The role of the RNA kinase CLP1 and ATP in mammalian tRNA splicing

Transfer RNAs (tRNAs) spielen eine wichtige Rolle in der Biologie, denn sie entschlüsseln die Erbinformation von Lebewesen, indem sie die Messenger RNA (mRNA) ablesen und die korrekten Aminosäuren an den Ort der Proteinsynthese bringen und an die wachsende Polypeptidkette anhängen. Bevor tRNAs ihre funktionelle L-Form annehmen, müssen sie am 5’ und 3’ Ende geschnitten und zahlreiche Nukleotide modifiziert werden. Außerdem enthalten manche tRNAs (Vorläufer-tRNAs, prä-tRNAs) intervenierende Sequenzen, die in einem zweistufigen Spleißprozess entfernt werden. Dabei schneidet zunächst die tRNA Spleißendonuklease, bestehend aus TSEN2, TSEN15, TSEN34, TSEN 54, sowie der RNA Kinase CLP1, 5’ und 3’ des Introns, wodurch zwei tRNA-Exonhälften entstehen. Im Hefe-pathway wird das 5’ Hydroxylende des 3’ Exons von CLP1 phosphoryliert und das 2’-3’ Cyclophosphatende des 5’ Exons wird von einer Phosphodiesterase geöffnet, wodurch die nunmehrigen 5’ Phosphat- und 3’ Hydroxylenden von einer tRNA-Ligase verbunden werden können. Das verbliebene 2’ Phosphat wird von einer Phosphotransferase entfernt. Im Säugerpathway, der in Wirbeltieren bevorzugt verwendet wird, werden die Exonhälften direkt nach dem Schneiden ligiert, d.h. ohne weitere Modifikation durch CLP1. In dieser Arbeit wird gezeigt, dass CLP1 nicht nur für die Exonphosphorylierung im Hefepathway zuständig ist, sondern bereits an der Schneidereaktion beteiligt ist und damit möglicherweise eine Funktion in beiden Pathways erfüllt. Wir konnten zeigen, dass Mausfibroblasten, aus denen CLP1 entfernt wurde („CLP1 knock-out“) oder die eine mutierte Version von CLP1 besitzen, dessen Kinasefunktion durch eine Lysin-zu-Alanin Punktmutation (K127A) zerstört wurde, eine stark verminderte prä-tRNA-Endonukleaseaktivität zeigen. Ein ähnlicher Effekt wurde mit einer durch kleine interferierende RNAs (siRNAs) ausgelösten mRNA-Depletion in HeLa-Zellen erzielt. Diese Reduktion der entstehenden Exonmengen wurde mittels TSEN2-Koimmunpräzipitation funktionell auf den Spleißendonukleasekomplex eingeschränkt. Durch die Depletion von ATP aus Zellextrakten und anschließende Immunpräzipitation oder direkt an TSEN2-Immunpräzipitaten wurde ebenfalls eine reduzierte prä-tRNA-Endonukleaseaktivität erreicht, die den beschriebenen CLP1-Effekten ähnelt. Die Rückgabe von ATP zu depletierten Proben bewirkte die Wiederherstellung hoher Exonmengen. Zur Charakterisierung des Prozessierungsdefekts wurde ein teilaufgereinigter CLP1-TSEN-Komplex durch eine Affinitätsreinigung einer mit Fusionsproteinen versehenen Version von Wildtyp- und K127A-CLP1 hergestellt. Die reduzierte prä-tRNA-Endonukleaseaktivität von K127A-CLP1 konnte bestätigt werden, wobei diese nicht auf die Abwesenheit eines oder mehrerer TSEN-Proteine zurückzuführen war, wie durch massenspektrometrische Analysen festgestellt werden konnte. Für eine effiziente prä-tRNA-Endonukleasereaktion war im verwendeten System ein β-γ-hydrolysierbares NTP nötig, was auch mit TSEN2-Immunpräzipitaten bestätigt werden konnte. Neben ATP konnte auch in Gegenwart von GTP, CTP oder UTP eine effiziente Prozessierung der prä-tRNA beobachtet werden. Durch die spezifische Inhibierung der Kinasefunktion von CLP1 im teilgereinigten Komplex durch eine synthetische Verbindung wurde ebenfalls eine reduzierte Endonukleaseaktivität beobachtet, was erneut für eine Beteiligung von CLP1 an diesem Prozess spricht. Die Charakterisierung der CLP1 K127A-Mutante hinsichtlich ihrer Fähigkeit, ATP zu binden bzw. zu hydrolysieren, erbrachte keine eindeutigen Resultate. Weiters konnten wir bisher nicht mit Sicherheit feststellen, ob eine Destabilisierung des Komplexes oder eine strukturelle Reorganisation die reduzierte prä-tRNA-Schneideaktivität verursacht. Daten aus Glyzeringradienten-Zentrifugationsexperimenten und die Wiederherstellung der Aktivität nach ATP-Zugabe zu depletierten Immunpräzipitaten widerlegen diese Hypothese. Im Gegensatz dazu fanden wir heraus, dass im teilgereinigten CLP1 K127A-Komplex TSEN2, TSEN34 und TSEN54 in drastisch reduzierten Mengen vorliegen, was wiederum für eine strukturelle Reorganisations des Komplexes spricht. In einer Analyse der mRNA-Mengen von Wildtyp- und K127A-Mausfibroblasten mittels quantitativer Echtzeit-PCR konnte überdies keine erniedrigte Konzentration von CLP1 oder TSEN-mRNAs festgestellt werden, die diesen Effekt erklären würden. Trotz der bereits erbrachten Erkenntnisse verbleiben noch zahlreiche offene Fragen bezüglich der reduzierten prä-tRNA-Endonukleaseaktivität. Einerseits muss geklärt werden, ob der Grund dafür tatsächlich in einer strukturellen Veränderung des Komplexes liegt, und falls nicht, muss die wirkliche Ursache gefunden werden. Andererseits könnte man Untersuchungen darüber anstellen, inwiefern CLP1 auch Proteinkinaseaktivität besitzt und dies die Exonentstehung beeinflussen könnte. Schließlich konnten wir auch Hinweise für eine Phosphorylierung der TSEN-Proteine finden, die den Ausgangspunkt eines völlig neuen Forschungsprojekts darstellen könnte.Transfer RNAs (tRNAs) play a fundamental role in biology. They decode the messenger RNA (mRNA) and carry amino acids to the sites of protein synthesis. Before they can adopt their mature, L-shaped conformation, they have to undergo numerous processing steps such as 5’ and 3’ end trimming and nucleotide modifications. A subset of vertebrate tRNAs is also transcribed as intron-containing precursor molecules (pre-tRNAs). The intervening sequence of this tRNA subset needs to be removed by a two-step splicing process. First, the tRNA splicing endonuclease (TSEN) complex, consisting of TSEN2, TSEN15, TSEN34, TSEN54, and the RNA kinase CLP1, cleaves the pre-tRNA at the 5‘ and 3‘ ends of the intron. This cleavage generates 5‘ and a 3‘ tRNA exon halves. In the yeast-like ligation pathway in human cells, the 5‘ hydroxyl terminus of the 3‘ exon is subsequently phosphorylated by CLP1, and the 2‘-3‘ cyclic phosphate at the 3’ end of the 5‘ exon is a substrate for a cyclic phosphodiesterase, allowing for a canonical 5' phosphate 3' hydroxyl ligation by a still elusive tRNA ligase. The remaining endogenous 2‘ phosphate is removed by a 2‘ phospho-transferase. In the animal pathway, which is predominantly used in vertebrates, the exon halves are directly ligated. Thus, CLP1 is dispensable for this type of ligation reaction. From the data presented in this work, we hypothesize that CLP1 not only performs phosphorylation of the 3’ exon half, but that ATP hydrolysis by CLP1 is additionally required for the preceding step, the generation of the exon halves by the TSEN complex. Therefore, CLP1 might be relevant for both tRNA splicing pathways. We show that extracts prepared from mouse embryonic fibroblasts (MEFs) that are devoid of CLP1 (“CLP1 knock-out”) or harbor a lysine-to-alanine point mutation (K127A) in CLP1 rendering the kinase inactive (“kinase-dead”) exhibit largely reduced pre-tRNA cleavage. In support of these data, knocking down CLP1 in HeLa cells by small interfering RNAs (siRNAs) resulted in a similar reduction in exon generation by the TSEN complex. The deficiency in pre-tRNA cleavage could be confirmed with TSEN2-immunoprecipitates from kinase-dead MEFs, pinpointing the defect to the pre-tRNA splicing endonuclease complex. When we removed ATP from TSEN2-immunoprecipitates from HeLa cells or performed co-immunoprecipitations with extracts depleted of ATP, we detected reduced tRNA exon generation, mimicking the effects previously observed upon interfering with CLP1’s kinase function. Interestingly, adding back ATP to the depleted immunoprecipitates restored pre-tRNA cleavage. In order to further elucidate the pre-tRNA cleavage deficiency phenotype, we used a semi-purified CLP1-TSEN complex obtained by a one-step purification of a tagged version of wild type and K127A CLP1. In addition to reduced exon generation with the mutant CLP1 complex, we found that the wild type complex requires a β-γ-hydrolyzable NTP for efficient pre-tRNA cleavage, confirming results obtained with TSEN2-immunoprecipitates. Mass spectrometry analysis of the semi-purified complexes confirmed the presence of the TSEN proteins in both CLP1 wild type and K127A samples. Furthermore, we found that GTP, CTP, and UTP could efficiently replace ATP in pre-tRNA cleavage. Additional evidence for CLP1’s role in pre-tRNA cleavage came when we added a specific CLP1 inhibitor to the semi-purified complex and subsequently observed reduced exon generation. However, the characterization of the CLP1 K127A mutant, regarding its ATP binding or hydrolysis competence, did not yield clear results. Moreover, we could not yet determine definitely if the disassembly of the complex or a structural rearrangement cause the cleavage deficiency. Data from glycerol gradient centrifugations and the results from addition of ATP to depleted immunoprecipitates argue against complex disassembly. In contrast, we found that in the CLP1 K127A semi-purified complex the levels of TSEN2, TSEN34, and TSEN54 were largely diminished, favoring the idea of complex destabilization. Quantitative real-time PCR results obtained by comparing wild type and kinase-dead MEFs disproved the hypothesis that the cleavage deficiency is caused by reduced TSEN or CLP1 mRNA levels. There remain several areas for further research on this topic. On the one hand, the reason for deficiency in pre-tRNA cleavage needs to be elucidated and the potential complex rearrangement must be clarified. On the other hand we could further investigate a putative protein kinase function of CLP1 and try to determine the biological role of a possible TSEN phosphorylation detected in the semi-purified endonuclease complex. For these two issues we could obtain only preliminary data so far; thus, they could be the starting point for new research projects

Risk factors and mortality associated with multimorbidity in people with stroke or transient ischaemic attack: a study of 8,751 UK Biobank participants

Background: Multimorbidity is common in stroke, but the risk factors and effects on mortality remain poorly understood. Objective: To examine multimorbidity and its associations with sociodemographic/lifestyle risk factors and all-cause mortality in UK Biobank participants with stroke or transient ischaemic attack (TIA). Design: Data were obtained from an anonymized community cohort aged 40–72 years. Overall, 42 comorbidities were self-reported by those with stroke or TIA. Relative risk ratios demonstrated associations between participant characteristics and number of comorbidities. Hazard ratios demonstrated associations between the number and type of comorbidities and all-cause mortality. Results were adjusted for age, sex, socioeconomic status, smoking, and alcohol intake. Data were linked to national mortality data. Median follow-up was 7 years. Results: Of 8,751 participants (mean age 60.9±6.7 years) with stroke or TIA, the all-cause mortality rate over 7 years was 8.4%. Over 85% reported ≥1 comorbidities. Age, socioeconomic deprivation, smoking and less frequent alcohol intake were associated with higher levels of multimorbidity. Increasing multimorbidity was associated with higher all-cause mortality. Mortality risk was double for those with ≥5 comorbidities compared to those with none. Having cancer, coronary heart disease, diabetes, or chronic obstructive pulmonary disease significantly increased mortality risk. Presence of any cardiometabolic comorbidity significantly increased mortality risk, as did any non-cardiometabolic comorbidity. Conclusions: In stroke survivors, the number of comorbidities may be a more helpful predictor of mortality than type of condition. Stroke guidelines should take greater account of comorbidities, and interventions are needed that improve outcomes for people with multimorbidity and stroke

Risk assessment and predicting outcomes in patients with depressive symptoms: a review of potential role of peripheral blood based biomarkers

Depression is one of the major global health challenges and a leading contributor of health related disability and costs. Depression is a heterogeneous disorder and current methods for assessing its severity in clinical practice rely on symptom count, however this approach is unreliable and inconsistent. The clinical evaluation of depressive symptoms is particularly challenging in primary care, where the majority of patients with depression are managed, due to the presence of co-morbidities. Current methods for risk assessment of depression do not accurately predict treatment response or clinical outcomes. Several biological pathways have been implicated in the pathophysiology of depression; however, accurate and predictive biomarkers remain elusive. We conducted a systematic review of the published evidence supporting the use of peripheral biomarkers to predict outcomes in depression, using Medline and Embase. Peripheral biomarkers in depression were found to be statistically significant predictors of mental health outcomes such as treatment response, poor outcome and symptom remission; and physical health outcomes such as increased incidence of cardiovascular events and deaths, and all-cause mortality. However, the available evidence has multiple methodological limitations which must be overcome to make any real clinical progress. Despite extensive research on the relationship of depression with peripheral biomarkers, its translational application in practice remains uncertain. In future, peripheral biomarkers identified with novel techniques and combining multiple biomarkers may have a potential role in depression risk assessment but further research is needed in this area

Multimorbidity and the COVID-19 pandemic – an urgent call to action

No abstract available

Multimorbidity and co-morbidity in atrial fibrillation and effects on survival: findings from UK Biobank cohort

Aims: To examine the number and type of co-morbid long-term health conditions (LTCs) and their associations with all-cause mortality in an atrial fibrillation (AF) population. Methods and results: Community cohort participants (UK Biobank n = 502 637) aged 37–73 years were recruited between 2006 and 2010. Self-reported LTCs (n = 42) identified in people with AF at baseline. All-cause mortality was available for a median follow-up of 7 years (interquartile range 76–93 months). Hazard ratios (HRs) examined associations between number and type of co-morbid LTC and all-cause mortality, adjusting for age, sex, socio-economic status, smoking, and anticoagulation status. Three thousand six hundred fifty-one participants (0.7% of the study population) reported AF; mean age was 61.9 years. The all-cause mortality rate was 6.7% (248 participants) at 7 years. Atrial fibrillation participants with ≥4 co-morbidities had a six-fold higher risk of mortality compared to participants without any LTC. Co-morbid heart failure was associated with higher risk of mortality [HR 2.96, 95% confidence interval (CI) 1.83–4.80], whereas the presence of co-morbid stroke did not have a significant association. Among non-cardiometabolic conditions, presence of chronic obstructive pulmonary disease (HR 3.31, 95% CI 2.14–5.11) and osteoporosis (HR 3.13, 95% CI 1.63–6.01) was associated with a higher risk of mortality. Conclusion: Survival in middle-aged to older individuals with self-reported AF is strongly correlated with level of multimorbidity. This group should be targeted for interventions to optimize their management, which in turn may potentially reduce the impact of their co-morbidities on survival. Future AF clinical guidelines need to place greater emphasis on the issue of co-morbidity

Digital support interventions for the self-management of low back pain: a systematic review

Background: Low back pain (LBP) is a common cause of disability and is ranked as the most burdensome health condition globally. Self-management, including components on increased knowledge, monitoring of symptoms, and physical activity, are consistently recommended in clinical guidelines as cost-effective strategies for LBP management and there is increasing interest in the potential role of digital health. Objective: The study aimed to synthesize and critically appraise published evidence concerning the use of interactive digital interventions to support self-management of LBP. The following specific questions were examined: (1) What are the key components of digital self-management interventions for LBP, including theoretical underpinnings? (2) What outcome measures have been used in randomized trials of digital self-management interventions in LBP and what effect, if any, did the intervention have on these? and (3) What specific characteristics or components, if any, of interventions appear to be associated with beneficial outcomes? Methods: Bibliographic databases searched from 2000 to March 2016 included Medline, Embase, CINAHL, PsycINFO, Cochrane Library, DoPHER and TRoPHI, Social Science Citation Index, and Science Citation Index. Reference and citation searching was also undertaken. Search strategy combined the following concepts: (1) back pain, (2) digital intervention, and (3) self-management. Only randomized controlled trial (RCT) protocols or completed RCTs involving adults with LBP published in peer-reviewed journals were included. Two reviewers independently screened titles and abstracts, full-text articles, extracted data, and assessed risk of bias using Cochrane risk of bias tool. An independent third reviewer adjudicated on disagreements. Data were synthesized narratively. Results: Of the total 7014 references identified, 11 were included, describing 9 studies: 6 completed RCTs and 3 protocols for future RCTs. The completed RCTs included a total of 2706 participants (range of 114-1343 participants per study) and varied considerably in the nature and delivery of the interventions, the duration/definition of LBP, the outcomes measured, and the effectiveness of the interventions. Participants were generally white, middle aged, and in 5 of 6 RCT reports, the majority were female and most reported educational level as time at college or higher. Only one study reported between-group differences in favor of the digital intervention. There was considerable variation in the extent of reporting the characteristics, components, and theories underpinning each intervention. None of the studies showed evidence of harm. Conclusions: The literature is extremely heterogeneous, making it difficult to understand what might work best, for whom, and in what circumstances. Participants were predominantly female, white, well educated, and middle aged, and thus the wider applicability of digital self-management interventions remains uncertain. No information on cost-effectiveness was reported. The evidence base for interactive digital interventions to support patient self-management of LBP remains weak

Examining the relationship between rheumatoid arthritis, multimorbidity and adverse health-related outcomes: a systematic review protocol

Background: Rheumatoid arthritis (RA) is a chronic autoimmune disorder characterised by articular inflammation and systemic complications. Multimorbidity (the presence of two or more long-term health conditions) is highly prevalent in people with RA but the effect of multimorbidity on mortality and other health-related outcomes is poorly understood. Objective: To determine what is known about the effect, if any, of multimorbidity on mortality and health-related outcomes in individuals with RA. Design: Systematic review of the literature. The following electronic medical databases will be searched: MEDLINE, Embase, Cumulative Index to Nursing and Allied Health Literature, PsycINFO, The Cochrane Library and Scopus. Included studies will be quality appraised using the Quality in Prognostic Studies tool developed by the Cochrane Prognosis Methods Group. A narrative synthesis of findings will be undertaken and meta-analyses considered, if appropriate. This protocol adheres to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols 2015 guidelines, ensuring the quality of the review. Conclusions: Understanding the influence of multimorbidity on mortality and other health-related outcomes in RA will provide an important basis of knowledge with the potential to improve future clinical management of RA. PROSPERO registration number: CRD42019137756

Impact of multimorbidity count on all-cause mortality and glycaemic outcomes in people with type 2 diabetes: a systematic review protocol

Introduction: Type 2 diabetes (T2D) is a leading health priority worldwide. Multimorbidity (MM) is a term describing the co-occurrence of two or more chronic diseases or conditions. The majority of people living with T2D have MM. The relationship between MM and mortality and glycaemia in people with T2D is not clear. Methods and analysis: Medline, Embase, Cumulative Index of Nursing and Allied Health Complete, The Cochrane Library, and SCOPUS will be searched with a prespecified search strategy. The searches will be limited to quantitative empirical studies in English with no restriction on publication date. One reviewer will perform title screening and two review authors will independently screen the abstract and full texts using Covidence software, with disagreements adjudicated by a third reviewer. Data will be extracted using a using a Population, Exposure, Comparator and Outcomes framework. Two reviewers will independently extract data and undertake the risk of bias (quality) assessment. Disagreements will be resolved by consensus. A narrative synthesis of the results will be conducted and meta-analysis considered if appropriate. Quality appraisal will be undertaken using the Newcastle-Ottawa quality assessment scale and the quality of the cumulative evidence of the included studies will be assessed using the Grading of Recommendations, Assessment, Development and Evaluation approach. This protocol was prepared in adherence to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols guidelines to ensure the quality of our review. Ethics and dissemination: This review will synthesise the existing evidence about the impact of MM on mortality and glycaemic outcomes in people living with T2D and increase our understanding of this subject and will inform future practice and policy. Findings will be disseminated via conference presentations, social media and peer-reviewed publication

Multimorbidity in stroke

No abstract available