Human promoter directionality is determined by transcriptional initiation and the opposing activities of INTS11 and CDK9.

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.

Plasma copeptin and markers of arterial disorder in patients with type 2 diabetes, a cross-sectional study.

OBJECTIVES: There is currently limited understanding of the relationship between copeptin, the midregional portion of proadrenomedullin (MRproADM) and the midregional fragment of the N-terminal of proatrial natriuretic peptide (MRproANP), and arterial disorders. Toe brachial index (TBI) and aortic pulse wave velocity (aPWV) are established parameters for detecting arterial disorders. This study evaluated whether copeptin, MRproADM, and MRproANP were associated with TBI and aPWV in patients with type 2 diabetes with no history of cardiovascular disease (CVD). METHODS: In the CARDIPP study, a cross-sectional analysis of 519 patients with type 2 diabetes aged 55-65 years with no history of CVD at baseline, had complete data on copeptin, MRproADM, MRproANP, TBI, and aPWV was performed. Linear regression analysis was used to investigate the associations between conventional CVD risk factors, copeptin, MRproADM, MRproANP, TBI, and aPWV. RESULTS: Copeptin was associated with TBI (ß-0.0020, CI-0.0035- (-0.0005), p = 0.010) and aPWV (ß 0.023, CI 0.002-0.044, p = 0.035). These associations were independent of age, sex, diabetes duration, mean 24-hour ambulatory systolic blood pressure, glycated hemoglobin A1c, total cholesterol, estimated glomerular filtration rate, body mass index, and active smoking. CONCLUSIONS: Plasma copeptin may be a helpful surrogate for identifying individuals at higher risk for arterial disorders. TRIAL REGISTRATION:  ClinicalTrials.gov identifier NCT010497377.

Recreational use of nitrous oxide causes seizure, pneumothorax, pneumomediastinum, and pneumopericardium: nitrous oxide and its harm, a case report.

Nitrous oxide, commonly known as 'laughing gas', has become a popular recreational drug. Whippets, small canisters containing gas in pressurized form, can be easily obtained from a food store. However, inhaling nitrous oxide from these canisters, which contain a 100% concentration, can lead to hypoxia, resulting in seizures or even death. Inhalation of nitrous oxide rarely causes pneumothorax, pneumomediastinum, and pneumopericardium. This case study highlights the potential dangers of recreational abuse of nitrous oxide.

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.

Association of physiological stress markers at the emergency department to readmission and death within 90 days: a prospective observational study.

Background: Predicting the risk of readmission or death in patients at the emergency department (ED) is essential in identifying patients who would benefit the most from interventions. We aimed to explore the prognostic value of mid-regional proadrenomedullin (MR-proADM), mid-regional pro-atrial natriuretic peptide (MR-proANP), copeptin, and high-sensitivity troponin T (hs-TnT) to identify patients with a higher risk of readmission and death among patients presenting with chest pain (CP) and/or shortness of breath (SOB) in the ED. Methods: This single-center prospective observational study included non-critically ill adult patients with a chief complaint of CP and/or SOB who visited the ED at Linköping University Hospital. Baseline data and blood samples were collected, and patients were followed up for 90 days after inclusion. The primary outcome was a composite of readmission and/or death from non-traumatic causes within 90 days of inclusion. Binary logistic regression was used and receiver operating characteristics (ROC) curves were constructed to determine the prognostic performance for predicting readmission and/or death within 90 days. Results: A total of 313 patients were included and 64 (20.4%) met the primary endpoint. MR-proADM > 0.75 pmol/L (odds ratio [OR]: 2.361 [95% confidence interval [CI]: 1.031 - 5.407], P = 0.042) and multimorbidity (OR: 2.647 [95% CI: 1.282 - 5.469], P = 0.009) were significantly associated with readmission and/or death within 90 days. MR-proADM increased predictive value in the ROC analysis to age, sex, and multimorbidity (P = 0.006). Conclusions: In non-critically ill patients with CP and/or SOB in the ED, MR-proADM and multimorbidity may be helpful for the prediction of the risk of readmission and/or death within 90 days.

[Spinal motion restriction in trauma care]. / Nya rekommendationer för spinal rörelsebegränsning vid trauma.

Nineteen Swedish professional organizations involved in trauma care have developed national recommendations for spinal motion restriction in trauma. The recommendations describe the best practice in spinal motion restriction for children, adults, and the elderly, both in prehospital care, in the emergency department and during transfer within and between hospitals. The background to the recommendations is presented, together with implications for the widespread educational system Advanced Trauma Life Support (ATLS).

Admission of patients with chest pain and/or breathlessness from the emergency department in relation to risk assessment and copeptin levels - an observational study.

Background: One of the most critical decisions that emergency department (ED) physicians make is the discharge versus admission of patients. We aimed to study the association of the decision in the ED to admit patients with chest pain and/or breathlessness to a ward with risk assessment using the Rapid Emergency Triage and Treatment System (RETTS), the National Early Warning Score (NEWS), and plasma levels of the biomarkers copeptin, midregional proadrenomedulin (MR-proADM), and midregional proatrial natriuretic peptide (MR-proANP). Methods: Patients presenting at the ED with chest pain and/or breathlessness with less than one week onset were enrolled. Patients were triaged according to RETTS. NEWS was calculated from the vital signs retrospectively. Results: Three hundred and thirty-four patients (167 males), mean age 63.8 ± 16.8 years, were included. Of which, 210 (62.8%) patients complained of chest pain, 65 (19.5%) of breathlessness, and 59 (17.7%) of both. Of these, 176 (52.7%) patients were admitted to a ward, and 158 (47.3%) patients were discharged from the ED. In binary logistic models, age, gender, vital signs (O2 saturation and heart rate), NEWS class, and copeptin were associated with admission to a ward from the ED. In receiver-operating-characteristics (ROC) analysis, copeptin had an incremental predictive value compared to NEWS alone (P = 0.002). Conclusions: Emergency physicians' decisions to admit patients with chest pain and/or breathlessness from the ED to a ward are related to age, O2 saturation, heart rate, NEWS category, and copeptin. As an independent predictive marker for admission, early analysis of copeptin might be beneficial when improving patient pathways at the ED.

ZC3H4 restricts non-coding transcription in human cells.

The human genome encodes thousands of non-coding RNAs. Many of these terminate early and are then rapidly degraded, but how their transcription is restricted is poorly understood. In a screen for protein-coding gene transcriptional termination factors, we identified ZC3H4. Its depletion causes upregulation and extension of hundreds of unstable transcripts, particularly antisense RNAs and those transcribed from so-called super-enhancers. These loci are occupied by ZC3H4, suggesting that it directly functions in their transcription. Consistently, engineered tethering of ZC3H4 to reporter RNA promotes its degradation by the exosome. ZC3H4 is predominantly metazoan -interesting when considering its impact on enhancer RNAs that are less prominent in single-celled organisms. Finally, ZC3H4 loss causes a substantial reduction in cell proliferation, highlighting its overall importance. In summary, we identify ZC3H4 as playing an important role in restricting non-coding transcription in multicellular organisms.

Integrator-Dependent and Allosteric/Intrinsic Mechanisms Ensure Efficient Termination of snRNA Transcription.

Many RNA polymerases terminate transcription using allosteric/intrinsic mechanisms, whereby protein alterations or nucleotide sequences promote their release from DNA. RNA polymerase II (Pol II) is somewhat different based on its behavior at protein-coding genes where termination additionally requires endoribonucleolytic cleavage and subsequent 5'→3' exoribonuclease activity. The Pol-II-transcribed small nuclear RNAs (snRNAs) also undergo endoribonucleolytic cleavage by the Integrator complex, which promotes their transcriptional termination. Here, we confirm the involvement of Integrator but show that Integrator-independent processes can terminate snRNA transcription both in its absence and naturally. This is often associated with exosome degradation of snRNA precursors that long-read sequencing analysis reveals as frequently terminating at T-runs located downstream of some snRNAs. This finding suggests a unifying vulnerability of RNA polymerases to such sequences given their well-known roles in terminating Pol III and bacterial RNA polymerase.

Toe brachial index predicts major acute cardiovascular events in patients with type 2 diabetes independently of arterial stiffness.

OBJECTIVE: Our aim was to analyze the predictive value of toe brachial index (TBI) as a risk marker for future major adverse cardiovascular events (MACE) and all-cause mortality in patients with type 2 diabetes (T2D). METHODS: TBI was measured in 741 patients with T2D in 2005-2008. Conventional risk factors for vascular disease as well as non-invasive measurements such as pulse-wave velocity (PWV) and intima-media thickness (IMT) of the carotid arteries were estimated. MACE was defined as cardiovascular death or hospitalization for non fatal myocardial infarction or non fatal stroke. Patients were followed for incidence of MACE using the national Swedish Cause of Death Registry and the Inpatient Register. RESULTS: During the follow-up for a period of 9 years MACE occurred in 97 patients and 85 patients died. TBI tertile, 1 versus 3, was significantly related to MACE (HR 2.67, 95%CI 1.60-4.50; p < 0.001) and to all-cause mortality (HR 1.98, 95%CI 1.16-3.83; p = 0.01). TBI tertile 1 as compared to TBI tertile 3 predicted MACE, but not all-cause mortality, independently of age, sex, diabetes duration and treatment, antihypertensive treatment, previous cardiovascular diseases, office systolic blood pressure, HbA1c, LDL cholesterol, estimated glomerular filtration rate, body mass index, current smoking PWV, IMT and carotid plaque presence (HR 3.39, 95%CI 1.53-7.51; p = 0.003 and HR 1.81, 95%CI 0.87-3.76; p = 0.1, respectively). CONCLUSIONS: Low TBI predicts an increased risk for MACE independently of arterial stiffness in patients with type 2 diabetes. TRIAL REGISTRATION: Clinical Trials.gov number NCT01049737. Registered January 14, 2010.

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.

Rapid Depletion of DIS3, EXOSC10, or XRN2 Reveals the Immediate Impact of Exoribonucleolysis on Nuclear RNA Metabolism and Transcriptional Control.

Cell-based studies of human ribonucleases traditionally rely on methods that deplete proteins slowly. We engineered cells in which the 3'→5' exoribonucleases of the exosome complex, DIS3 and EXOSC10, can be rapidly eliminated to assess their immediate roles in nuclear RNA biology. The loss of DIS3 has the greatest impact, causing the substantial accumulation of thousands of transcripts within 60 min. These transcripts include enhancer RNAs, promoter upstream transcripts (PROMPTs), and products of premature cleavage and polyadenylation (PCPA). These transcripts are unaffected by the rapid loss of EXOSC10, suggesting that they are rarely targeted to it. More direct detection of EXOSC10-bound transcripts revealed its substrates to prominently include short 3' extended ribosomal and small nucleolar RNAs. Finally, the 5'→3' exoribonuclease, XRN2, has little activity on exosome substrates, but its elimination uncovers different mechanisms for the early termination of transcription from protein-coding gene promoters.

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.

Poly(A) Polymerase and the Nuclear Poly(A) Binding Protein, PABPN1, Coordinate the Splicing and Degradation of a Subset of Human Pre-mRNAs.

Most human protein-encoding transcripts contain multiple introns that are removed by splicing. Although splicing catalysis is frequently cotranscriptional, some introns are excised after polyadenylation. Accumulating evidence suggests that delayed splicing has regulatory potential, but the mechanisms are still not well understood. Here we identify a terminal poly(A) tail as being important for a subset of intron excision events that follow cleavage and polyadenylation. In these cases, splicing is promoted by the nuclear poly(A) binding protein, PABPN1, and poly(A) polymerase (PAP). PABPN1 promotes intron excision in the context of 3'-end polyadenylation but not when bound to internal A-tracts. Importantly, the ability of PABPN1 to promote splicing requires its RNA binding and, to a lesser extent, PAP-stimulatory functions. Interestingly, an N-terminal alanine expansion in PABPN1 that is thought to cause oculopharyngeal muscular dystrophy cannot completely rescue the effects of PABPN1 depletion, suggesting that this pathway may have relevance to disease. Finally, inefficient polyadenylation is associated with impaired recruitment of splicing factors to affected introns, which are consequently degraded by the exosome. Our studies uncover a new function for polyadenylation in controlling the expression of a subset of human genes via pre-mRNA splicing.

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.

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.

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.