DDX21: The link between m6A and R-loops.

In this issue of Molecular Cell, Hao et al.1 demonstrate that the RNA helicase DDX21 recruits the m6A methyltransferase complex to R-loops, ensuring proper transcription termination and genome stability.

DDX21 mediates co-transcriptional RNA m6A modification to promote transcription termination and genome stability.

N6-methyladenosine (m6A) is a crucial RNA modification that regulates diverse biological processes in human cells, but its co-transcriptional deposition and functions remain poorly understood. Here, we identified the RNA helicase DDX21 with a previously unrecognized role in directing m6A modification on nascent RNA for co-transcriptional regulation. DDX21 interacts with METTL3 for co-recruitment to chromatin through its recognition of R-loops, which can be formed co-transcriptionally as nascent transcripts hybridize onto the template DNA strand. Moreover, DDX21's helicase activity is needed for METTL3-mediated m6A deposition onto nascent RNA following recruitment. At transcription termination regions, this nexus of actions promotes XRN2-mediated termination of RNAPII transcription. Disruption of any of these steps, including the loss of DDX21, METTL3, or their enzymatic activities, leads to defective termination that can induce DNA damage. Therefore, we propose that the R-loop-DDX21-METTL3 nexus forges the missing link for co-transcriptional modification of m6A, coordinating transcription termination and genome stability.

Structural basis of Integrator-dependent RNA polymerase II termination.

The Integrator complex can terminate RNA polymerase II (Pol II) in the promoter-proximal region of genes. Previous work has shed light on how Integrator binds to the paused elongation complex consisting of Pol II, the DRB sensitivity-inducing factor (DSIF) and the negative elongation factor (NELF) and how it cleaves the nascent RNA transcript1, but has not explained how Integrator removes Pol II from the DNA template. Here we present three cryo-electron microscopy structures of the complete Integrator-PP2A complex in different functional states. The structure of the pre-termination complex reveals a previously unresolved, scorpion-tail-shaped INTS10-INTS13-INTS14-INTS15 module that may use its 'sting' to open the DSIF DNA clamp and facilitate termination. The structure of the post-termination complex shows that the previously unresolved subunit INTS3 and associated sensor of single-stranded DNA complex (SOSS) factors prevent Pol II rebinding to Integrator after termination. The structure of the free Integrator-PP2A complex in an inactive closed conformation2 reveals that INTS6 blocks the PP2A phosphatase active site. These results lead to a model for how Integrator terminates Pol II transcription in three steps that involve major rearrangements.

Compatibility of termination mechanisms in bacterial transcription with inference on eukaryotic models.

Transcription termination has evolved to proceed through diverse mechanisms. For several classes of terminators, multiple models have been debatably proposed. Recent single-molecule studies on bacterial terminators have resolved several long-standing controversies. First, termination mode or outcome is twofold rather than single. RNA is released alone before DNA or together with DNA from RNA polymerase (RNAP), i.e. with RNA release for termination, RNAP retains on or dissociates off DNA, respectively. The concomitant release, described in textbooks, results in one-step decomposition of transcription complexes, and this 'decomposing termination' prevails at ρ factor-dependent terminators. Contrastingly, the sequential release was recently discovered abundantly from RNA hairpin-dependent intrinsic terminations. RNA-only release allows RNAP to diffuse on DNA in both directions and recycle for reinitiation. This 'recycling termination' enables one-dimensional reinitiation, which would be more expeditious than three-dimensional reinitiation by RNAP dissociated at decomposing termination. Second, while both recycling and decomposing terminations occur at a hairpin-dependent terminator, four termination mechanisms compatibly operate at a ρ-dependent terminator with ρ in alternative modes and even intrinsically without ρ. RNA-bound catch-up ρ mediates recycling termination first and decomposing termination later, while RNAP-prebound stand-by ρ invokes only decomposing termination slowly. Without ρ, decomposing termination occurs slightly and sluggishly. These four mechanisms operate on distinct timescales, providing orderly fail-safes. The stand-by mechanism is benefited by terminational pause prolongation and modulated by accompanying riboswitches more greatly than the catch-up mechanisms. Conclusively, any mechanism alone is insufficient to perfect termination, and multiple mechanisms operate compatibly to achieve maximum possible efficiency under separate controls.

Structural basis of exoribonuclease-mediated mRNA transcription termination.

Efficient termination is required for robust gene transcription. Eukaryotic organisms use a conserved exoribonuclease-mediated mechanism to terminate the mRNA transcription by RNA polymerase II (Pol II)1-5. Here we report two cryogenic electron microscopy structures of Saccharomyces cerevisiae Pol II pre-termination transcription complexes bound to the 5'-to-3' exoribonuclease Rat1 and its partner Rai1. Our structures show that Rat1 displaces the elongation factor Spt5 to dock at the Pol II stalk domain. Rat1 shields the RNA exit channel of Pol II, guides the nascent RNA towards its active centre and stacks three nucleotides at the 5' terminus of the nascent RNA. The structures further show that Rat1 rotates towards Pol II as it shortens RNA. Our results provide the structural mechanism for the Rat1-mediated termination of mRNA transcription by Pol II in yeast and the exoribonuclease-mediated termination of mRNA transcription in other eukaryotes.

Rat1 promotes premature transcription termination at R-loops.

Certain DNA sequences can adopt a non-B form in the genome that interfere with DNA-templated processes, including transcription. Among the sequences that are intrinsically difficult to transcribe are those that tend to form R-loops, three-stranded nucleic acid structures formed by a DNA-RNA hybrid and the displaced ssDNA. Here we compared the transcription of an endogenous gene with and without an R-loop-forming sequence inserted. We show that, in agreement with previous in vivo and in vitro analyses, transcription elongation is delayed by R-loops in yeast. Importantly, we demonstrate that the Rat1 transcription terminator factor facilitates transcription throughout such structures by inducing premature termination of arrested RNAPIIs. We propose that RNase H degrades the RNA moiety of the hybrid, providing an entry site for Rat1. Thus, we have uncovered an unanticipated function of Rat1 as a transcription restoring factor opening up the possibility that it may also promote transcription through other genomic DNA structures intrinsically difficult to transcribe. If R-loop-mediated transcriptional stress is not relieved by Rat1, it will cause genomic instability, probably through the increase of transcription-replication conflicts, a deleterious situation that could lead to cancer.

Single-molecule characterization of Sen1 translocation properties provides insights into eukaryotic factor-dependent transcription termination.

Sen1 is an essential helicase for factor-dependent transcription termination in Saccharomyces cerevisiae, whose molecular-motor mechanism has not been well addressed. Here, we use single-molecule experimentation to better understand the molecular-motor determinants of its action on RNA polymerase II (Pol II) complex. We quantify Sen1 translocation activity on single-stranded DNA (ssDNA), finding elevated translocation rates, high levels of processivity and ATP affinities. Upon deleting the N- and C-terminal domains, or further deleting different parts of the prong subdomain, which is an essential element for transcription termination, Sen1 displays changes in its translocation properties, such as slightly reduced translocation processivities, enhanced translocation rates and statistically identical ATP affinities. Although these parameters fulfil the requirements for Sen1 translocating along the RNA transcript to catch up with a stalled Pol II complex, we observe significant reductions in the termination efficiencies as well as the factions of the formation of the previously described topological intermediate prior to termination, suggesting that the prong may preserve an interaction with Pol II complex during factor-dependent termination. Our results underscore a more detailed rho-like mechanism of Sen1 and a critical interaction between Sen1 and Pol II complex for factor-dependent transcription termination in eukaryotes.

IntS6 and the Integrator phosphatase module tune the efficiency of select premature transcription termination events.

The metazoan-specific Integrator complex catalyzes 3' end processing of small nuclear RNAs (snRNAs) and premature termination that attenuates the transcription of many protein-coding genes. Integrator has RNA endonuclease and protein phosphatase activities, but it remains unclear if both are required for complex function. Here, we show IntS6 (Integrator subunit 6) over-expression blocks Integrator function at a subset of Drosophila protein-coding genes, although having no effect on snRNAs or attenuation of other loci. Over-expressed IntS6 titrates protein phosphatase 2A (PP2A) subunits, thereby only affecting gene loci where phosphatase activity is necessary for Integrator function. IntS6 functions analogous to a PP2A regulatory B subunit as over-expression of canonical B subunits, which do not bind Integrator, is also sufficient to inhibit Integrator activity. These results show that the phosphatase module is critical at only a subset of Integrator-regulated genes and point to PP2A recruitment as a tunable step that modulates transcription termination efficiency.

Herpes simplex virus 1 immediate early transcription initiation, pause-release, elongation, and termination in the presence and absence of ICP4.

IMPORTANCE: Infection with herpes simplex virus 1 (HSV-1) leads to lifelong infection due to the virus's remarkable ability to control transcription of its own genome, resulting in two transcriptional programs: lytic (highly active) and latent (restricted). The lytic program requires immediate early (IE) proteins to first repress transcription of late viral genes, which then undergo sequential de-repression, leading to a specific sequence of gene expression. Here, we show that the IE ICP4 functions to regulate the cascade by limiting RNA polymerase initiation at immediate early times. However, late viral genes that initiate too early in the absence of ICP4 do not yield mRNA as transcription stalls within gene bodies. It follows that other regulatory steps intercede to prevent elongation of genes at the incorrect time, demonstrating the precise control HSV-1 exerts over its own transcription.

R-loop-dependent promoter-proximal termination ensures genome stability.

The proper regulation of transcription is essential for maintaining genome integrity and executing other downstream cellular functions1,2. Here we identify a stable association between the genome-stability regulator sensor of single-stranded DNA (SOSS)3 and the transcription regulator Integrator-PP2A (INTAC)4-6. Through SSB1-mediated recognition of single-stranded DNA, SOSS-INTAC stimulates promoter-proximal termination of transcription and attenuates R-loops associated with paused RNA polymerase II to prevent R-loop-induced genome instability. SOSS-INTAC-dependent attenuation of R-loops is enhanced by the ability of SSB1 to form liquid-like condensates. Deletion of NABP2 (encoding SSB1) or introduction of cancer-associated mutations into its intrinsically disordered region leads to a pervasive accumulation of R-loops, highlighting a genome surveillance function of SOSS-INTAC that enables timely termination of transcription at promoters to constrain R-loop accumulation and ensure genome stability.

Internal transcription termination widely regulates differential expression of operon-organized genes including ribosomal protein and RNA polymerase genes in an archaeon.

Genes organized within operons in prokaryotes benefit from coordinated expression. However, within many operons, genes are expressed at different levels, and the mechanisms for this remain obscure. By integrating PacBio-seq, dRNA-seq, Term-seq and Illumina-seq data of a representative archaeon Methanococcus maripaludis, internal transcription termination sites (ioTTSs) were identified within 38% of operons. Higher transcript and protein abundances were found for genes upstream than downstream of ioTTSs. For representative operons, these differences were confirmed by northern blotting, qRT-PCR and western blotting, demonstrating that these ioTTS terminations were functional. Of special interest, mutation of ioTTSs in ribosomal protein (RP)-RNA polymerase (RNAP) operons not only elevated expression of the downstream RNAP genes but also decreased production of the assembled RNAP complex, slowed whole cell transcription and translation, and inhibited growth. Overexpression of the RNAP subunits with a shuttle vector generated the similar physiological effects. Therefore, ioTTS termination is a general and physiologically significant regulatory mechanism of the operon gene expression. Because the RP-RNAP operons are found to be widely distributed in archaeal species, this regulatory mechanism could be commonly employed in archaea.

Knockout of protein phosphatase 1 in Leishmania major reveals its role during RNA polymerase II transcription termination.

The genomes of kinetoplastids are organized into polycistronic transcription units that are flanked by a modified DNA base (base J, beta-D-glucosyl-hydroxymethyluracil). Previous work established a role of base J in promoting RNA polymerase II (Pol II) termination in Leishmania major and Trypanosoma brucei. We recently identified a PJW/PP1 complex in Leishmania containing a J-binding protein (JBP3), PP1 phosphatase 1, PP1 interactive-regulatory protein (PNUTS) and Wdr82. Analyses suggested the complex regulates transcription termination by recruitment to termination sites via JBP3-base J interactions and dephosphorylation of proteins, including Pol II, by PP1. However, we never addressed the role of PP1, the sole catalytic component, in Pol II transcription termination. We now demonstrate that deletion of the PP1 component of the PJW/PP1 complex in L. major, PP1-8e, leads to readthrough transcription at the 3'-end of polycistronic gene arrays. We show PP1-8e has in vitro phosphatase activity that is lost upon mutation of a key catalytic residue and associates with PNUTS via the conserved RVxF motif. Additionally, purified PJW complex with associated PP1-8e, but not complex lacking PP1-8e, led to dephosphorylation of Pol II, suggesting a direct role of PNUTS/PP1 holoenzymes in regulating transcription termination via dephosphorylating Pol II in the nucleus.

Duf89 abets lncRNA control of fission yeast phosphate homeostasis via its antagonism of precocious lncRNA transcription termination.

Fission yeast phosphate homeostasis gene pho1 is actively repressed during growth in phosphate-rich medium by transcription in cis of a long noncoding (lnc) RNA from the 5' flanking prt(nc-pho1) gene. Pho1 expression is: (i) derepressed by genetic maneuvers that favor precocious lncRNA 3'-processing and termination, in response to DSR and PAS signals in prt; and (ii) hyperrepressed in genetic backgrounds that dampen 3'-processing/termination efficiency. Governors of 3'-processing/termination include the RNA polymerase CTD code, the CPF (cleavage and polyadenylation factor) complex, termination factors Seb1 and Rhn1, and the inositol pyrophosphate signaling molecule 1,5-IP8 Here, we present genetic and biochemical evidence that fission yeast Duf89, a metal-dependent phosphatase/pyrophosphatase, is an antagonist of precocious 3'-processing/termination. We show that derepression of pho1 in duf89Δ cells correlates with squelching the production of full-length prt lncRNA and is erased or attenuated by: (i) DSR/PAS mutations in prt; (ii) loss-of-function mutations in components of the 3'-processing and termination machinery; (iii) elimination of the CTD Thr4-PO4 mark; (iv) interdicting CTD prolyl isomerization by Pin1; (v) inactivating the Asp1 kinase that synthesizes IP8; and (vi) loss of the putative IP8 sensor Spx1. The findings that duf89Δ is synthetically lethal with pho1-derepressive mutations CTD-S7A and aps1Δ-and that this lethality is rescued by CTD-T4A, CPF/Rhn1/Pin1 mutations, and spx1Δ-implicate Duf89 more broadly as a collaborator in cotranscriptional regulation of essential fission yeast genes. The duf89-D252A mutation, which abolishes Duf89 phosphohydrolase activity, phenocopied duf89 +, signifying that duf89Δ phenotypes are a consequence of Duf89 protein absence, not absence of Duf89 catalysis.

Human senataxin is a bona fide R-loop resolving enzyme and transcription termination factor.

Prolonged pausing of the transcription machinery may lead to the formation of three-stranded nucleic acid structures, called R-loops, typically resulting from the annealing of the nascent RNA with the template DNA. Unscheduled persistence of R-loops and RNA polymerases may interfere with transcription itself and other essential processes such as DNA replication and repair. Senataxin (SETX) is a putative helicase, mutated in two neurodegenerative disorders, which has been implicated in the control of R-loop accumulation and in transcription termination. However, understanding the precise role of SETX in these processes has been precluded by the absence of a direct characterisation of SETX biochemical activities. Here, we purify and characterise the helicase domain of SETX in parallel with its yeast orthologue, Sen1. Importantly, we show that SETX is a bona fide helicase with the ability to resolve R-loops. Furthermore, SETX has retained the transcription termination activity of Sen1 but functions in a species-specific manner. Finally, subsequent characterisation of two SETX variants harbouring disease-associated mutations shed light into the effect of such mutations on SETX folding and biochemical properties. Altogether, these results broaden our understanding of SETX function in gene expression and the maintenance of genome integrity and provide clues to elucidate the molecular basis of SETX-associated neurodegenerative diseases.

H3K4me3 regulates RNA polymerase II promoter-proximal pause-release.

Trimethylation of histone H3 lysine 4 (H3K4me3) is associated with transcriptional start sites and has been proposed to regulate transcription initiation1,2. However, redundant functions of the H3K4 SET1/COMPASS methyltransferase complexes complicate the elucidation of the specific role of H3K4me3 in transcriptional regulation3,4. Here, using mouse embryonic stem cells as a model system, we show that acute ablation of shared subunits of the SET1/COMPASS complexes leads to a complete loss of all H3K4 methylation. Turnover of H3K4me3 occurs more rapidly than that of H3K4me1 and H3K4me2 and is dependent on KDM5 demethylases. Notably, acute loss of H3K4me3 does not have detectable effects on transcriptional initiation but leads to a widespread decrease in transcriptional output, an increase in RNA polymerase II (RNAPII) pausing and slower elongation. We show that H3K4me3 is required for the recruitment of the integrator complex subunit 11 (INTS11), which is essential for the eviction of paused RNAPII and transcriptional elongation. Thus, our study demonstrates a distinct role for H3K4me3 in transcriptional pause-release and elongation rather than transcriptional initiation.

Bacteria require phase separation for fitness in the mammalian gut.

Therapeutic manipulation of the gut microbiota holds great potential for human health. The mechanisms bacteria use to colonize the gut therefore present valuable targets for clinical intervention. We now report that bacteria use phase separation to enhance fitness in the mammalian gut. We establish that the intrinsically disordered region (IDR) of the broadly and highly conserved transcription termination factor Rho is necessary and sufficient for phase separation in vivo and in vitro in the human commensal Bacteroides thetaiotaomicron. Phase separation increases transcription termination by Rho in an IDR-dependent manner. Moreover, the IDR is critical for gene regulation in the gut. Our findings expose phase separation as vital for host-commensal bacteria interactions and relevant for novel clinical applications.

Transcriptional pause extension benefits the stand-by rather than catch-up Rho-dependent termination.

Transcriptional pause is essential for all types of termination. In this single-molecule study on bacterial Rho factor-dependent terminators, we confirm that the three Rho-dependent termination routes operate compatibly together in a single terminator, and discover that their termination efficiencies depend on the terminational pauses in unexpected ways. Evidently, the most abundant route is that Rho binds nascent RNA first and catches up with paused RNA polymerase (RNAP) and this catch-up Rho mediates simultaneous releases of transcript RNA and template DNA from RNAP. The fastest route is that the catch-up Rho effects RNA-only release and leads to 1D recycling of RNAP on DNA. The slowest route is that the RNAP-prebound stand-by Rho facilitates only the simultaneous rather than sequential releases. Among the three routes, only the stand-by Rho's termination efficiency positively correlates with pause duration, contrary to a long-standing speculation, invariably in the absence or presence of NusA/NusG factors, competitor RNAs or a crowding agent. Accordingly, the essential terminational pause does not need to be long for the catch-up Rho's terminations, and long pauses benefit only the stand-by Rho's terminations. Furthermore, the Rho-dependent termination of mgtA and ribB riboswitches is controlled mainly by modulation of the stand-by rather than catch-up termination.

Knowing when to stop: Transcription termination on protein-coding genes by eukaryotic RNAPII.

Gene expression is controlled in a dynamic and regulated manner to allow for the consistent and steady expression of some proteins as well as the rapidly changing production of other proteins. Transcription initiation has been a major focus of study because it is highly regulated. However, termination of transcription also plays an important role in controlling gene expression. Transcription termination on protein-coding genes is intimately linked with 3' end cleavage and polyadenylation of transcripts, and it generally results in the production of a mature mRNA that is exported from the nucleus. Termination on many non-coding genes can also result in the production of a mature transcript. Termination is dynamically regulated-premature termination and transcription readthrough occur in response to a number of cellular signals, and these can have varied consequences on gene expression. Here, we review eukaryotic transcription termination by RNA polymerase II (RNAPII), focusing on protein-coding genes.

Structural basis for intrinsic transcription termination.

Efficient and accurate termination is required for gene transcription in all living organisms1,2. Cellular RNA polymerases in both bacteria and eukaryotes can terminate their transcription through a factor-independent termination pathway3,4-called intrinsic termination transcription in bacteria-in which RNA polymerase recognizes terminator sequences, stops nucleotide addition and releases nascent RNA spontaneously. Here we report a set of single-particle cryo-electron microscopy structures of Escherichia coli transcription intrinsic termination complexes representing key intermediate states of the event. The structures show how RNA polymerase pauses at terminator sequences, how the terminator RNA hairpin folds inside RNA polymerase, and how RNA polymerase rewinds the transcription bubble to release RNA and then DNA. These macromolecular snapshots define a structural mechanism for bacterial intrinsic termination and a pathway for RNA release and DNA collapse that is relevant for factor-independent termination by all RNA polymerases.

Structural basis of Rho-dependent transcription termination.

Rho is a ring-shaped hexameric ATP-dependent molecular motor. Together with the transcription elongation factor NusG, Rho mediates factor-dependent transcription termination and transcription-translation-coupling quality control in Escherichia coli1-4. Here we report the preparation of complexes that are functional in factor-dependent transcription termination from Rho, NusG, RNA polymerase (RNAP), and synthetic nucleic acid scaffolds, and we report cryogenic electron microscopy structures of the complexes. The structures show that functional factor-dependent pre-termination complexes contain a closed-ring Rho hexamer; have RNA threaded through the central channel of Rho; have 60 nucleotides of RNA interacting sequence-specifically with the exterior of Rho and 6 nucleotides of RNA interacting sequence-specifically with the central channel of Rho; have Rho oriented relative to RNAP such that ATP-dependent translocation by Rho exerts mechanical force on RNAP; and have NusG bridging Rho and RNAP. The results explain five decades of research on Rho and provide a foundation for understanding Rho's function.