The role of the yeast cleavage and polyadenylation factor subunit Ydh1p/Cft2p in pre‐mRNA 3′‐end formation

Cleavage and polyadenylation factor (CPF) is a multi‐protein complex that functions in pre‐mRNA 3′‐end formation and in the RNA polymerase II (RNAP II) transcription cycle. Ydh1p/Cft2p is an essential component of CPF but its precise role in 3′‐end processing remained unclear. We found that mutations in YDH1 inhibited both the cleavage and the polyadenylation steps of the 3′‐end formation reaction in vitro. Recently, we demonstrated that an important function of CPF lies in the recognition of poly(A) site sequences and RNA binding analyses suggesting that Ydh1p/Cft2p interacts with the poly(A) site region. Here we show that mutant ydh1 strains are deficient in the recognition of the ACT1 cleavage site in vivo. The C‐terminal domain (CTD) of RNAP II plays a major role in coupling 3′‐end processing and transcription. We provide evidence that Ydh1p/Cft2p interacts with the CTD of RNAP II, several other subunits of CPF and with Pcf11p, a component of CF IA. We propose that Ydh1p/Cft2p contributes to the formation of important interaction surfaces that mediate the dynamic association of CPF with RNAP II, the recognition of poly(A) site sequences and the assembly of the polyadenylation machinery on the RNA substrat

Anaemia is associated with higher disease activity in axial spondyloarthritis but is not an independent predictor of spinal radiographic progression: data from the Swiss Clinical Quality Management Registry

OBJECTIVE As anaemia represents a biomarker for increased radiographic damage in rheumatoid arthritis, we aimed to investigate whether it independently predicts spinal radiographic progression in axial spondyloarthritis (axSpA). METHODS AxSpA patients with available haemoglobin levels from the prospective Swiss Clinical Quality Management Registry were included for comparison of patients with and without anaemia. Spinal radiographic progression was assessed according to the modified Stoke Ankylosing Spondylitis Spinal Score (mSASSS) in patients with ankylosing spondylitis (AS) if ≥ 2 sets of spinal radiographs were available every 2 years. The relationship between anaemia and progression (defined as an increase ≥ 2 mSASSS units in 2 years) was analysed with generalized estimating equation models after adjustment for the Ankylosing Spondylitis Disease Activity Score (ASDAS) and potential confounding, as well as after multiple imputations of missing values. RESULTS A total of 212/2522 axSpA patients presented with anaemia (9%). Anaemic patients had higher clinical disease activity, higher acute phase reactants and more severe impairments in physical function, mobility and quality of life. In the subgroup of patients with AS (N = 433), a comparable mSASSS progression was found in anaemic and non-anaemic patients (OR 0.69, 95% CI 0.25 to 1.96, p = 0.49). Age, male sex, baseline radiographic damage and ASDAS were associated with enhanced progression. The results were confirmed in complete case analyses and with progression defined as the formation of ≥ 1 syndesmophyte in 2 years. CONCLUSION Although anaemia was associated with higher disease activity in axSpA, it did not additionally contribute to the prediction of spinal radiographic progression. Key Points • Anaemia is associated with higher disease activity and more severely impaired physical function, mobility and quality of life in axSpA. • Anaemia does not provide an additional value to ASDAS for prediction of spinal radiographic progression

Anaemia is associated with higher disease activity in axial spondyloarthritis but is not an independent predictor of spinal radiographic progression: data from the Swiss Clinical Quality Management Registry.

OBJECTIVE As anaemia represents a biomarker for increased radiographic damage in rheumatoid arthritis, we aimed to investigate whether it independently predicts spinal radiographic progression in axial spondyloarthritis (axSpA). METHODS AxSpA patients with available haemoglobin levels from the prospective Swiss Clinical Quality Management Registry were included for comparison of patients with and without anaemia. Spinal radiographic progression was assessed according to the modified Stoke Ankylosing Spondylitis Spinal Score (mSASSS) in patients with ankylosing spondylitis (AS) if ≥ 2 sets of spinal radiographs were available every 2 years. The relationship between anaemia and progression (defined as an increase ≥ 2 mSASSS units in 2 years) was analysed with generalized estimating equation models after adjustment for the Ankylosing Spondylitis Disease Activity Score (ASDAS) and potential confounding, as well as after multiple imputations of missing values. RESULTS A total of 212/2522 axSpA patients presented with anaemia (9%). Anaemic patients had higher clinical disease activity, higher acute phase reactants and more severe impairments in physical function, mobility and quality of life. In the subgroup of patients with AS (N = 433), a comparable mSASSS progression was found in anaemic and non-anaemic patients (OR 0.69, 95% CI 0.25 to 1.96, p = 0.49). Age, male sex, baseline radiographic damage and ASDAS were associated with enhanced progression. The results were confirmed in complete case analyses and with progression defined as the formation of ≥ 1 syndesmophyte in 2 years. CONCLUSION Although anaemia was associated with higher disease activity in axSpA, it did not additionally contribute to the prediction of spinal radiographic progression. Key Points • Anaemia is associated with higher disease activity and more severely impaired physical function, mobility and quality of life in axSpA. • Anaemia does not provide an additional value to ASDAS for prediction of spinal radiographic progression

Subclinical giant cell arteritis in new onset polymyalgia rheumatica:A systematic review and meta-analysis of individual patient data

Objectives: To determine the prevalence and predictors of subclinical giant cell arteritis (GCA) in patients with newly diagnosed polymyalgia rheumatica (PMR). Methods: PubMed, Embase, and Web of Science Core Collection were systematically searched (date of last search July 14, 2021) for any published information on any consecutively recruited cohort reporting the prevalence of GCA in steroid-naïve patients with PMR without cranial or ischemic symptoms. We combined prevalences across populations in a random-effect meta-analysis. Potential predictors of subclinical GCA were identified by mixed-effect logistic regression using individual patient data (IPD) from cohorts screened with PET/(CT). Results: We included 13 cohorts with 566 patients from studies published between 1965 to 2020. Subclinical GCA was diagnosed by temporal artery biopsy in three studies, ultrasound in three studies, and PET/(CT) in seven studies. The pooled prevalence of subclinical GCA across all studies was 23% (95% CI 14%-36%, I2=84%) for any screening method and 29% in the studies using PET/(CT) (95% CI 13%-53%, I2=85%) (n=266 patients). For seven cohorts we obtained IPD for 243 patients screened with PET/(CT). Inflammatory back pain (OR 2.73, 1.32-5.64), absence of lower limb pain (OR 2.35, 1.05-5.26), female sex (OR 2.31, 1.17-4.58), temperature >37° (OR 1.83, 0.90-3.71), weight loss (OR 1.83, 0.96-3.51), thrombocyte count (OR 1.51, 1.05-2.18), and haemoglobin level (OR 0.80, 0.64-1.00) were most strongly associated with subclinical GCA in the univariable analysis but not C-reactive protein (OR 1.00, 1.00-1.01) or erythrocyte sedimentation rate (OR 1.01, 1.00-1.02). A prediction model calculated from these variables had an area under the curve of 0.66 (95% CI 0.55-0.75). Conclusion: More than a quarter of patients with PMR may have subclinical GCA. The prediction model from the most extensive IPD set has only modest diagnostic accuracy. Hence, a paradigm shift in the assessment of PMR patients in favour of implementing imaging studies should be discussed

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

© 2024 The Authors. Journal of Extracellular Vesicles, published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY), https://creativecommons.org/licenses/by/4.0/Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly.Peer reviewe

EULAR recommendations for the management of rheumatoid arthritis with synthetic and biological disease-modifying antirheumatic drugs: 2016 update

Recent insights in rheumatoid arthritis (RA) necessitated updating the European League Against Rheumatism (EULAR) RA management recommendations. A large international Task Force based decisions on evidence from 3 systematic literature reviews, developing 4 overarching principles and 12 recommendations (vs 3 and 14, respectively, in 2013). The recommendations address conventional synthetic (cs) disease-modifying antirheumatic drugs (DMARDs) (methotrexate (MTX), leflunomide, sulfasalazine); glucocorticoids (GC); biological (b) DMARDs (tumour necrosis factor (TNF)-inhibitors (adalimumab, certolizumab pegol, etanercept, golimumab, infliximab), abatacept, rituximab, tocilizumab, clazakizumab, sarilumab and sirukumab and biosimilar (bs) DMARDs) and targeted synthetic (ts) DMARDs (Janus kinase (Jak) inhibitors tofacitinib, baricitinib). Monotherapy, combination therapy, treatment strategies (treat-to-target) and the targets of sustained clinical remission (as defined by the American College of Rheumatology-(ACR)-EULAR Boolean or index criteria) or low disease activity are discussed. Cost aspects were taken into consideration. As first strategy, the Task Force recommends MTX (rapid escalation to 25 mg/week) plus short-term GC, aiming at >50% improvement within 3 and target attainment within 6 months. If this fails stratification is recommended. Without unfavourable prognostic markers, switching to—or adding—another csDMARDs (plus short-term GC) is suggested. In the presence of unfavourable prognostic markers (autoantibodies, high disease activity, early erosions, failure of 2 csDMARDs), any bDMARD (current practice) or Jak-inhibitor should be added to the csDMARD. If this fails, any other bDMARD or tsDMARD is recommended. If a patient is in sustained remission, bDMARDs can be tapered. For each recommendation, levels of evidence and Task Force agreement are provided, both mostly very high. These recommendations intend informing rheumatologists, patients, national rheumatology societies, hospital officials, social security agencies and regulators about EULAR's most recent consensus on the management of RA, aimed at attaining best outcomes with current therapies

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its ‘Minimal Information for Studies of Extracellular Vesicles’, which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly

Characterization of factors involved in the coupling of 3' end processing and splicing and in the 3' end formation of mRNA precursors

Eukaryotic mRNA precursors are processed at their 5’ and 3’ ends and are spliced prior to their export from the nucleus to the cytoplasm. Although all three processing reactions can be studied separately in vitro, they are coupled in vivo. 3’ end processing of most mammalian pre-mRNAs involves endonucleolytic cleavage followed by polyadenylation of the upstream cleavage product. Cleavage and polyadenylation specificity factor (CPSF) is a multiprotein complex, which together with cleavage factor Im and IIm (CF Im, CF IIm), cleavage stimulatory factor (CstF), poly(A) polymerase (PAP) and nuclear poly(A) binding protein 1 (PABPN1) is required for 3’ end formation. We have found that the U2 snRNA and subunits of the splicing factors 3a and 3b (SF3a, SF3b), which are components of the U2 snRNP, were also present in highly purified CPSF fractions. GST pull-down experiments indicated a direct interaction of CPSF subunits with SF3b49 and SF3b130. Furthermore, antibodies directed against CPSF100 co-immunoprecipitated subunits of SF3a and SF3b and the U2 snRNA Taken together our results show that subunits of CPSF and the U2 snRNP directly interact with each other. In order to analyze whether this interaction plays a role in the coupling of 3’ end processing and splicing, we depleted CPSF subunits from HeLa cell nuclear extract and tested the extracts for splicing activity. CPSF100-depleted extract showed no detectable cleavage activity and its splicing activity was significantly reduced in coupled assays but not in un-coupled assays. Moreover, pre-mRNAs containing mutations in the binding site of SF3b were not only less efficiently spliced but they also showed reduced cleavage activity. Interestingly, efficient cleavage required the presence of the U2 snRNA in coupled but not in un-coupled assays. Based on our studies, we propose that the interactions between CPSF and U2 snRNP contribute to the coupling of splicing and 3’ end formation. Furthermore, we depleted CPSF100 and the U2 snRNP subunits SF3b155 and SF3b130 by means of RNAi. We observed that knock down of both SF3b proteins caused high lethality of the cells indicating that these polypeptides are essential. However, depletion of CPSF100 did not result in a significant increase in cell mortality, suggesting that the protein is either not essential that the knock down was not efficient enough to result in cell lethality or that CPSF100 shares redundant functions with another protein. We were able to show that SF3b155 and SF3b130 are required for efficient splicing in vivo but did not detect a splicing deficiency in CPSF100 depleted cells. Knock down of neither of the proteins resulted in an observable 3’ end processing deficiency. Further work is required to address the question if the U2 snRNP and CPSF couple splicing and 3’ end processing in vivo. Splicing and 3’ end formation are highly conserved mechanisms from mammals to yeast and the two organisms share homologues of most of the proteins involved in the two reactions. To test whether the coupling mechanism mediated by CPSF and the U2 snRNP is conserved between different organisms, we focused on the yeast system. The essential protein Rse1p is the yeast homolog of SF3b130. We show that the rse1-1 strain is sensitive to cordycepin, which suggests that Rse1p might be involved in 3’ end processing. Furthermore, Rse1p and 3’ end processing factors interacted genetically and Northern blot analysis suggested that strains carrying mutations in Rse1p and subunits of CPF had increased levels of unspliced pre-mRNA at restrictive temperature compared to the single mutants. We therefore suggest that the coupling of 3’ end processing and splicing mediated by CPSF and U2 snRNP is conserved between mammals and yeast. Precursor tRNAs (pre-tRNAs) must undergo a number of processing steps before they become mature tRNAs and some tRNAs contain introns. tRNA splicing is a three step reaction and each step requires an individual set of proteins. In the first step the pretRNA is cut at its two splice sites. This reaction is catalyzed by the so called tRNA splicing endonuclease complex. Recently this complex was purified from mammalian cells and interestingly hClp1 (a subunit of the 3’ end processing factor CF IIm) was identified as one of its components. Furthermore, hSen2 a subunit of the endonuclease complex was shown to be required for efficient 3’ end processing in vivo. A model was proposed suggesting that the tRNA endonuclease complex is involved in 3’ end processing. In collaboration with S. Paushkin and C. Trotta we continued to investigate if this model is indeed correct. We found that biochemically purified CF IIm indeed carried tRNA endonuclease activity. However, tRNA endonuclease complexes were not able to reconstitute cleavage activity of CF IIm-depleted HeLa nuclear extract, unless they were purified with His-Flag tagged hClp1. Taken all our results into account we cannot exclude that the tRNA endonuclease complex is indeed involved in 3’ end processing. However, we believe that the evidence supporting this model is rather weak. We think it is more likely that hClp1 is part of the tRNA endonuclease complex as well as a subunit of CF IIm, and that the two complexes are not functionally related. As mentioned earlier, 3’ end processing is highly conserved form mammals to yeast. In S. cerevisiae cleavage and polyadenylation factor (CPF) is a multiprotein complex, which together with the cleavage factor IA (CF IA) and the cleavage factor IB (CF IB) is required for both the cleavage and the polyadenylation step of the 3’ end formation reaction. Ydh1p/Cft2p is an essential component of CPF. Cleavage and polyadenylation reactions revealed that the protein is required for both reactions to occur in vitro. Previously, it was demonstrated that an important function of CPF lies in the recognition of poly(A) site sequences and previous RNA binding analyses with recombinant Ydh1p/Cft2p suggested that the protein may interact with the CYC1 poly(A) site region. In accordance, we found that mutant ydh1 strains were deficient in recognition of the ACT1 cleavage site in vivo. Transcription by RNA polymerase II (RNAP II) and 3’ end processing reactions are tightly linked. The C-terminal domain (CTD) of RNAP II plays a major role in coupling the two events, as it tethers the factors involved in polyadenylation to the polymerase. We provide evidence that Ydh1p/Cft2p interacts with the CTD, several subunits of CPF and with Pcf11p, a component of CF IA. We propose that Ydh1p/Cft2p contributes to the formation of important interaction surfaces that mediate the dynamic association of CPF with RNAP II, the recognition of poly(A) site sequences and the assembly of the polyadenylation machinery on the RNA substrate

The role of the yeast cleavage and polyadenylation factor subunit Ydh1p/Cft2p in pre-mRNA 3′-end formation

Cleavage and polyadenylation factor (CPF) is a multi-protein complex that functions in pre-mRNA 3′-end formation and in the RNA polymerase II (RNAP II) transcription cycle. Ydh1p/Cft2p is an essential component of CPF but its precise role in 3′-end processing remained unclear. We found that mutations in YDH1 inhibited both the cleavage and the polyadenylation steps of the 3′-end formation reaction in vitro. Recently, we demonstrated that an important function of CPF lies in the recognition of poly(A) site sequences and RNA binding analyses suggesting that Ydh1p/Cft2p interacts with the poly(A) site region. Here we show that mutant ydh1 strains are deficient in the recognition of the ACT1 cleavage site in vivo. The C-terminal domain (CTD) of RNAP II plays a major role in coupling 3′-end processing and transcription. We provide evidence that Ydh1p/Cft2p interacts with the CTD of RNAP II, several other subunits of CPF and with Pcf11p, a component of CF IA. We propose that Ydh1p/Cft2p contributes to the formation of important interaction surfaces that mediate the dynamic association of CPF with RNAP II, the recognition of poly(A) site sequences and the assembly of the polyadenylation machinery on the RNA substrate

Early axial spondyloarthritis according to the ASAS consensus definition: characterisation of patients and effectiveness of a first TNF inhibitor in a large observational registry

OBJECTIVE To characterise the population fulfilling the Assessment of SpondyloArthritis international Society (ASAS) consensus definition of early axial spondyloarthritis (axSpA) and to determine the effectiveness of a first tumour necrosis factor inhibitor (TNFi) in early versus established axSpA in a large observational registry. METHODS A total of 3064 patients with axSpA in the Swiss Clinical Quality Management registry with data on duration of axial symptoms were included (≤2 years=early axSpA, N=658; >2 years=established axSpA, N=2406). Drug retention was analysed in patients starting a first TNFi in early axSpA (N=250) versus established axSpA (N=874) with multiple-adjusted Cox proportional hazards models. Adjusted logistic regression analyses were used to determine the achievement of the ASAS criteria for 40% improvement (ASAS40) at 1 year. RESULTS Sex distribution, disease activity, impairments of function and health-related quality of life were comparable between patients with early and established axSpA. Patients with established disease were older, had more prevalent axial radiographical damage and had a higher impairment of mobility. A comparable TNFi retention was found in early versus established disease after adjustment for age, sex, human leucocyte antigen-B27 status, education, body mass index, smoking, elevated C reactive protein and sacroiliac inflammation on MRI (HR 1.05, 95% CI 0.78 to 1.42). The adjusted ASAS40 response was similar in the two groups (OR 1.09, 95% CI 0.67 to 1.78). Results were confirmed in the population fulfilling the ASAS classification criteria. CONCLUSION Considering the recent ASAS definition of early axSpA, TNFi effectiveness seems comparable in early versus established disease