction precede neuron loss in MAPT mutation cerebral organoids.

Frontotemporal dementia (FTD) because of MAPT mutation causes pathological accumulation of tau and glutamatergic cortical neuronal death by unknown mechanisms. We used human induced pluripotent stem cell (iPSC)-derived cerebral organoids expressing tau-V337M and isogenic corrected controls to discover early alterations because of the mutation that precede neurodegeneration. At 2 months, mutant organoids show upregulated expression of MAPT, glutamatergic signaling pathways, and regulators, including the RNA-binding protein ELAVL4, and increased stress granules. Over the following 4 months, mutant organoids accumulate splicing changes, disruption of autophagy function, and build-up of tau and P-tau-S396. By 6 months, tau-V337M organoids show specific loss of glutamatergic neurons as seen in individuals with FTD. Mutant neurons are susceptible to glutamate toxicity, which can be rescued pharmacologically by the PIKFYVE kinase inhibitor apilimod. Our results demonstrate a sequence of events that precede neurodegeneration, revealing molecular pathways associated with glutamate signaling as potential targets for therapeutic intervention in FTD.

RNA promotes the formation of spatial compartments in the nucleus.

RNA, DNA, and protein molecules are highly organized within three-dimensional (3D) structures in the nucleus. Although RNA has been proposed to play a role in nuclear organization, exploring this has been challenging because existing methods cannot measure higher-order RNA and DNA contacts within 3D structures. To address this, we developed RNA & DNA SPRITE (RD-SPRITE) to comprehensively map the spatial organization of RNA and DNA. These maps reveal higher-order RNA-chromatin structures associated with three major classes of nuclear function: RNA processing, heterochromatin assembly, and gene regulation. These data demonstrate that hundreds of ncRNAs form high-concentration territories throughout the nucleus, that specific RNAs are required to recruit various regulators into these territories, and that these RNAs can shape long-range DNA contacts, heterochromatin assembly, and gene expression. These results demonstrate a mechanism where RNAs form high-concentration territories, bind to diffusible regulators, and guide them into compartments to regulate essential nuclear functions.

ERα is an RNA-binding protein sustaining tumor cell survival and drug resistance.

Estrogen receptor α (ERα) is a hormone receptor and key driver for over 70% of breast cancers that has been studied for decades as a transcription factor. Unexpectedly, we discover that ERα is a potent non-canonical RNA-binding protein. We show that ERα RNA binding function is uncoupled from its activity to bind DNA and critical for breast cancer progression. Employing genome-wide cross-linking immunoprecipitation (CLIP) sequencing and a functional CRISPRi screen, we find that ERα-associated mRNAs sustain cancer cell fitness and elicit cellular responses to stress. Mechanistically, ERα controls different steps of RNA metabolism. In particular, we demonstrate that ERα RNA binding mediates alternative splicing of XBP1 and translation of the eIF4G2 and MCL1 mRNAs, which facilitates survival upon stress conditions and sustains tamoxifen resistance of cancer cells. ERα is therefore a multifaceted RNA-binding protein, and this activity transforms our knowledge of post-transcriptional regulation underlying cancer development and drug response.

Detection and Degradation of Stalled Nascent Chains via Ribosome-Associated Quality Control.

Stalled protein synthesis produces defective nascent chains that can harm cells. In response, cells degrade these nascent chains via a process called ribosome-associated quality control (RQC). Here, we review the irregularities in the translation process that cause ribosomes to stall as well as how cells use RQC to detect stalled ribosomes, ubiquitylate their tethered nascent chains, and deliver the ubiquitylated nascent chains to the proteasome. We additionally summarize how cells respond to RQC failure.

RNA Splicing by the Spliceosome.

The spliceosome removes introns from messenger RNA precursors (pre-mRNA). Decades of biochemistry and genetics combined with recent structural studies of the spliceosome have produced a detailed view of the mechanism of splicing. In this review, we aim to make this mechanism understandable and provide several videos of the spliceosome in action to illustrate the intricate choreography of splicing. The U1 and U2 small nuclear ribonucleoproteins (snRNPs) mark an intron and recruit the U4/U6.U5 tri-snRNP. Transfer of the 5' splice site (5'SS) from U1 to U6 snRNA triggers unwinding of U6 snRNA from U4 snRNA. U6 folds with U2 snRNA into an RNA-based active site that positions the 5'SS at two catalytic metal ions. The branch point (BP) adenosine attacks the 5'SS, producing a free 5' exon. Removal of the BP adenosine from the active site allows the 3'SS to bind, so that the 5' exon attacks the 3'SS to produce mature mRNA and an excised lariat intron.

How Is Precursor Messenger RNA Spliced by the Spliceosome?

Splicing of the precursor messenger RNA, involving intron removal and exon ligation, is mediated by the spliceosome. Together with biochemical and genetic investigations of the past four decades, structural studies of the intact spliceosome at atomic resolution since 2015 have led to mechanistic delineation of RNA splicing with remarkable insights. The spliceosome is proven to be a protein-orchestrated metalloribozyme. Conserved elements of small nuclear RNA (snRNA) constitute the splicing active site with two catalytic metal ions and recognize three conserved intron elements through duplex formation, which are delivered into the splicing active site for branching and exon ligation. The protein components of the spliceosome stabilize the conformation of the snRNA, drive spliceosome remodeling, orchestrate the movement of the RNA elements, and facilitate the splicing reaction. The overall organization of the spliceosome and the configuration of the splicing active site are strictly conserved between human and yeast.

Regulation and functions of non-m6A mRNA modifications.

Nucleobase modifications are prevalent in eukaryotic mRNA and their discovery has resulted in the emergence of epitranscriptomics as a research field. The most abundant internal (non-cap) mRNA modification is N6-methyladenosine (m6A), the study of which has revolutionized our understanding of post-transcriptional gene regulation. In addition, numerous other mRNA modifications are gaining great attention because of their major roles in RNA metabolism, immunity, development and disease. In this Review, we focus on the regulation and function of non-m6A modifications in eukaryotic mRNA, including pseudouridine (Ψ), N6,2'-O-dimethyladenosine (m6Am), N1-methyladenosine (m1A), inosine, 5-methylcytidine (m5C), N4-acetylcytidine (ac4C), 2'-O-methylated nucleotide (Nm) and internal N7-methylguanosine (m7G). We highlight their regulation, distribution, stoichiometry and known roles in mRNA metabolism, such as mRNA stability, translation, splicing and export. We also discuss their biological consequences in physiological and pathological processes. In addition, we cover research techniques to further study the non-m6A mRNA modifications and discuss their potential future applications.

SnapShot: Splicing Alterations in Cancer.

RNA splicing, the spliceosome-catalyzed process by which pre-messenger RNA (pre-mRNA) is processed to mature mRNA, is altered in a number of ways in cancer. Tumor-specific splicing alterations are created by mutations that disrupt splicing-regulatory elements within genes and impair splicing recognition or by altering the RNA-binding preferences of individual splicing factors. This SnapShot summarizes our current understanding of splicing-factor alterations in cancers. To view this SnapShot, open or download the PDF.

Initiation of Parental Genome Reprogramming in Fertilized Oocyte by Splicing Kinase SRPK1-Catalyzed Protamine Phosphorylation.

The paternal genome undergoes a massive exchange of histone with protamine for compaction into sperm during spermiogenesis. Upon fertilization, this process is potently reversed, which is essential for parental genome reprogramming and subsequent activation; however, it remains poorly understood how this fundamental process is initiated and regulated. Here, we report that the previously characterized splicing kinase SRPK1 initiates this life-beginning event by catalyzing site-specific phosphorylation of protamine, thereby triggering protamine-to-histone exchange in the fertilized oocyte. Interestingly, protamine undergoes a DNA-dependent phase transition to gel-like condensates and SRPK1-mediated phosphorylation likely helps open up such structures to enhance protamine dismissal by nucleoplasmin (NPM2) and enable the recruitment of HIRA for H3.3 deposition. Remarkably, genome-wide assay for transposase-accessible chromatin sequencing (ATAC-seq) analysis reveals that selective chromatin accessibility in both sperm and MII oocytes is largely erased in early pronuclei in a protamine phosphorylation-dependent manner, suggesting that SRPK1-catalyzed phosphorylation initiates a highly synchronized reorganization program in both parental genomes.