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In bacteria, small non-coding RNAs (sRNAs) bind to target mRNAs and regulate their translation and/or stability. In the polycistronic galETKM operon of Escherichia coli, binding of the Spot 42 sRNA to the operon transcript leads to the generation of galET mRNA. The mechanism of this regulation has remained unclear. We show that sRNA-mRNA base pairing at the beginning of the galK gene leads to both transcription termination and transcript cleavage within galK, and generates galET mRNAs with two different 3’-OH ends. Transcription termination requires Rho, and transcript cleavage requires the endonuclease RNase E. The sRNA-mRNA base-paired segments required for generating the two galET species are different, indicating different sequence requirements for the two events. The use of two targets in an mRNA, each of which causes a different outcome, appears to be a novel mode of action for a sRNA. Considering the prevalence of potential sRNA targets at cistron junctions, the generation of new mRNA species by the mechanisms reported here might be a widespread mode of bacterial gene regulation., Author summary sRNAs are regulators of gene expression in all forms of life. In bacteria such as E. coli, sRNAs base-pair with mRNA, which can have many consequences such as premature transcription termination by Rho and degradation of mRNA by the endoribonuclease RNase E. Here we show that the two processes can occur on the same sRNA-mRNA pair and produce shorter but stable mRNA that can be of functional significance. Thus, in sRNA regulation of mRNA, transcript cleavage can be an additional mechanism to well-known transcription termination to generate new mRNA species. The choice between the mechanisms is dictated by where on the mRNA the base pairing with sRNA takes place.

Transcriptional terminators signal where transcribing RNA polymerases (RNAPs) should halt and disassociate from DNA. However, because termination is stochastic, two different forms of transcript could be produced: one ending at the terminator and the other reading through. An ability to control the abundance of these transcript isoforms would offer bioengineers a mechanism to regulate multi-gene constructs at the level of transcription. Here, we explore this possibility by repurposing terminators as ‘transcriptional valves’ that can tune the proportion of RNAP read-through. Using one-pot combinatorial DNA assembly, we iteratively construct 1780 transcriptional valves for T7 RNAP and show how nanopore-based direct RNA sequencing (dRNA-seq) can be used to characterize entire libraries of valves simultaneously at a nucleotide resolution in vitro and unravel genetic design principles to tune and insulate termination. Finally, we engineer valves for multiplexed regulation of CRISPR guide RNAs. This work provides new avenues for controlling transcription and demonstrates the benefits of long-read sequencing for exploring complex sequence-function landscapes., Transcriptional terminators are generally viewed as hard endpoints for transcribing RNA polymerases. Here, the authors reimagine terminators as transcriptional valves with predictable read through. They engineer and characterize 1780 valves and use them for multiplexed gene regulation.

Pseudomonas aeruginosa produces serious chronic infections in hospitalized patients and immunocompromised individuals, including patients with cystic fibrosis. The molecular mechanisms by which P. aeruginosa responds to antibiotics and other stresses to promote persistent infections may provide new avenues for therapeutic intervention. Azithromycin (AZM), an antibiotic frequently used in cystic fibrosis treatment, is thought to improve clinical outcomes through a number of mechanisms including impaired biofilm growth and quorum sensing (QS). The mechanisms underlying the transcriptional response to AZM remain unclear. Here, we interrogated the P. aeruginosa transcriptional response to AZM using a fast, cost-effective genome-wide approach to quantitate RNA 3’ ends (3pMap). We also identified hundreds of P. aeruginosa genes with high incidence of premature 3’ end formation indicative of riboregulation in their transcript leaders using 3pMap. AZM treatment of planktonic and biofilm cultures alters the expression of hundreds of genes, including those involved in QS, biofilm formation, and virulence. Strikingly, most genes downregulated by AZM in biofilms had increased levels of intragenic 3’ ends indicating premature transcription termination, transcriptional pausing, or accumulation of stable intermediates resulting from the action of nucleases. Reciprocally, AZM reduced premature intragenic 3’ end termini in many upregulated genes. Most notably, reduced termination accompanied robust induction of obgE, a GTPase involved in persister formation in P. aeruginosa. Our results support a model in which AZM-induced changes in 3’ end formation alter the expression of central regulators which in turn impairs the expression of QS, biofilm formation and stress response genes, while upregulating genes associated with persistence., Author summary Pseudomonas aeruginosa is a common source of hospital-acquired infections and causes prolonged illness in patients with cystic fibrosis. P. aeruginosa infections are often treated with the macrolide antibiotic azithromycin, which changes the expression of many genes involved in infection. By examining such expression changes at nucleotide resolution, we found azithromycin treatment alters the locations of mRNA 3’ ends suggesting most downregulated genes are subject to premature 3’ end formation. We further identified candidate RNA regulatory elements that P. aeruginosa may use to control gene expression. Our work provides new insights in P. aeruginosa gene regulation and its response to antibiotics.

Transcription termination has important regulatory functions, impacting mRNA stability, localization and translation potential. Failure to appropriately terminate transcription can also lead to read-through transcription and the synthesis of antisense RNAs which can have profound impact on gene expression. The Transcription-Export (THO/TREX) protein complex plays an important role in coupling transcription with splicing and export of mRNA. However, little is known about the role of the THO/TREX complex in the control of transcription termination. In this work, we show that two proteins of the THO/TREX complex, namely TREX COMPONENT 1 (TEX1 or THO3) and HYPER RECOMBINATION1 (HPR1 or THO1) contribute to the correct transcription termination at several loci in Arabidopsis thaliana. We first demonstrate this by showing defective termination in tex1 and hpr1 mutants at the nopaline synthase (NOS) terminator present in a T-DNA inserted between exon 1 and 3 of the PHO1 locus in the pho1-7 mutant. Read-through transcription beyond the NOS terminator and splicing-out of the T-DNA resulted in the generation of a near full-length PHO1 mRNA (minus exon 2) in the tex1 pho1-7 and hpr1 pho1-7 double mutants, with enhanced production of a truncated PHO1 protein that retained phosphate export activity. Consequently, the strong reduction of shoot growth associated with the severe phosphate deficiency of the pho1-7 mutant was alleviated in the tex1 pho1-7 and hpr1 pho1-7 double mutants. Additionally, we show that RNA termination defects in tex1 and hpr1 mutants leads to 3’UTR extensions in several endogenous genes. These results demonstrate that THO/TREX complex contributes to the regulation of transcription termination., Author summary Production of messenger RNAs (mRNAs) involves numerous steps including initiation of transcription, elongation, splicing, termination, as well as export out of the nucleus. All these steps are highly coordinated and failure in any steps has a profound impact on the level and identity of mRNAs produced. The THO/TREX protein complex is associated with nascent RNAs and contributes to several mRNA biogenesis steps, including splicing and export. However, the contribution of the THO/TREX complex to mRNA termination was poorly defined. We have identified a role for two components of the THO/TREX complex, namely the proteins TEX1 and HPR1, in the control of transcription termination in the plant Arabidopsis thaliana. We show that the tex1 and hpr1 mutants have defects in terminating mRNA at the nopaline synthase (NOS) terminator found in a T-DNA insertion mutant leading to the transcriptional read-through pass the NOS terminator. We also show that tex1 and hpr1 mutants have defects in mRNA termination at several endogenous genes, leading to the production of 3’UTR extensions. Together, these results highlight a role for the THO/TREX complex in mRNA termination.

The intestinal pathogen Clostridioides difficile exhibits heterogeneity in motility and toxin production. This phenotypic heterogeneity is achieved through phase variation by site-specific recombination via the DNA recombinase RecV, which reversibly inverts the “flagellar switch” upstream of the flgB operon. A recV mutation prevents flagellar switch inversion and results in phenotypically locked strains. The orientation of the flagellar switch influences expression of the flgB operon post-transcription initiation, but the specific molecular mechanism is unknown. Here, we report the isolation and characterization of spontaneous suppressor mutants in the non-motile, non-toxigenic recV flg OFF background that regained motility and toxin production. The restored phenotypes corresponded with increased expression of flagellum and toxin genes. The motile suppressor mutants contained single-nucleotide polymorphisms (SNPs) in rho, which encodes the bacterial transcription terminator Rho factor. Analyses using transcriptional reporters indicate that Rho contributes to heterogeneity in flagellar gene expression by preferentially terminating transcription of flg OFF mRNA within the 5’ leader sequence. Additionally, Rho is important for initial colonization of the intestine in a mouse model of infection, which may in part be due to the sporulation and growth defects observed in the rho mutants. Together these data implicate Rho factor as a regulator of gene expression affecting phase variation of important virulence factors of C. difficile., Author summary Phenotypic heterogeneity maintained by phase variation allows bacterial subpopulations to overcome potentially detrimental stresses in the environment, contributing to bacterial survival. Phase variation of flagella and toxins in C. difficile suggests that maintaining heterogeneity of their production may be important for survival and virulence. In this study, we identified Rho as a trans-acting factor that mediates the differential gene expression that imparts heterogeneity in flagellum and toxin production. These results reveal a new role for Rho-mediated transcription termination in regulation of gene expression.

Modifications to transcriptional regulators play a major role in adaptation. Here, we compared the effects of multiple beneficial mutations within and between Escherichia coli rpoB, the gene encoding the RNA polymerase β subunit, and rho, which encodes a transcriptional terminator. These two genes have harbored adaptive mutations in numerous E. coli evolution experiments but particularly in our previous large-scale thermal stress experiment, where the two genes characterized alternative adaptive pathways. To compare the effects of beneficial mutations, we engineered four advantageous mutations into each of the two genes and measured their effects on fitness, growth, gene expression and transcriptional termination at 42.2 °C. Among the eight mutations, two rho mutations had no detectable effect on relative fitness, suggesting they were beneficial only in the context of epistatic interactions. The remaining six mutations had an average relative fitness benefit of ∼20%. The rpoB mutations affected the expression of ∼1,700 genes; rho mutations affected the expression of fewer genes but most (83%) were a subset of those altered by rpoB mutants. Across the eight mutants, relative fitness correlated with the degree to which a mutation restored gene expression back to the unstressed, 37.0 °C state. The beneficial mutations in the two genes did not have identical effects on fitness, growth or gene expression, but they caused parallel phenotypic effects on gene expression and genome-wide transcriptional termination., Molecular Biology and Evolution, 34 (11), ISSN:0737-4038, ISSN:1537-1719

Base J, β-D-glucosyl-hydroxymethyluracil, is a modification of thymine DNA base involved in RNA Polymerase (Pol) II transcription termination in kinetoplastid protozoa. Little is understood regarding how specific thymine residues are targeted for J-modification or the mechanism of J regulated transcription termination. To identify proteins involved in J-synthesis, we expressed a tagged version of the J-glucosyltransferase (JGT) in Leishmania tarentolae, and identified four co-purified proteins by mass spectrometry: protein phosphatase (PP1), a homolog of Wdr82, a potential PP1 regulatory protein (PNUTS) and a protein containing a J-DNA binding domain (named JBP3). Gel shift studies indicate JBP3 is a J-DNA binding protein. Reciprocal tagging, co-IP and sucrose gradient analyses indicate PP1, JGT, JBP3, Wdr82 and PNUTS form a multimeric complex in kinetoplastids, similar to the mammalian PTW/PP1 complex involved in transcription termination via PP1 mediated dephosphorylation of Pol II. Using RNAi and analysis of Pol II termination by RNA-seq and RT-PCR, we demonstrate that ablation of PNUTS, JBP3 and Wdr82 lead to defects in Pol II termination at the 3’-end of polycistronic gene arrays in Trypanosoma brucei. Mutants also contain increased antisense RNA levels upstream of transcription start sites, suggesting an additional role of the complex in regulating termination of bi-directional transcription. In addition, PNUTS loss causes derepression of silent Variant Surface Glycoprotein genes involved in host immune evasion. Our results suggest a novel mechanistic link between base J and Pol II polycistronic transcription termination in kinetoplastids., Author summary Trypanosoma brucei is a parasitic protozoan that causes African sleeping sickness in humans. The genome of T. brucei is organized into polycistronic gene clusters that contain multiple genes that are co-transcribed from a single promoter. We have recently described the presence of a modified DNA base J and variant of histone H3 (H3.V) at transcription termination sites within gene clusters where the loss of base J and H3.V leads to read-through transcription and the expression of downstream genes. We now identify a novel stable multimeric complex containing a J binding protein (JBP3), base J glucosyltransferase (JGT), PP1 phosphatase, PP1 interactive-regulatory protein (PNUTS) and Wdr82, which we refer to as PJW/PP1. A similar complex (PTW/PP1) has been shown to be involved in Pol II termination in humans and yeast. We demonstrate that PNUTS, JBP3 and Wdr82 mutants lead to read-through transcription in T. brucei. Our data suggest the PJW/PP1 complex regulates termination by recruitment to termination sites via JBP3-base J interactions and dephosphorylation of specific proteins (including Pol II and termination factors) by PP1. These findings significantly expand our understanding of mechanisms underlying transcription termination in eukaryotes, including organisms that utilize polycistronic transcription and novel epigenetic marks such as base J and H3.V. The studies also provide the first direct mechanistic link between J modification of DNA at termination sites and regulated Pol II termination and gene expression in kinetoplastids.

RNA Polymerase II (RNAPII) transcription termination is regulated by the phosphorylation status of the C-terminal domain (CTD). The phosphatase Rtr1 has been shown to regulate serine 5 phosphorylation on the CTD; however, its role in the regulation of RNAPII termination has not been explored. As a consequence of RTR1 deletion, interactions within the termination machinery and between the termination machinery and RNAPII were altered as quantified by Disruption-Compensation (DisCo) network analysis. Of note, interactions between RNAPII and the cleavage factor IA (CF1A) subunit Pcf11 were reduced in rtr1Δ, whereas interactions with the CTD and RNA-binding termination factor Nrd1 were increased. Globally, rtr1Δ leads to decreases in numerous noncoding RNAs that are linked to the Nrd1, Nab3 and Sen1 (NNS) -dependent RNAPII termination pathway. Genome-wide analysis of RNAPII and Nrd1 occupancy suggests that loss of RTR1 leads to increased termination at noncoding genes. Additionally, premature RNAPII termination increases globally at protein-coding genes with a decrease in RNAPII occupancy occurring just after the peak of Nrd1 recruitment during early elongation. The effects of rtr1Δ on RNA expression levels were lost following deletion of the exosome subunit Rrp6, which works with the NNS complex to rapidly degrade a number of noncoding RNAs following termination. Overall, these data suggest that Rtr1 restricts the NNS-dependent termination pathway in WT cells to prevent premature termination of mRNAs and ncRNAs. Rtr1 facilitates low-level elongation of noncoding transcripts that impact RNAPII interference thereby shaping the transcriptome., Author summary Many cellular RNAs including those that encode for proteins are produced by the enzyme RNA Polymerase II. In this work, we have defined a new role for the phosphatase Rtr1 in the regulation of RNA Polymerase II progression from the start of transcription to the 3’ end of the gene where the nascent RNA from protein-coding genes is typically cleaved and polyadenylated. Deletion of the gene that encodes RTR1 leads to changes in the interactions between RNA polymerase II and the termination machinery. Rtr1 loss also causes early termination of RNA Polymerase II at many of its target gene types, including protein coding genes and noncoding RNAs. Evidence suggests that the premature termination observed in RTR1 knockout cells occurs through the termination factor and RNA binding protein Nrd1 and its binding partner Nab3. Deletion of RRP6, a known component of the Nrd1-Nab3 termination coupled RNA degradation pathway, is epistatic to RTR1 suggesting that Rrp6 is required to terminate and/or degrade many of the noncoding RNAs that have increased turnover in RTR1 deletion cells. These findings suggest that Rtr1 normally promotes elongation of RNA Polymerase II transcripts through prevention of Nrd1-directed termination.

Evolutionarily conserved NusG protein enhances bacterial RNA polymerase processivity but can also promote transcription termination by binding to, and stimulating the activity of, Rho factor. Rho terminates transcription upon anchoring to cytidine-rich motifs, the so-called Rho utilization sites (Rut) in nascent RNA. Both NusG and Rho have been implicated in the silencing of horizontally-acquired A/T-rich DNA by nucleoid structuring protein H-NS. However, the relative roles of the two proteins in H-NS-mediated gene silencing remain incompletely defined. In the present study, a Salmonella strain carrying the nusG gene under the control of an arabinose-inducible repressor was used to assess the genome-wide response to NusG depletion. Results from two complementary approaches, i) screening lacZ protein fusions generated by random transposition and ii) transcriptomic analysis, converged to show that loss of NusG causes massive upregulation of Salmonella pathogenicity islands (SPIs) and other H-NS-silenced loci. A similar, although not identical, SPI-upregulated profile was observed in a strain with a mutation in the rho gene, Rho K130Q. Surprisingly, Rho mutation Y80C, which affects Rho’s primary RNA binding domain, had either no effect or made H-NS-mediated silencing of SPIs even tighter. Thus, while corroborating the notion that bound H-NS can trigger Rho-dependent transcription termination in vivo, these data suggest that H-NS-elicited termination occurs entirely through a NusG-dependent pathway and is less dependent on Rut site binding by Rho. We provide evidence that through Rho recruitment, and possibly through other still unidentified mechanisms, NusG prevents pervasive transcripts from elongating into H-NS-silenced regions. Failure to perform this function causes the feedforward activation of the entire Salmonella virulence program. These findings provide further insight into NusG/Rho contribution in H-NS-mediated gene silencing and underscore the importance of this contribution for the proper functioning of a global regulatory response in growing bacteria. The complete set of transcriptomic data is freely available for viewing through a user-friendly genome browser interface., Author summary Most virulence genes of pathogenic Enterobacteria lie within genomic islands acquired through horizontal transfer in the course of evolution. While essential during infection, expression of many of these genes is detrimental for growth outside the host. Hence, mechanisms have evolved that prevent transcription of this foreign DNA, except when needed. A major mechanism uses Histone-like protein H-NS which binds to AT-rich nucleation sites, a hallmark of horizontally acquired DNA, forming oligomeric structures that silence transcription over extended regions. This silencing activity is all the more important as A/T-rich sequences are intrinsically prone to spurious transcription initiation events. Nonetheless, H-NS-silenced regions remain exposed to invading transcripts that either escape repression or originate from neighboring DNA. Results presented here, support the proposal that NusG, a conserved transcription elongation factor, plays a key role in ensuring that elongation complexes come to a stop when running into a patch of oligomerized H-NS. NusG performs this function by stimulating the activity of transcription termination factor Rho. We show that alteration of this mechanism in Salmonella has profound physiological consequences, most likely because unstopped transcripts invade H-NS-silenced pathogenicity islands and trigger a feed-forward regulatory cascade leading to the gratuitous activation of these islands and other co-regulated loci.

Many bacterial species use cyclic diguanylate (c-di-GMP) to control their physiology and behaviors in response to extracellular stimuli. C-di-GMP primarily mediates the switch between a free-living, motile state and a nonmotile lifestyle, with the latter often entailing surface adherence and biofilm development. This paradigm is conserved across diverse gram-positive and gram-negative species, though the specific extracellular signals that impact c-di-GMP levels and the response mechanisms employed vary. Because c-di-GMP affects motility and adherence, it consequently can influence bacterial pathogenicity. In addition, a number of pathogenic bacteria have engaged c-di-GMP signaling to control specific virulence mechanisms, including effector secretion mechanisms and toxin biosynthesis.

Lytic herpes simplex virus 1 (HSV-1) infection triggers disruption of transcription termination (DoTT) of most cellular genes, resulting in extensive intergenic transcription. Similarly, cellular stress responses lead to gene-specific transcription downstream of genes (DoG). In this study, we performed a detailed comparison of DoTT/DoG transcription between HSV-1 infection, salt and heat stress in primary human fibroblasts using 4sU-seq and ATAC-seq. Although DoTT at late times of HSV-1 infection was substantially more prominent than DoG transcription in salt and heat stress, poly(A) read-through due to DoTT/DoG transcription and affected genes were significantly correlated between all three conditions, in particular at earlier times of infection. We speculate that HSV-1 either directly usurps a cellular stress response or disrupts the transcription termination machinery in other ways but with similar consequences. In contrast to previous reports, we found that inhibition of Ca2+ signaling by BAPTA-AM did not specifically inhibit DoG transcription but globally impaired transcription. Most importantly, HSV-1-induced DoTT, but not stress-induced DoG transcription, was accompanied by a strong increase in open chromatin downstream of the affected poly(A) sites. In its extent and kinetics, downstream open chromatin essentially matched the poly(A) read-through transcription. We show that this does not cause but rather requires DoTT as well as high levels of transcription into the genomic regions downstream of genes. This raises intriguing new questions regarding the role of histone repositioning in the wake of RNA Polymerase II passage downstream of impaired poly(A) site recognition., Author summary Recently, we reported that productive herpes simplex virus 1 (HSV-1) infection leads to disruption of transcription termination (DoTT) of most but not all cellular genes. This results in extensive transcription beyond poly(A) sites and into downstream genes. Subsequently, cellular stress responses were found to trigger transcription downstream of genes (DoG) for >10% of protein-coding genes. Here, we directly compared the two phenomena in HSV-1 infection, salt and heat stress and observed significant overlaps between the affected genes. We speculate that HSV-1 either directly usurps a cellular stress response or disrupts the transcription termination machinery in other ways with similar consequences. In addition, we show that inhibition of calcium signaling does not specifically inhibit stress-induced DoG transcription but globally impairs RNA polymerase I, II and III transcription. Finally, HSV-1-induced DoTT, but not stress-induced DoG transcription, was accompanied by a strong increase in chromatin accessibility downstream of affected poly(A) sites. In its kinetics and extent, this essentially matched poly(A) read-through transcription but does not cause but rather requires DoTT. We hypothesize that this results from impaired histone repositioning when RNA Polymerase II enters downstream intergenic regions of genes affected by DoTT.

Many RNA-binding proteins possess domains with a biased amino acid content. A common property of these low complexity domains (LCDs) is that they assemble into an ordered amyloid form, juxtaposing RNA recognition motifs in a subcellular compartment in which RNA metabolism is focused. Yeast Nab3 is one such protein that contains RNA-binding domains and a low complexity, glutamine/proline-rich, prion-like domain that can self-assemble. Nab3 also contains a region of structural homology to human hnRNP-C that resembles a leucine zipper which can oligomerize. Here we show that the LCD and the human hnRNP-C homology domains of Nab3 were experimentally separable, as cells were viable with either segment, but not when both were missing. In exploiting the lethality of deleting these regions of Nab3, we were able to test if heterologous prion-like domains known to assemble into amyloid, could substitute for the native sequence. Those from the hnRNP-like protein Hrp1, the canonical prion Sup35, or the epsin-related protein Ent2, could rescue viability and enable the new Nab3 chimeric protein to support transcription termination. Other low complexity domains from RNA-binding, termination-related proteins or a yeast prion, could not. As well, an unbiased genetic selection revealed a new protein sequence that could rescue the loss of Nab3’s essential domain via multimerization. This new sequence and Sup35’s prion domain could also rescue the lethal loss of Hrp1’s prion-like domain when substituted for it. This suggests there are different cross-functional classes of amyloid-forming LCDs and that appending merely any assembly-competent LCD to Nab3 does not restore function or rescue viability. The analysis has revealed the functional complexity of LCDs and provides a means by which the differing classes of LCD can be dissected and understood.

In eukaryotes, RNA species originating from pervasive transcription are regulators of various cellular processes, from the expression of individual genes to the control of cellular development and oncogenesis. In prokaryotes, the function of pervasive transcription and its output on cell physiology is still unknown. Most bacteria possess termination factor Rho, which represses pervasive, mostly antisense, transcription. Here, we investigate the biological significance of Rho-controlled transcription in the Gram-positive model bacterium Bacillus subtilis. Rho inactivation strongly affected gene expression in B. subtilis, as assessed by transcriptome and proteome analysis of a rho–null mutant during exponential growth in rich medium. Subsequent physiological analyses demonstrated that a considerable part of Rho-controlled transcription is connected to balanced regulation of three mutually exclusive differentiation programs: cell motility, biofilm formation, and sporulation. In the absence of Rho, several up-regulated sense and antisense transcripts affect key structural and regulatory elements of these differentiation programs, thereby suppressing motility and biofilm formation and stimulating sporulation. We dissected how Rho is involved in the activity of the cell fate decision-making network, centered on the master regulator Spo0A. We also revealed a novel regulatory mechanism of Spo0A activation through Rho-dependent intragenic transcription termination of the protein kinase kinB gene. Altogether, our findings indicate that distinct Rho-controlled transcripts are functional and constitute a previously unknown built-in module for the control of cell differentiation in B. subtilis. In a broader context, our results highlight the recruitment of the termination factor Rho, for which the conserved biological role is probably to repress pervasive transcription, in highly integrated, bacterium-specific, regulatory networks., Author summary Bacillus subtilis is a widely used model to study cell differentiation in the bacterial world. This soil-dwelling bacterium can engage in several alternative developmental programs, which generate distinct cell types adapted to different lifestyles, to cope with its complex and changing natural environment. The underlying differentiation control mechanisms are interconnected and tightly regulated, because these physiological and morphological cellular states are mutually exclusive and a correct choice is crucial. Here, we describe a previously unrecognized mechanism that regulates cell fate decisions in B. subtilis. It is based on the elements of pervasive genome-wide transcription controlled by the termination factor Rho. Pervasive transcription originating from non-defined or cryptic signals is spread throughout bacterial transcriptomes, but its physiological role is not yet well understood. We show that the elements of the Rho-controlled transcriptome affect specific developmental programs in B. subtilis: cell motility, biofilm formation, and sporulation, by directly or indirectly targeting expression of the key factors of cellular differentiation. Our results illuminate how Rho plays a prominent role in complex and organism-specific regulatory networks by controlling pervasive transcription. These findings rank Rho among the global transcriptional regulators of B. subtilis and invite systematic exploration of its role in other microorganisms.

Condensin is an essential component of chromosome dynamics, including mitotic chromosome condensation and segregation, DNA repair, and development. Genome-wide localization of condensin is known to correlate with transcriptional activity. The functional relationship between condensin accumulation and transcription sites remains unclear, however. By constructing the auxin-inducible degron strain of condensin, herein we demonstrate that condensin does not affect transcription itself. Instead, RNA processing at transcriptional termination appears to define condensin accumulation sites during mitosis, in the fission yeast Schizosaccharomyces pombe . Combining the auxin-degron strain with the nda3 β-tubulin cold-sensitive (cs) mutant enabled us to inactivate condensin in mitotically arrested cells, without releasing the cells into anaphase. Transcriptional activation and termination were not affected by condensin's degron-mediated depletion, at heat-shock inducible genes or mitotically activated genes. On the other hand, condensin accumulation sites shifted approximately 500 bp downstream in the auxin-degron of 5′-3′ exoribonuclease Dhp1, in which transcripts became aberrantly elongated, suggesting that condensin accumulates at transcriptionally terminated DNA regions. Growth defects in mutant strains of 3′-processing ribonuclease and polyA cleavage factors were additive in condensin temperature-sensitive (ts) mutants. Considering condensin's in vitro activity to form double-stranded DNAs from unwound, single-stranded DNAs or DNA-RNA hybrids, condensin-mediated processing of mitotic transcripts at the 3′-end may be a prerequisite for faithful chromosome segregation.

Summary Accurate regulation of mRNA termination is required for correct gene expression. Here, we describe a role for SCAF4 and SCAF8 as anti-terminators, suppressing the use of early, alternative polyadenylation (polyA) sites. The SCAF4/8 proteins bind the hyper-phosphorylated RNAPII C-terminal repeat domain (CTD) phosphorylated on both Ser2 and Ser5 and are detected at early, alternative polyA sites. Concomitant knockout of human SCAF4 and SCAF8 results in altered polyA selection and subsequent early termination, leading to expression of truncated mRNAs and proteins lacking functional domains and is cell lethal. While SCAF4 and SCAF8 work redundantly to suppress early mRNA termination, they also have independent, non-essential functions. SCAF8 is an RNAPII elongation factor, whereas SCAF4 is required for correct termination at canonical, distal transcription termination sites in the presence of SCAF8. Together, SCAF4 and SCAF8 coordinate the transition between elongation and termination, ensuring correct polyA site selection and RNAPII transcriptional termination in human cells., Graphical Abstract, Highlights • Human SCAF4 and SCAF8 couple RNAPII Ser2P- and Ser5P-binding and RNA processing • SCAF4 and SCAF8 bind nascent RNA upstream of early polyadenylation sites • SCAF4 and SCAF8 prevent early mRNA transcript cleavage and polyadenylation • Lack of SCAF4 and SCAF8 result in truncated protein products and is cell lethal, Eukaryotic anti-terminator proteins suppress usage of early polyadenylation sites to prevent production of truncated proteins.

Highlights • Transcriptional directionality is controlled by premature transcription termination. • Transcriptional directionality is enforced by gene looping. • mRNA-specific termination signals and factors are required for gene looping., Bidirectional promoters are a common feature of many eukaryotic organisms from yeast to humans. RNA Polymerase II that is recruited to this type of promoter can start transcribing in either direction using alternative DNA strands as the template. Such promiscuous transcription can lead to the synthesis of unwanted transcripts that may have negative effects on gene expression. Recent studies have identified transcription termination and gene looping as critical players in the enforcement of promoter directionality. Interestingly, both mechanisms share key components. Here, we focus on recent findings relating to the transcriptional output of bidirectional promoters.

Enhancement of transcription by a splicing-competent intron is an evolutionarily conserved feature among eukaryotes. The molecular mechanism underlying the phenomenon, however, is not entirely clear. Here we show that the intron is an important regulator of promoter directionality. Employing strand-specific transcription run-on (TRO) analysis, we show that the transcription of mRNA is favored over the upstream anti-sense transcripts (uaRNA) initiating from the promoter in the presence of an intron. Mutation of either the 5′ or 3′ splice site resulted in the reversal of promoter directionality, thereby suggesting that it is not merely the 5′ splice site but the entire splicing-competent intron that regulates transcription directionality. ChIP analysis revealed the recruitment of termination factors near the promoter region in the presence of an intron. Removal of intron or the mutation of splice sites adversely affected the promoter localization of termination factors. We have earlier demonstrated that the intron-mediated enhancement of transcription is dependent on gene looping. Here we show that gene looping is crucial for the recruitment of termination factors in the promoter-proximal region of an intron-containing gene. In a looping-defective mutant, despite normal splicing, the promoter occupancy of factors required for poly(A)-dependent termination of transcription was compromised. This was accompanied by a concomitant loss of transcription directionality. On the basis of these results, we propose that the intron-dependent gene looping places the terminator-bound factors in the vicinity of the promoter region for termination of the promoter-initiated upstream antisense transcription, thereby conferring promoter directionality., Author Summary Eukaryotic genes differ from their prokaryotic counterparts in having intervening non-coding sequences called introns. The precise biological role of introns in eukaryotic systems remains unclear even more than forty years after their initial discovery. One function of intron that has been remarkably conserved during evolution is their ability to enhance the transcription of genes that harbor them. How does the intron regulate transcription, however, is not known. Here we show that the intron enhances gene expression by affecting direction of the promoter-initiated transcription. In the presence of an intron, polymerase tends to transcribe the downstream coding region producing mRNA, while in the absence of a splicing-competent intron polymerase starts transcribing promoter upstream region producing upstream antisense RNA (uaRNA). Intron-mediated promoter directionality was dependent on gene looping, which is the interaction off the promoter and terminator region of a gene in a transcription-dependent manner. We show that the intron-dependent gene looping facilitates the recruitment of termination factors in the promoter-proximal region. The recruited termination factors stop uaRNA synthesis thereby conferring directionality to the promoter-bound polymerase.

Epigenetic gene silencing plays a critical role in regulating gene expression and contributes to organismal development and cell fate acquisition in eukaryotes. In fission yeast, Schizosaccharomyces pombe, heterochromatin-associated gene silencing is known to be mediated by RNA processing pathways including RNA interference (RNAi) and a 3’-5’ exoribonuclease complex, the exosome. Here, we report a new RNA-processing pathway that contributes to epigenetic gene silencing and assembly of heterochromatin mediated by 5’-3’ exoribonuclease Dhp1/Rat1/Xrn2. Dhp1 mutation causes defective gene silencing both at peri-centromeric regions and at the silent mating type locus. Intriguingly, mutation in either of the two well-characterized Dhp1-interacting proteins, the Din1 pyrophosphohydrolase or the Rhn1 transcription termination factor, does not result in silencing defects at the main heterochromatic regions. We demonstrate that Dhp1 interacts with heterochromatic factors and is essential in the sequential steps of establishing silencing in a manner independent of both RNAi and the exosome. Genomic and genetic analyses suggest that Dhp1 is involved in post-transcriptional silencing of repetitive regions through its RNA processing activity. The results describe the unexpected role of Dhp1/Rat1/Xrn2 in chromatin-based silencing and elucidate how various RNA-processing pathways, acting together or independently, contribute to epigenetic regulation of the eukaryotic genome., Author Summary Epigenetic mechanisms regulate when, where, and how an organism uses the genetic information stored in its genome. They are essential to many cellular processes, such as the regulation of gene expression, genome organization, and cell-fate determination. They also govern growth, development, and ultimately human health. Heterochromatin constitutes silenced chromatic domains, in which gene silencing occurs through epigenetic mechanisms. RNA processing pathways, such as RNA interference (RNAi) and the exosome, are known to mediate the silencing of genes via degradation of unwanted or aberrant transcripts. In this study, we describe a new RNA processing mechanism in epigenetic silencing using fission yeast, a premier model for studying these processes. With genetic, cell biology, and genomic approaches, we uncovered a previously unrecognized function of Dhp1, a highly conserved 5’-3’ exoribonuclease and ortholog of budding yeast Rat1 and metazoan Xrn2. We show that Dhp1 mediates a novel RNA processing mechanism in epigenetic silencing which occurs independently of both RNAi and the exosome. Our results clarify how multiple RNA processing pathways are involved in the regulation of eukaryotic gene expression and chromatin organization.

The Baculovirus Expression Vector System (BEVS) is widely used to produce high amounts of recombinant proteins. Nevertheless, generating recombinant baculovirus in high quality is rather time-consuming and labor-intensive. Alternatively, virus-free expression in insect cells did not achieve similar expression levels for most proteins so far. The transactivation method is a promising approach for protein expression in Sf21 cells. It combines advantages of BEVS and plasmid-based expression by activating strong virus-dependent promoters on a transfected plasmid by baculoviral coinfection. Here, we identified expression elements required for transactivation. Therefore, we designed several vectors comprising different viral promoters or promoter combinations and tested them for eGFP expression using the automated BioLector microcultivation system. Remarkably, only the combination of the very late promoter p10 together with the homologous region 5 (hr5) could boost expression during transactivation. Other elements, like p10 alone or the late viral promoter polH, did not respond to transactivation. A new combination of hr5 and p10 with the strongest immediate early OpMNPV viral promoter OpIE2 improved the yield of eGFP by ~25% in comparison to the previous applied hr5-IE1-p10 expression cassette. Furthermore, we observed a strong influence of the transcription termination sequence and vector backbone on the level of expression. Finally, the expression levels for transactivation, BEVS and solely plasmid-based expression were compared for the marker protein eGFP, underlining the potential of transactivation for fast recombinant protein expression in Sf21 cells. In conclusion, essential elements for transactivation could be identified. The optimal elements were applied to generate an improved vector applicable in virus-free plasmid-based expression, transactivation and BEVS.