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.

A restrictor complex of ZC3H4, WDR82, and ARS2 integrates with PNUTS to control unproductive transcription.

The transcriptional termination of unstable non-coding RNAs (ncRNAs) is poorly understood compared to coding transcripts. We recently identified ZC3H4-WDR82 ("restrictor") as restricting human ncRNA transcription, but how it does this is unknown. Here, we show that ZC3H4 additionally associates with ARS2 and the nuclear exosome targeting complex. The domains of ZC3H4 that contact ARS2 and WDR82 are required for ncRNA restriction, suggesting their presence in a functional complex. Consistently, ZC3H4, WDR82, and ARS2 co-transcriptionally control an overlapping population of ncRNAs. ZC3H4 is proximal to the negative elongation factor, PNUTS, which we show enables restrictor function and is required to terminate the transcription of all major RNA polymerase II transcript classes. In contrast to short ncRNAs, longer protein-coding transcription is supported by U1 snRNA, which shields transcripts from restrictor and PNUTS at hundreds of genes. These data provide important insights into the mechanism and control of transcription by restrictor and PNUTS.

A unified allosteric/torpedo mechanism for transcriptional termination on human protein-coding genes.

The allosteric and torpedo models have been used for 30 yr to explain how transcription terminates on protein-coding genes. The former invokes termination via conformational changes in the transcription complex and the latter proposes that degradation of the downstream product of poly(A) signal (PAS) processing is important. Here, we describe a single mechanism incorporating features of both models. We show that termination is completely abolished by rapid elimination of CPSF73, which causes very extensive transcriptional readthrough genome-wide. This is because CPSF73 functions upstream of modifications to the elongation complex and provides an entry site for the XRN2 torpedo. Rapid depletion of XRN2 enriches these events that we show are underpinned by protein phosphatase 1 (PP1) activity, the inhibition of which extends readthrough in the absence of XRN2. Our results suggest a combined allosteric/torpedo mechanism, in which PP1-dependent slowing down of polymerases over termination regions facilitates their pursuit/capture by XRN2 following PAS processing.

A modular architecture for organizing, processing and sharing neurophysiology data.

We describe an architecture for organizing, integrating and sharing neurophysiology data within a single laboratory or across a group of collaborators. It comprises a database linking data files to metadata and electronic laboratory notes; a module collecting data from multiple laboratories into one location; a protocol for searching and sharing data and a module for automatic analyses that populates a website. These modules can be used together or individually, by single laboratories or worldwide collaborations.

Xrn2 accelerates termination by RNA polymerase II, which is underpinned by CPSF73 activity.

Termination is a ubiquitous phase in every transcription cycle but is incompletely understood and a subject of debate. We used gene editing as a new approach to address its mechanism through engineered conditional depletion of the 5' → 3' exonuclease Xrn2 or the polyadenylation signal (PAS) endonuclease CPSF73 (cleavage and polyadenylation specificity factor 73). The ability to rapidly control Xrn2 reveals a clear and general role for it in cotranscriptional degradation of 3' flanking region RNA and transcriptional termination. This defect is characterized genome-wide at high resolution using mammalian native elongating transcript sequencing (mNET-seq). An Xrn2 effect on termination requires prior RNA cleavage, and we provide evidence for this by showing that catalytically inactive CPSF73 cannot restore termination to cells lacking functional CPSF73. Notably, Xrn2 plays no significant role in either Histone or small nuclear RNA (snRNA) gene termination even though both RNA classes undergo 3' end cleavage. In sum, efficient termination on most protein-coding genes involves CPSF73-mediated RNA cleavage and cotranscriptional degradation of polymerase-associated RNA by Xrn2. However, as CPSF73 loss caused more extensive readthrough transcription than Xrn2 elimination, it likely plays a more underpinning role in termination.

Emergent RNA-RNA interactions can promote stability in a facultative phototrophic endosymbiosis.

Eukaryote-eukaryote endosymbiosis was responsible for the spread of chloroplast (plastid) organelles. Stability is required for the metabolic and genetic integration that drives the establishment of new organelles, yet the mechanisms that act to stabilize emergent endosymbioses-between two fundamentally selfish biological organisms-are unclear. Theory suggests that enforcement mechanisms, which punish misbehavior, may act to stabilize such interactions by resolving conflict. However, how such mechanisms can emerge in a facultative endosymbiosis has yet to be explored. Here, we propose that endosymbiont-host RNA-RNA interactions, arising from digestion of the endosymbiont population, can result in a cost to host growth for breakdown of the endosymbiosis. Using the model facultative endosymbiosis between Paramecium bursaria and Chlorella spp., we demonstrate that this mechanism is dependent on the host RNA-interference (RNAi) system. We reveal through small RNA (sRNA) sequencing that endosymbiont-derived messenger RNA (mRNA) released upon endosymbiont digestion can be processed by the host RNAi system into 23-nt sRNA. We predict multiple regions of shared sequence identity between endosymbiont and host mRNA, and demonstrate through delivery of synthetic endosymbiont sRNA that exposure to these regions can knock down expression of complementary host genes, resulting in a cost to host growth. This process of host gene knockdown in response to endosymbiont-derived RNA processing by host RNAi factors, which we term "RNAi collisions," represents a mechanism that can promote stability in a facultative eukaryote-eukaryote endosymbiosis. Specifically, by imposing a cost for breakdown of the endosymbiosis, endosymbiont-host RNA-RNA interactions may drive maintenance of the symbiosis across fluctuating ecological conditions.

Termination of Transcription by RNA Polymerase II: BOOM!

RNA polymerase II (Pol II) transcribes hundreds of thousands of transcription units - a reaction always brought to a close by its termination. Because Pol II transcribes multiple gene types, its termination occurs in a variety of ways, with the polymerase being responsive to different inputs. Moreover, it is not just a default process occurring at the end of genes. Promoter-proximal and premature termination is common and might in turn regulate gene expression levels. Although some transcription termination mechanisms have been debated for decades, research is only just underway on emergent processes. We provide an updated view of transcription termination in human cells, highlighting common themes and some interesting differences between the contexts in which it occurs.

3' end formation of pre-mRNA and phosphorylation of Ser2 on the RNA polymerase II CTD are reciprocally coupled in human cells.

3' end formation of pre-mRNAs is coupled to their transcription via the C-terminal domain (CTD) of RNA polymerase II (Pol II). Nearly all protein-coding transcripts are matured by cleavage and polyadenylation (CPA), which is frequently misregulated in disease. Understanding how transcription is coordinated with CPA in human cells is therefore very important. We found that the CTD is heavily phosphorylated on Ser2 (Ser2p) at poly(A) (pA) signals coincident with recruitment of the CstF77 CPA factor. Depletion of the Ser2 kinase Cdk12 impairs Ser2p, CstF77 recruitment, and CPA, strongly suggesting that the processes are linked, as they are in budding yeast. Importantly, we additionally show that the high Ser2p signals at the 3' end depend on pA signal function. Down-regulation of CPA results in the loss of a 3' Ser2p peak, whereas a new peak is formed when CPA is induced de novo. Finally, high Ser2p signals are generated by Pol II pausing, which is a well-known feature of pA site recognition. Thus, a reciprocal relationship between early steps in pA site processing and Ser2p ensures efficient 3' end formation.

Trk receptor signaling and sensory neuron fate are perturbed in human neuropathy caused by Gars mutations.

Charcot-Marie-Tooth disease type 2D (CMT2D) is a peripheral nerve disorder caused by dominant, toxic, gain-of-function mutations in the widely expressed, housekeeping gene, GARS The mechanisms underlying selective nerve pathology in CMT2D remain unresolved, as does the cause of the mild-to-moderate sensory involvement that distinguishes CMT2D from the allelic disorder distal spinal muscular atrophy type V. To elucidate the mechanism responsible for the underlying afferent nerve pathology, we examined the sensory nervous system of CMT2D mice. We show that the equilibrium between functional subtypes of sensory neuron in dorsal root ganglia is distorted by Gars mutations, leading to sensory defects in peripheral tissues and correlating with overall disease severity. CMT2D mice display changes in sensory behavior concordant with the afferent imbalance, which is present at birth and nonprogressive, indicating that sensory neuron identity is prenatally perturbed and that a critical developmental insult is key to the afferent pathology. Through in vitro experiments, mutant, but not wild-type, GlyRS was shown to aberrantly interact with the Trk receptors and cause misactivation of Trk signaling, which is essential for sensory neuron differentiation and development. Together, this work suggests that both neurodevelopmental and neurodegenerative mechanisms contribute to CMT2D pathogenesis, and thus has profound implications for the timing of future therapeutic treatments.

A novel SOD1-ALS mutation separates central and peripheral effects of mutant SOD1 toxicity.

Transgenic mouse models expressing mutant superoxide dismutase 1 (SOD1) have been critical in furthering our understanding of amyotrophic lateral sclerosis (ALS). However, such models generally overexpress the mutant protein, which may give rise to phenotypes not directly relevant to the disorder. Here, we have analysed a novel mouse model that has a point mutation in the endogenous mouse Sod1 gene; this mutation is identical to a pathological change in human familial ALS (fALS) which results in a D83G change in SOD1 protein. Homozgous Sod1(D83G/D83G) mice develop progressive degeneration of lower (LMN) and upper motor neurons, likely due to the same unknown toxic gain of function as occurs in human fALS cases, but intriguingly LMN cell death appears to stop in early adulthood and the mice do not become paralyzed. The D83 residue coordinates zinc binding, and the D83G mutation results in loss of dismutase activity and SOD1 protein instability. As a result, Sod1(D83G/D83G) mice also phenocopy the distal axonopathy and hepatocellular carcinoma found in Sod1 null mice (Sod1(-/-)). These unique mice allow us to further our understanding of ALS by separating the central motor neuron body degeneration and the peripheral effects from a fALS mutation expressed at endogenous levels.

The Global Regulatory Cyclic AMP Receptor Protein (CRP) Controls Multifactorial Fluoroquinolone Susceptibility in Salmonella enterica Serovar Typhimurium.

Fluoroquinolone antibiotics are prescribed for the treatment of Salmonella enterica infections, but resistance to this family of antibiotics is growing. Here we report that loss of the global regulatory protein cyclic AMP (cAMP) receptor protein (CRP) or its allosteric effector, cAMP, reduces susceptibility to fluoroquinolones. A Δcrp mutation was synergistic with the primary fluoroquinolone resistance allele gyrA83, thus able to contribute to clinically relevant resistance. Decreased susceptibility to fluoroquinolones could be partly explained by decreased expression of the outer membrane porin genes ompA and ompF with a concomitant increase in the expression of the ciprofloxacin resistance efflux pump gene acrB in Δcrp cells. Expression of gyrAB, which encode the DNA supercoiling enzyme GyrAB, which is blocked by fluoroquinolones, and expression of topA, which encodes the dominant supercoiling-relaxing enzyme topoisomerase I, were unchanged in Δcrp cells. Yet Δcrp cells maintained a more relaxed state of DNA supercoiling, correlating with an observed increase in topoisomerase IV (parCE) expression. Surprisingly, the Δcrp mutation had the unanticipated effect of enhancing fitness in the presence of fluoroquinolone antibiotics, which can be explained by the observation that exposure of Δcrp cells to ciprofloxacin had the counterintuitive effect of restoring wild-type levels of DNA supercoiling. Consistent with this, Δcrp cells did not become elongated or induce the SOS response when challenged with ciprofloxacin. These findings implicate the combined action of multiple drug resistance mechanisms in Δcrp cells: reduced permeability and elevated efflux of fluoroquinolones coupled with a relaxed DNA supercoiling state that buffers cells against GyrAB inhibition by fluoroquinolones.

Persistent microglial activation and synaptic loss with behavioral abnormalities in mouse offspring exposed to CASPR2-antibodies in utero.

Gestational transfer of maternal antibodies against fetal neuronal proteins may be relevant to some neurodevelopmental disorders, but until recently there were no proteins identified. We recently reported a fivefold increase in CASPR2-antibodies in mid-gestation sera from mothers of children with intellectual and motor disabilities. Here, we exposed mice in utero to purified IgG from patients with CASPR2-antibodies (CASPR2-IgGs) or from healthy controls (HC-IgGs). CASPR2-IgG but not HC-IgG bound to fetal brain parenchyma, from which CASPR2-antibodies could be eluted. CASPR2-IgG exposed neonates achieved milestones similarly to HC-IgG exposed controls but, when adult, the CASPR2-IgG exposed progeny showed marked social interaction deficits, abnormally located glutamatergic neurons in layers V-VI of the somatosensory cortex, a 16% increase in activated microglia, and a 15-52% decrease in glutamatergic synapses in layers of the prefrontal and somatosensory cortices. Thus, in utero exposure to CASPR2-antibodies led to permanent behavioral, cellular, and synaptic abnormalities. These findings support a pathogenic role for maternal antibodies in human neurodevelopmental conditions, and CASPR2 as a potential target.

Transcriptional termination enhances protein expression in human cells.

Transcriptional termination of mammalian RNA polymerase II (Pol II) requires a poly(A) (pA) signal and, often, a downstream terminator sequence. Termination is triggered following recognition of the pA signal by Pol II and subsequent pre-mRNA cleavage, which occurs either at the pA site or in transcripts from terminator elements. Although this process has been extensively studied, it is generally considered inconsequential to the level of gene expression. However, our results demonstrate that termination acts as a driving force for optimal gene expression. We show that this effect is general but most dramatic where weak or noncanonical pA signals are present. We establish that termination of Pol II increases the efficiency of pre-mRNA processing that is completed posttranscriptionally. As such, transcripts escape from nuclear surveillance.

Co-transcriptional degradation of aberrant pre-mRNA by Xrn2.

Eukaryotic protein-coding genes are transcribed as pre-mRNAs that are matured by capping, splicing and cleavage and polyadenylation. Although human pre-mRNAs can be long and complex, containing multiple introns and many alternative processing sites, they are usually processed co-transcriptionally. Mistakes during nuclear mRNA maturation could lead to potentially harmful transcripts that are important to eliminate. However, the processes of human pre-mRNA degradation are not well characterised in the human nucleus. We have studied how aberrantly processed pre-mRNAs are degraded and find a role for the 5'→3' exonuclease, Xrn2. Xrn2 associates with and co-transcriptionally degrades nascent ß-globin transcripts, mutated to inhibit splicing or 3' end processing. Importantly, we provide evidence that many endogenous pre-mRNAs are also co-transcriptionally degraded by Xrn2 when their processing is inhibited by Spliceostatin A. Our data therefore establish a previously unknown function for Xrn2 and an important further aspect of pre-mRNA metabolism that occurs co-transcriptionally.

Molecular dissection of mammalian RNA polymerase II transcriptional termination.

Transcriptional termination of mammalian RNA polymerase II (Pol II) is an essential but little-understood step in protein-coding gene expression. Mechanistically, termination by all DNA-dependent RNA polymerases can be reduced to two steps, namely release of the RNA transcript and release of the DNA template. Using a simple nuclear fractionation procedure, we have monitored transcript and template release in the context of both natural and artificial Pol II terminator sequences. We describe the timing and relationship between these events and in so doing establish the roles of the poly(A) signal, cotranscriptional RNA cleavage events, and 5'-3' exonucleolytic RNA degradation in the mammalian Pol II termination process.

Hyperbaric oxygen for blast-related postconcussion syndrome: three-month outcomes.

OBJECTIVE: Mild traumatic brain injury (mTBI) and postconcussion syndrome (PCS) are common among military combatants. Hyperbaric oxygen (HBO2 ) is a proposed treatment for these conditions, but it has not been rigorously studied. The objective of this study was to determine the effects of HBO2 by 3 months post compression at 2 commonly employed dosing levels to treat PCS; whether specific subgroups may have benefited; and if no overall effect was found, whether benefit is masked by other conditions. METHODS: This randomized, double-blind, sham-controlled study was conducted at the Naval Air Station in Pensacola, Florida on 61 male Marines with a history of mTBI and PCS. Intervention consisted of 40 once daily 60-minute hyperbaric chamber compressions at 2.0 atmospheres absolute (ATA) at 1 of 3 randomly preassigned oxygen fractions, resulting in respective blinded groups with an oxygen-breathing exposure equivalent to (1) surface air (sham), (2) 100% oxygen at 1.5ATA, or (3) 100% oxygen at 2.0ATA. The main outcome measure was the Rivermead Post-Concussion Questionnaire-16 (RPQ-16) collected before compressions and at 2 later points. RESULTS: The interaction of time by intervention group was not significant for improvement on the RPQ-16. Nor was there evidence of efficacy on the RPQ-16 for any subgroup. No significant time by intervention interaction was found for any functional, cognitive, or psychomotor secondary outcome measure at an unadjusted 0.05 significance level. INTERPRETATION: Using a randomized control trial design and analysis including a sham, results showed no evidence of efficacy by 3 months post-compression to treat the symptomatic, cognitive, or behavioral sequelae of PCS after combat-related mTBI.

Splicing-coupled 3' end formation requires a terminal splice acceptor site, but not intron excision.

Splicing of human pre-mRNA is reciprocally coupled to 3' end formation by terminal exon definition, which occurs co-transcriptionally. It is required for the final maturation of most human pre-mRNAs and is therefore important to understand. We have used several strategies to block splicing at specific stages in vivo and studied their effect on 3' end formation. We demonstrate that a terminal splice acceptor site is essential to establish coupling with the poly(A) signal in a chromosomally integrated ß-globin gene. This is in part to alleviate the suppression of 3' end formation by U1 small nuclear RNA, which is known to bind pre-mRNA at the earliest stage of spliceosome assembly. Interestingly, blocks to splicing that are subsequent to terminal splice acceptor site function, but before catalysis, have little observable effect on 3' end formation. These data suggest that early stages of spliceosome assembly are sufficient to functionally couple splicing and 3' end formation, but that on-going intron removal is less critical.

The effect of hyperbaric oxygen on persistent postconcussion symptoms.

BACKGROUND: The high incidence of persistent postconcussion symptoms in service members with combat-related mild traumatic brain injury has prompted research in the use of hyperbaric oxygen (HBO2) for management. OBJECTIVE: The effects of HBO2 on persistent postconcussion symptoms in 60 military service members with at least 1 combat-related mild traumatic brain injury were examined in a single-center, double-blind, randomized, sham-controlled, prospective trial at the Naval Medicine Operational Training Center at Naval Air Station Pensacola. METHODS: Over a 10-week period, subjects received a series of 40, once-daily, hyperbaric chamber compressions at 2.0 atmospheres absolute (ATA). During each session, subjects breathed 1 of 3 preassigned oxygen fractions (10.5%, 75%, or 100%) for 60 minutes, resulting in an oxygen exposure equivalent to breathing surface air, 100% oxygen at 1.5 ATA, or 100% oxygen at 2.0 ATA, respectively. Individual, subscale and total item responses on the Rivermead Postconcussion Symptom Questionnaire and individual and total Posttraumatic Disorder Checklist-Military Version were measured just prior to intervention and immediately postintervention. RESULTS: Between-group testing of pre- and postintervention means revealed no significant differences on individual or total scores on the Posttraumatic Disorder Checklist-Military Version or Rivermead Postconcussion Symptom Questionnaire, demonstrating a successful randomization and no significant main effect for HBO2 at 1.5 or 2.0 ATA equivalent compared with the sham compression. Within-group testing of pre- and postintervention means revealed significant differences on several individual items for each group and difference in the Posttraumatic Disorder Checklist-Military Version total score for the 2.0 ATA HBO2 group. DISCUSSION: The primary analyses of between group differences found no evidence of efficacy for HBO2. The scattered within group differences are threatened by Type 2 errors and could be explained by nonspecific effects. CONCLUSION: This study demonstrated that HBO2 at either 1.5 or 2.0 ATA equivalent had no effect on postconcussion symptoms after mild traumatic brain injury when compared with sham compression.

ZC3H4/Restrictor exerts a stranglehold on pervasive transcription.

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.