Transcription elongation defects link oncogenic SF3B1 mutations to targetable alterations in chromatin landscape.

Transcription and splicing of pre-messenger RNA are closely coordinated, but how this functional coupling is disrupted in human diseases remains unexplored. Using isogenic cell lines, patient samples, and a mutant mouse model, we investigated how cancer-associated mutations in SF3B1 alter transcription. We found that these mutations reduce the elongation rate of RNA polymerase II (RNAPII) along gene bodies and its density at promoters. The elongation defect results from disrupted pre-spliceosome assembly due to impaired protein-protein interactions of mutant SF3B1. The decreased promoter-proximal RNAPII density reduces both chromatin accessibility and H3K4me3 marks at promoters. Through an unbiased screen, we identified epigenetic factors in the Sin3/HDAC/H3K4me pathway, which, when modulated, reverse both transcription and chromatin changes. Our findings reveal how splicing factor mutant states behave functionally as epigenetic disorders through impaired transcription-related changes to the chromatin landscape. We also present a rationale for targeting the Sin3/HDAC complex as a therapeutic strategy.

ALKBH5 modulates hematopoietic stem and progenitor cell energy metabolism through m6A modification-mediated RNA stability control.

N6-methyladenosine (m6A) RNA modification controls numerous cellular processes. To what extent these post-transcriptional regulatory mechanisms play a role in hematopoiesis has not been fully elucidated. We here show that the m6A demethylase alkB homolog 5 (ALKBH5) controls mitochondrial ATP production and modulates hematopoietic stem and progenitor cell (HSPC) fitness in an m6A-dependent manner. Loss of ALKBH5 results in increased RNA methylation and instability of oxoglutarate-dehydrogenase (Ogdh) messenger RNA and reduction of OGDH protein levels. Limited OGDH availability slows the tricarboxylic acid (TCA) cycle with accumulation of α-ketoglutarate (α-KG) and conversion of α-KG into L-2-hydroxyglutarate (L-2-HG). L-2-HG inhibits energy production in both murine and human hematopoietic cells in vitro. Impaired mitochondrial energy production confers competitive disadvantage to HSPCs and limits clonogenicity of Mll-AF9-induced leukemia. Our study uncovers a mechanism whereby the RNA m6A demethylase ALKBH5 regulates the stability of metabolic enzyme transcripts, thereby controlling energy metabolism in hematopoiesis and leukemia.

Acetyl-methyllysine marks chromatin at active transcription start sites.

Lysine residues in histones and other proteins can be modified by post-translational modifications that encode regulatory information1. Lysine acetylation and methylation are especially important for regulating chromatin and gene expression2-4. Pathways involving these post-translational modifications are targets for clinically approved therapeutics to treat human diseases. Lysine methylation and acetylation are generally assumed to be mutually exclusive at the same residue. Here we report cellular lysine residues that are both methylated and acetylated on the same side chain to form Nε-acetyl-Nε-methyllysine (Kacme). We show that Kacme is found on histone H4 (H4Kacme) across a range of species and across mammalian tissues. Kacme is associated with marks of active chromatin, increased transcriptional initiation and is regulated in response to biological signals. H4Kacme can be installed by enzymatic acetylation of monomethyllysine peptides and is resistant to deacetylation by some HDACs in vitro. Kacme can be bound by chromatin proteins that recognize modified lysine residues, as we demonstrate with the crystal structure of acetyllysine-binding protein BRD2 bound to a histone H4Kacme peptide. These results establish Kacme as a cellular post-translational modification with the potential to encode information distinct from methylation and acetylation alone and demonstrate that Kacme has all the hallmarks of a post-translational modification with fundamental importance to chromatin biology.

Catalytic and non-catalytic mechanisms of histone H4 lysine 20 methyltransferase SUV420H1.

SUV420H1 di- and tri-methylates histone H4 lysine 20 (H4K20me2/H4K20me3) and plays crucial roles in DNA replication, repair, and heterochromatin formation. It is dysregulated in several cancers. Many of these processes were linked to its catalytic activity. However, deletion and inhibition of SUV420H1 have shown distinct phenotypes, suggesting that the enzyme likely has uncharacterized non-catalytic activities. Our cryoelectron microscopy (cryo-EM), biochemical, biophysical, and cellular analyses reveal how SUV420H1 recognizes its nucleosome substrates, and how histone variant H2A.Z stimulates its catalytic activity. SUV420H1 binding to nucleosomes causes a dramatic detachment of nucleosomal DNA from the histone octamer, which is a non-catalytic activity. We hypothesize that this regulates the accessibility of large macromolecular complexes to chromatin. We show that SUV420H1 can promote chromatin condensation, another non-catalytic activity that we speculate is needed for its heterochromatin functions. Together, our studies uncover and characterize the catalytic and non-catalytic mechanisms of SUV420H1, a key histone methyltransferase that plays an essential role in genomic stability.

Improving the study of RNA dynamics through advances in RNA-seq with metabolic labeling and nucleotide-recoding chemistry.

RNA metabolic labeling using 4-thiouridine (s4U) captures the dynamics of RNA synthesis and decay. The power of this approach is dependent on appropriate quantification of labeled and unlabeled sequencing reads, which can be compromised by the apparent loss of s4U-labeled reads in a process we refer to as dropout. Here we show that s4U-containing transcripts can be selectively lost when RNA samples are handled under sub-optimal conditions, but that this loss can be minimized using an optimized protocol. We demonstrate a second cause of dropout in nucleotide recoding and RNA sequencing (NR-seq) experiments that is computational and downstream of library preparation. NR-seq experiments involve chemically converting s4U from a uridine analog to a cytidine analog and using the apparent T-to-C mutations to identify the populations of newly synthesized RNA. We show that high levels of T-to-C mutations can prevent read alignment with some computational pipelines, but that this bias can be overcome using improved alignment pipelines. Importantly, kinetic parameter estimates are affected by dropout independent of the NR chemistry employed, and all chemistries are practically indistinguishable in bulk, short-read RNA-seq experiments. Dropout is an avoidable problem that can be identified by including unlabeled controls, and mitigated through improved sample handing and read alignment that together improve the robustness and reproducibility of NR-seq experiments.

Systematic detection of tertiary structural modules in large RNAs and RNP interfaces by Tb-seq.

Compact RNA structural motifs control many aspects of gene expression, but we lack methods for finding these structures in the vast expanse of multi-kilobase RNAs. To adopt specific 3-D shapes, many RNA modules must compress their RNA backbones together, bringing negatively charged phosphates into close proximity. This is often accomplished by recruiting multivalent cations (usually Mg2+), which stabilize these sites and neutralize regions of local negative charge. Coordinated lanthanide ions, such as terbium (III) (Tb3+), can also be recruited to these sites, where they induce efficient RNA cleavage, thereby revealing compact RNA 3-D modules. Until now, Tb3+ cleavage sites were monitored via low-throughput biochemical methods only applicable to small RNAs. Here we present Tb-seq, a high-throughput sequencing method for detecting compact tertiary structures in large RNAs. Tb-seq detects sharp backbone turns found in RNA tertiary structures and RNP interfaces, providing a way to scan transcriptomes for stable structural modules and potential riboregulatory motifs.

bakR: uncovering differential RNA synthesis and degradation kinetics transcriptome-wide with Bayesian hierarchical modeling.

Differential expression analysis of RNA sequencing (RNA-seq) data can identify changes in cellular RNA levels, but provides limited information about the kinetic mechanisms underlying such changes. Nucleotide recoding RNA-seq methods (NR-seq; e.g., TimeLapse-seq, SLAM-seq, etc.) address this shortcoming and are widely used approaches to identify changes in RNA synthesis and degradation kinetics. While advanced statistical models implemented in user-friendly software (e.g., DESeq2) have ensured the statistical rigor of differential expression analyses, no such tools that facilitate differential kinetic analysis with NR-seq exist. Here, we report the development of Bayesian analysis of the kinetics of RNA (bakR; https:// github.com/simonlabcode/bakR), an R package to address this need. bakR relies on Bayesian hierarchical modeling of NR-seq data to increase statistical power by sharing information across transcripts. Analyses of simulated data confirmed that bakR implementations of the hierarchical model outperform attempts to analyze differential kinetics with existing models. bakR also uncovers biological signals in real NR-seq data sets and provides improved analyses of existing data sets. This work establishes bakR as an important tool for identifying differential RNA synthesis and degradation kinetics.

Internally controlled RNA sequencing comparisons using nucleoside recoding chemistry.

Quantitative comparisons of RNA levels from different samples can lead to new biological understanding if they are able to distinguish biological variation from variable sample preparation. These challenges are pronounced in comparisons that require complex biochemical manipulations (e.g. isolating polysomes to study translation). Here, we present Transcript Regulation Identified by Labeling with Nucleoside Analogues in Cell Culture (TILAC), an internally controlled approach for quantitative comparisons of RNA content. TILAC uses two metabolic labels, 4-thiouridine (s4U) and 6-thioguanosine (s6G), to differentially label RNAs in cells, allowing experimental and control samples to be pooled prior to downstream biochemical manipulations. TILAC leverages nucleoside recoding chemistry to generate characteristic sequencing signatures for each label and uses statistical modeling to compare the abundance of RNA transcripts between samples. We verified the performance of TILAC in transcriptome-scale experiments involving RNA polymerase II inhibition and heat shock. We then applied TILAC to quantify changes in mRNA association with actively translating ribosomes during sodium arsenite stress and discovered a set of transcripts that are translationally upregulated, including MCM2 and DDX5. TILAC is broadly applicable to uncover differences between samples leading to improved biological insights.

Targeted Degradation of mRNA Decapping Enzyme DcpS by a VHL-Recruiting PROTAC.

The RNA decapping scavenger protein, DcpS, has recently been identified as a dependency in acute myeloid leukemia (AML). The potent DcpS inhibitor RG3039 attenuates AML cell viability, and shRNA knockdown of DcpS is also antiproliferative. Importantly, DcpS was found to be non-essential in normal human hematopoietic cells, which opens a therapeutic window for AML treatment by DcpS modulation. Considering this strong DcpS dependence in AML cell lines, we explored PROTAC-mediated degradation as an alternative strategy to modulate DcpS activity. Herein, we report the development of JCS-1, a PROTAC exhibiting effective degradation of DcpS at nanomolar concentrations. JCS-1 non-covalently binds DcpS with a RG3039-based warhead and recruits the E3 ligase VHL, which induces potent, rapid, and sustained DcpS degradation in several AML cell lines. JCS-1 serves as a chemical biology tool to interrogate DcpS degradation and associated changes in RNA processes in different cellular contexts, which may be an attractive strategy for the treatment of AML and other DcpS-dependent genetic disorders.

Functional elements of the cis-regulatory lincRNA-p21.

The p53-induced long noncoding RNA (lncRNA) lincRNA-p21 is proposed to act in cis to promote p53-dependent expression of the neighboring cell cycle gene, Cdkn1a/p21. The molecular mechanism through which the transcribed lincRNA-p21 regulatory locus activates p21 expression remains poorly understood. To elucidate the functional elements of cis-regulation, we generate a series of genetic models that disrupt DNA regulatory elements, the transcription of lincRNA-p21, or the accumulation of mature lincRNA-p21. Unexpectedly, we determine that full-length transcription, splicing, and accumulation of lincRNA-p21 are dispensable for the chromatin organization of the locus and for cis-regulation. Instead, we find that production of lincRNA-p21 through conserved regions in exon 1 of lincRNA-p21 promotes cis-activation. These findings demonstrate that the activation of nascent transcription from this lncRNA locus, but not the generation or accumulation of a mature lncRNA transcript, is necessary to enact local gene expression control.

Precision analysis of mutant U2AF1 activity reveals deployment of stress granules in myeloid malignancies.

Splicing factor mutations are common among cancers, recently emerging as drivers of myeloid malignancies. U2AF1 carries hotspot mutations in its RNA-binding motifs; however, how they affect splicing and promote cancer remain unclear. The U2AF1/U2AF2 heterodimer is critical for 3' splice site (3'SS) definition. To specifically unmask changes in U2AF1 function in vivo, we developed a crosslinking and immunoprecipitation procedure that detects contacts between U2AF1 and the 3'SS AG at single-nucleotide resolution. Our data reveal that the U2AF1 S34F and Q157R mutants establish new 3'SS contacts at -3 and +1 nucleotides, respectively. These effects compromise U2AF2-RNA interactions, resulting predominantly in intron retention and exon exclusion. Integrating RNA binding, splicing, and turnover data, we predicted that U2AF1 mutations directly affect stress granule components, which was corroborated by single-cell RNA-seq. Remarkably, U2AF1-mutant cell lines and patient-derived MDS/AML blasts displayed a heightened stress granule response, pointing to a novel role for biomolecular condensates in adaptive oncogenic strategies.

Noncoding RNAs: biology and applications-a Keystone Symposia report.

The human transcriptome contains many types of noncoding RNAs, which rival the number of protein-coding species. From long noncoding RNAs (lncRNAs) that are over 200 nucleotides long to piwi-interacting RNAs (piRNAs) of only 20 nucleotides, noncoding RNAs play important roles in regulating transcription, epigenetic modifications, translation, and cell signaling. Roles for noncoding RNAs in disease mechanisms are also being uncovered, and several species have been identified as potential drug targets. On May 11-14, 2021, the Keystone eSymposium "Noncoding RNAs: Biology and Applications" brought together researchers working in RNA biology, structure, and technologies to accelerate both the understanding of RNA basic biology and the translation of those findings into clinical applications.

STL-seq reveals pause-release and termination kinetics for promoter-proximal paused RNA polymerase II transcripts.

Despite the critical regulatory function of promoter-proximal pausing, the influence of pausing kinetics on transcriptional control remains an active area of investigation. Here, we present Start-TimeLapse-seq (STL-seq), a method that captures the genome-wide kinetics of short, capped RNA turnover and reveals principles of regulation at the pause site. By measuring the rates of release into elongation and premature termination through the inhibition of pause release, we determine that pause-release rates are highly variable, and most promoter-proximal paused RNA polymerase II molecules prematurely terminate (∼80%). The preferred regulatory mechanism upon a hormonal stimulus (20-hydroxyecdysone) is to influence pause-release rather than termination rates. Transcriptional shutdown occurs concurrently with the induction of promoter-proximal termination under hyperosmotic stress, but paused transcripts from TATA box-containing promoters remain stable, demonstrating an important role for cis-acting DNA elements in pausing. STL-seq dissects the kinetics of pause release and termination, providing an opportunity to identify mechanisms of transcriptional regulation.

Hyperosmotic stress alters the RNA polymerase II interactome and induces readthrough transcription despite widespread transcriptional repression.

Stress-induced readthrough transcription results in the synthesis of downstream-of-gene (DoG)-containing transcripts. The mechanisms underlying DoG formation during cellular stress remain unknown. Nascent transcription profiles during DoG induction in human cell lines using TT-TimeLapse sequencing revealed widespread transcriptional repression upon hyperosmotic stress. Yet, DoGs are produced regardless of the transcriptional level of their upstream genes. ChIP sequencing confirmed that stress-induced redistribution of RNA polymerase (Pol) II correlates with the transcriptional output of genes. Stress-induced alterations in the Pol II interactome are observed by mass spectrometry. While certain cleavage and polyadenylation factors remain Pol II associated, Integrator complex subunits dissociate from Pol II under stress leading to a genome-wide loss of Integrator on DNA. Depleting the catalytic subunit of Integrator using siRNAs induces hundreds of readthrough transcripts, whose parental genes partially overlap those of stress-induced DoGs. Our results provide insights into the mechanisms underlying DoG production and how Integrator activity influences DoG transcription.

Genome-wide CRISPR Screens Reveal Host Factors Critical for SARS-CoV-2 Infection.

Identification of host genes essential for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may reveal novel therapeutic targets and inform our understanding of coronavirus disease 2019 (COVID-19) pathogenesis. Here we performed genome-wide CRISPR screens in Vero-E6 cells with SARS-CoV-2, Middle East respiratory syndrome CoV (MERS-CoV), bat CoV HKU5 expressing the SARS-CoV-1 spike, and vesicular stomatitis virus (VSV) expressing the SARS-CoV-2 spike. We identified known SARS-CoV-2 host factors, including the receptor ACE2 and protease Cathepsin L. We additionally discovered pro-viral genes and pathways, including HMGB1 and the SWI/SNF chromatin remodeling complex, that are SARS lineage and pan-coronavirus specific, respectively. We show that HMGB1 regulates ACE2 expression and is critical for entry of SARS-CoV-2, SARS-CoV-1, and NL63. We also show that small-molecule antagonists of identified gene products inhibited SARS-CoV-2 infection in monkey and human cells, demonstrating the conserved role of these genetic hits across species. This identifies potential therapeutic targets for SARS-CoV-2 and reveals SARS lineage-specific and pan-CoV host factors that regulate susceptibility to highly pathogenic CoVs.

Discovery of cellular substrates of human RNA-decapping enzyme DCP2 using a stapled bicyclic peptide inhibitor.

DCP2 is an RNA-decapping enzyme that controls the stability of human RNAs that encode factors functioning in transcription and the immune response. While >1,800 human DCP2 substrates have been identified, compensatory expression changes secondary to genetic ablation of DCP2 have complicated a complete mapping of its regulome. Cell-permeable, selective chemical inhibitors of DCP2 could provide a powerful tool to study DCP2 specificity. Here, we report phage display selection of CP21, a bicyclic peptide ligand to DCP2. CP21 has high affinity and selectivity for DCP2 and inhibits DCP2 decapping activity toward selected RNA substrates in human cells. CP21 increases formation of P-bodies, liquid condensates enriched in intermediates of RNA decay, in a manner that resembles the deletion or mutation of DCP2. We used CP21 to identify 76 previously unreported DCP2 substrates. This work demonstrates that DCP2 inhibition can complement genetic approaches to study RNA decay.

The NBDY Microprotein Regulates Cellular RNA Decapping.

Proteogenomic identification of translated small open reading frames in humans has revealed thousands of microproteins, or polypeptides of fewer than 100 amino acids, that were previously invisible to geneticists. Hundreds of microproteins have been shown to be essential for cell growth and proliferation, and many regulate macromolecular complexes. One such regulatory microprotein is NBDY, a 68-amino acid component of the human cytoplasmic RNA decapping complex. Heterologously expressed NBDY was previously reported to regulate cytoplasmic ribonucleoprotein granules known as P-bodies and reporter gene stability, but the global effect of endogenous NBDY on the cellular transcriptome remained undefined. In this work, we demonstrate that endogenous NBDY directly interacts with the human RNA decapping complex through EDC4 and DCP1A and localizes to P-bodies. Global profiling of RNA stability changes in NBDY knockout (KO) cells reveals dysregulated stability of more than 1400 transcripts. DCP2 substrate transcript half-lives are both increased and decreased in NBDY KO cells, which correlates with 5' UTR length. NBDY deletion additionally alters the stability of non-DCP2 target transcripts, possibly as a result of downregulated expression of nonsense-mediated decay factors in NBDY KO cells. We present a comprehensive model of the regulation of RNA stability by NBDY.

Genome-wide CRISPR screen reveals host genes that regulate SARS-CoV-2 infection.

Identification of host genes essential for SARS-CoV-2 infection may reveal novel therapeutic targets and inform our understanding of COVID-19 pathogenesis. Here we performed a genome-wide CRISPR screen with SARS-CoV-2 and identified known SARS-CoV-2 host factors including the receptor ACE2 and protease Cathepsin L. We additionally discovered novel pro-viral genes and pathways including the SWI/SNF chromatin remodeling complex and key components of the TGF-ß signaling pathway. Small molecule inhibitors of these pathways prevented SARS-CoV-2-induced cell death. We also revealed that the alarmin HMGB1 is critical for SARS-CoV-2 replication. In contrast, loss of the histone H3.3 chaperone complex sensitized cells to virus-induced death. Together this study reveals potential therapeutic targets for SARS-CoV-2 and highlights host genes that may regulate COVID-19 pathogenesis.

Global Profiling of Cellular Substrates of Human Dcp2.

Decapping is the first committed step in 5'-to-3' RNA decay, and in the cytoplasm of human cells, multiple decapping enzymes regulate the stabilities of distinct subsets of cellular transcripts. However, the complete set of RNAs regulated by any individual decapping enzyme remains incompletely mapped, and no consensus sequence or property is currently known to unambiguously predict decapping enzyme substrates. Dcp2 was the first-identified and best-studied eukaryotic decapping enzyme, but it has been shown to regulate the stability of <400 transcripts in mammalian cells to date. Here, we globally profile changes in the stability of the human transcriptome in Dcp2 knockout cells via TimeLapse-seq. We find that P-body enrichment is the strongest correlate of Dcp2-dependent decay and that modification with m6A exhibits an additive effect with P-body enrichment for Dcp2 targeting. These results are consistent with a model in which P-bodies represent sites where translationally repressed transcripts are sorted for decay by soluble cytoplasmic decay complexes through additional molecular marks.

Author Correction: Enhanced nucleotide chemistry and toehold nanotechnology reveals lncRNA spreading on chromatin.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.