Eukaryotic genomes are widely transcribed by RNA polymerase II (pol II) both within genes and in intergenic regions. POL II elongation complexes comprising the polymerase, the DNA template and nascent RNA transcript must be extremely processive in order to transcribe the longest genes which are over 1 megabase long and take many hours to traverse. Dedicated termination mechanisms are required to disrupt these highly stable complexes. Transcription termination occurs not only at the 3\' ends of genes once a full length transcript has been made, but also within genes and in promiscuously transcribed intergenic regions. Termination at these latter positions is termed \"premature\" because it is not triggered in response to a specific signal that marks the 3\' end of a gene, like a polyA site. One purpose of premature termination is to remove polymerases from intergenic regions where they are \"not wanted\" because they may interfere with transcription of overlapping genes or the progress of replication forks. Premature termination has recently been appreciated to occur at surprisingly high rates within genes where it is speculated to serve regulatory or quality control functions. In this review I summarize current understanding of the different mechanisms of premature termination and its potential functions.

The transcription termination process is an important part of the gene expression process in the cell. It has been studied extensively, but many aspects of the mechanism are not well understood. The widespread availability of experimental RNA-seq data from high-throughput experiments provides a unique opportunity to infer the end of the transcription units genome wide. This data is available for both Rho-dependent and Rho-independent termination pathways that drive transcription termination in bacteria. Our book chapter gives an overview of the current knowledge of Rho-independent transcription termination mechanisms and the prediction approaches currently deployed to infer the termination sites. Thereafter, we describe our method that uses cluster hairpins to detect Rho-independent transcription termination sites. These clusters are a group of hairpins that lies at <15 bp from each other and are together capable of enforcing the termination process. The idea of a group of hairpins being extensively used for transcription termination is new, and results show that at least 52% of the total cases are of this type, while in the remaining cases, a single strong hairpin is capable of driving transcription termination. The reads derived from the RNA-seq data for corresponding bacteria have been used to validate the predicted sites. The predictions that match these RNA-seq derived sites have higher confidence, and we find almost 98% of the predicted sites, including alternate termination sites, to match the RNA-seq data. We discuss the features of predicted hairpins in detail for a better understanding of the Rho-independent transcription termination mechanism in bacteria. We also explain how users can use the tools developed by us to do transcription terminator predictions and design their experiments through genome-level visualization of the transcription termination sites from the precomputed INTERPIN database.

INTS11 and CPSF73 are metal-dependent endonucleases for Integrator and pre-mRNA 3\'-end processing, respectively. Here, we show that the INTS11 binding partner BRAT1/CG7044, a factor important for neuronal fitness, stabilizes INTS11 in the cytoplasm and is required for Integrator function in the nucleus. Loss of BRAT1 in neural organoids leads to transcriptomic disruption and precocious expression of neurogenesis-driving transcription factors. The structures of the human INTS9-INTS11-BRAT1 and Drosophila dIntS11-CG7044 complexes reveal that the conserved C terminus of BRAT1/CG7044 is captured in the active site of INTS11, with a cysteine residue directly coordinating the metal ions. Inspired by these observations, we find that UBE3D is a binding partner for CPSF73, and UBE3D likely also uses a conserved cysteine residue to directly coordinate the active site metal ions. Our studies have revealed binding partners for INTS11 and CPSF73 that behave like cytoplasmic chaperones with a conserved impact on the nuclear functions of these enzymes.

The regulation of transcription by RNA polymerase II (RNAPII) underpins all cellular processes and is perturbed in thousands of diseases. In humans, RNAPII transcribes ∼20000 protein-coding genes and engages in apparently futile non-coding transcription at thousands of other sites. Despite being so ubiquitous, this transcription is usually attenuated soon after initiation and the resulting products are immediately degraded by the nuclear exosome. We and others have recently described a new complex, \"Restrictor\", which appears to control such unproductive transcription. Underpinned by the RNA binding protein, ZC3H4, Restrictor curtails unproductive/pervasive transcription genome-wide. Here, we discuss these recent discoveries and speculate on some of the many unknowns regarding Restrictor function and mechanism.

RNA polymerase II (RNAPII) transcription initiates bidirectionally at many human protein-coding genes. Sense transcription usually dominates and leads to messenger RNA production, whereas antisense transcription rapidly terminates. The basis for this directionality is not fully understood. Here, we show that sense transcriptional initiation is more efficient than in the antisense direction, which establishes initial promoter directionality. After transcription begins, the opposing functions of the endonucleolytic subunit of Integrator, INTS11, and cyclin-dependent kinase 9 (CDK9) maintain directionality. Specifically, INTS11 terminates antisense transcription, whereas sense transcription is protected from INTS11-dependent attenuation by CDK9 activity. Strikingly, INTS11 attenuates transcription in both directions upon CDK9 inhibition, and the engineered recruitment of CDK9 desensitises transcription to INTS11. Therefore, the preferential initiation of sense transcription and the opposing activities of CDK9 and INTS11 explain mammalian promoter directionality.

The interconnections between co-transcriptional regulation, chromatin environment, and transcriptional output remain poorly understood. Here, we investigate the mechanism underlying RNA 3\' processing-mediated Polycomb silencing of Arabidopsis FLOWERING LOCUS C (FLC). We show a requirement for ANTHESIS PROMOTING FACTOR 1 (APRF1), a homolog of yeast Swd2 and human WDR82, known to regulate RNA polymerase II (RNA Pol II) during transcription termination. APRF1 interacts with TYPE ONE SERINE/THREONINE PROTEIN PHOSPHATASE 4 (TOPP4) (yeast Glc7/human PP1) and LUMINIDEPENDENS (LD), the latter showing structural features found in Ref2/PNUTS, all components of the yeast and human phosphatase module of the CPF 3\' end-processing machinery. LD has been shown to co-associate in vivo with the histone H3 K4 demethylase FLOWERING LOCUS D (FLD). This work shows how the APRF1/LD-mediated polyadenylation/termination process influences subsequent rounds of transcription by changing the local chromatin environment at FLC.

OBJECTIVE: Transcription termination fine-tunes gene expression and contributes to the specification of RNA function in eukaryotic cells. Transcription termination of HBV is subject to the recognition of the canonical polyadenylation signal (cPAS) common to all viral transcripts. However, the regulation of this cPAS and its impact on viral gene expression and replication is currently unknown.
METHODS: To unravel the regulation of HBV transcript termination, we implemented a 3\' RACE (rapid amplification of cDNA ends)-PCR assay coupled to single molecule sequencing both in in vitro-infected hepatocytes and in chronically infected patients.
RESULTS: The detection of a previously unidentified transcriptional readthrough indicated that the cPAS was not systematically recognized during HBV replication in vitro and in vivo. Gene expression downregulation experiments demonstrated a role for the RNA helicases DDX5 and DDX17 in promoting viral transcriptional readthrough, which was, in turn, associated with HBV RNA destabilization and decreased HBx protein expression. RNA and chromatin immunoprecipitation, together with mutation of the cPAS sequence, suggested a direct role of DDX5 and DDX17 in functionally linking cPAS recognition to transcriptional readthrough, HBV RNA stability and replication.
CONCLUSIONS: Our findings identify DDX5 and DDX17 as crucial determinants of HBV transcriptional fidelity and as host restriction factors for HBV replication.
UNASSIGNED: HBV covalently closed circular (ccc)DNA degradation or functional inactivation remains the holy grail for the achievement of HBV cure. Transcriptional fidelity is a cornerstone in the regulation of gene expression. Here, we demonstrate that two helicases, DDX5 and DDX17, inhibit recognition of the HBV polyadenylation signal and thereby transcriptional termination, thus decreasing HBV RNA stability and acting as restriction factors for efficient cccDNA transcription and viral replication. The observation that DDX5 and DDX17 are downregulated in patients chronically infected with HBV suggests a role for these helicases in HBV persistence in vivo. These results open new perspectives for researchers aiming at identifying new targets to neutralise cccDNA transcription.

Splicing factors are vital for the regulation of RNA splicing, but some have also been implicated in regulating transcription. The underlying molecular mechanisms of their involvement in transcriptional processes remain poorly understood.
Here, we describe a direct role of splicing factor RBM22 in coordinating multiple steps of RNA Polymerase II (RNAPII) transcription in human cells. The RBM22 protein widely occupies the RNAPII-transcribed gene locus in the nucleus. Loss of RBM22 promotes RNAPII pause release, reduces elongation velocity, and provokes transcriptional readthrough genome-wide, coupled with production of transcripts containing sequences from downstream of the gene. RBM22 preferentially binds to the hyperphosphorylated, transcriptionally engaged RNAPII and coordinates its dynamics by regulating the homeostasis of the 7SK-P-TEFb complex and the association between RNAPII and SPT5 at the chromatin level.
Our results uncover the multifaceted role of RBM22 in orchestrating the transcriptional program of RNAPII and provide evidence implicating a splicing factor in both RNAPII elongation kinetics and termination control.

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.

African swine fever virus (ASFV) is a nucleocytoplasmic large DNA virus (NCLDV) that encodes its own host-like RNA polymerase (RNAP) and factors required to produce mature mRNA. The formation of accurate mRNA 3\' ends by ASFV RNAP depends on transcription termination, likely enabled by a combination of sequence motifs and transcription factors, although these are poorly understood. The termination of any RNAP is rarely 100% efficient, and the transcriptional \"readthrough\" at terminators can generate long mRNAs which may interfere with the expression of downstream genes. ASFV transcriptome analyses reveal a landscape of heterogeneous mRNA 3\' termini, likely a combination of bona fide termination sites and the result of mRNA degradation and processing. While short-read sequencing (SRS) like 3\' RNA-seq indicates an accumulation of mRNA 3\' ends at specific sites, it cannot inform about which promoters and transcription start sites (TSSs) directed their synthesis, i.e., information about the complete and unprocessed mRNAs at nucleotide resolution.
Here, we report a rigorous analysis of full-length ASFV transcripts using long-read sequencing (LRS). We systematically compared transcription termination sites predicted from SRS 3\' RNA-seq with 3\' ends mapped by LRS during early and late infection.
Using in-vitro transcription assays, we show that recombinant ASFV RNAP terminates transcription at polyT stretches in the non-template strand, similar to the archaeal RNAP or eukaryotic RNAPIII, unaided by secondary RNA structures or predicted viral termination factors. Our results cement this T-rich motif (U-rich in the RNA) as a universal transcription termination signal in ASFV. Many genes share the usage of the same terminators, while genes can also use a range of terminators to generate transcript isoforms varying enormously in length. A key factor in the latter phenomenon is the highly abundant terminator readthrough we observed, which is more prevalent during late compared with early infection.
This indicates that ASFV mRNAs under the control of late gene promoters utilize different termination mechanisms and factors to early promoters and/or that cellular factors influence the viral transcriptome landscape differently during the late stages of infection.