Convergence of multiple RNA-silencing pathways on GW182/TNRC6.

The RNA-binding protein TRIM71/LIN-41 is a phylogenetically conserved developmental regulator that functions in mammalian stem cell reprogramming, brain development, and cancer. TRIM71 recognizes target mRNAs through hairpin motifs and silences them through molecular mechanisms that await identification. Here, we uncover that TRIM71 represses its targets through RNA-supported interaction with TNRC6/GW182, a core component of the miRNA-induced silencing complex (miRISC). We demonstrate that AGO2, TRIM71, and UPF1 each recruit TNRC6 to specific sets of transcripts to silence them. As cellular TNRC6 levels are limiting, competition occurs among the silencing pathways, such that the loss of AGO proteins or of AGO binding to TNRC6 enhances the activities of the other pathways. We conclude that a miRNA-like silencing activity is shared among different mRNA silencing pathways and that the use of TNRC6 as a central hub provides a means to integrate their activities.

The RNA hairpin binder TRIM71 modulates alternative splicing by repressing MBNL1.

TRIM71/LIN-41, a phylogenetically conserved regulator of development, controls stem cell fates. Mammalian TRIM71 exhibits both RNA-binding and protein ubiquitylation activities, but the functional contribution of either activity and relevant primary targets remain poorly understood. Here, we demonstrate that TRIM71 shapes the transcriptome of mouse embryonic stem cells (mESCs) predominantly through its RNA-binding activity. We reveal that TRIM71 binds targets through 3' untranslated region (UTR) hairpin motifs and that it acts predominantly by target degradation. TRIM71 mutations implicated in etiogenesis of human congenital hydrocephalus impair target silencing. We identify a set of primary targets consistently regulated in various human and mouse cell lines, including MBNL1 (Muscleblind-like protein 1). MBNL1 promotes cell differentiation through regulation of alternative splicing, and we demonstrate that TRIM71 promotes embryonic splicing patterns through MBNL1 repression. Hence, repression of MBNL1-dependent alternative splicing may contribute to TRIM71's function in regulating stem cell fates.

HbA1c-dependent projection of long-term renal outcomes.

BACKGROUND: Diabetes mellitus is a major risk factor for the development of chronic kidney disease (CKD). There is limited data addressing the value of glycated hemoglobin (HbA1c) to predict renal outcomes independent of diabetes status. METHODS: This single-center retrospective observational study presents data of 19,285 subjects, irrespective of initial CKD or diabetes status. The primary endpoint was defined as the time to manifestation of moderate CKD (estimated glomerular filtration rate [eGFR] <60 mL/min/1.73 m2 ) in subjects with eGFR ≥60 mL/min/1.73 m2 at baseline. The secondary endpoint was defined as time to progression of CKD (eGFR <30 mL/min/1.73 m2 ) in subjects with eGFR 30-60 mL/min/1.73 m2 . Multivariate time-to-event and logistic regression models were applied to estimate the influences of HbA1c, sex, age, eGFR, triglycerides, and cholesterol on both endpoints. RESULTS: Lowest baseline HbA1c levels were associated with the slowest decline of kidney function (median time to manifestation of moderate CKD for HbA1c <5.7%: 15.9 years [95% confidence interval (CI): 15.2-16.7]; for HbA1c 5.7%-6.5%: 14.5 years [95% CI: 14.0-15.1]; for HbA1c 6.5%-8.5%: 11.1 years [95% CI: 10.4-11.7]; for HbA1c >8.5%: 8.3 years [95% CI: 7.8-9.2]; p < 0.001). Similar results were observed for the secondary endpoint. Covariate-adjusted time-to-event analysis demonstrated an almost linear correlation between continuous baseline HbA1c levels and the probabilities of reaching both endpoints. CONCLUSIONS: HbA1c levels are a strong predictor for eGFR decline, irrespective of diabetes status or CKD stage, demonstrating a tight concentration-dependent relationship. This association becomes apparent in the prediabetic HbA1c range and remains constant over the entire HbA1c spectrum.

Mutual regulation of tumour vessel normalization and immunostimulatory reprogramming.

Blockade of angiogenesis can retard tumour growth, but may also paradoxically increase metastasis. This paradox may be resolved by vessel normalization, which involves increased pericyte coverage, improved tumour vessel perfusion, reduced vascular permeability, and consequently mitigated hypoxia. Although these processes alter tumour progression, their regulation is poorly understood. Here we show that type 1 T helper (TH1) cells play a crucial role in vessel normalization. Bioinformatic analyses revealed that gene expression features related to vessel normalization correlate with immunostimulatory pathways, especially T lymphocyte infiltration or activity. To delineate the causal relationship, we used various mouse models with vessel normalization or T lymphocyte deficiencies. Although disruption of vessel normalization reduced T lymphocyte infiltration as expected, reciprocal depletion or inactivation of CD4+ T lymphocytes decreased vessel normalization, indicating a mutually regulatory loop. In addition, activation of CD4+ T lymphocytes by immune checkpoint blockade increased vessel normalization. TH1 cells that secrete interferon-γ are a major population of cells associated with vessel normalization. Patient-derived xenograft tumours growing in immunodeficient mice exhibited enhanced hypoxia compared to the original tumours in immunocompetent humans, and hypoxia was reduced by adoptive TH1 transfer. Our findings elucidate an unexpected role of TH1 cells in vasculature and immune reprogramming. TH1 cells may be a marker and a determinant of both immune checkpoint blockade and anti-angiogenesis efficacy.

Clinical covariates influencing clinical outcomes in primary membranous nephropathy.

BACKGROUND: Primary membranous nephropathy (PMN) frequently causes nephrotic syndrome and declining kidney function. Disease progression is likely modulated by patient-specific and therapy-associated factors awaiting characterization. These cofactors may facilitate identification of risk groups and could result in more individualized therapy recommendations. METHODS: In this single-center retrospective observational study, we analyze the effect of patient-specific and therapy-associated covariates on proteinuria, hypoalbuminemia, and estimated glomerular filtration rate (eGFR) in 74 patients diagnosed with antibody positive PMN and nephrotic-range proteinuria (urine-protein-creatinine-ratio [UPCR] ≥ 3.5 g/g), treated at the University of Freiburg Medical Center between January 2000 - November 2022. The primary endpoint was defined as time to proteinuria / serum-albumin response (UPCR ≤ 0.5 g/g or serum-albumin ≥ 3.5 g/dl), the secondary endpoint as time to permanent eGFR decline (≥ 40% relative to baseline). RESULTS: The primary endpoint was reached after 167 days. The secondary endpoint was reached after 2413 days. Multivariate time-to-event analyses showed significantly faster proteinuria / serum-albumin response for higher serum-albumin levels (HR 2.7 [95% CI: 1.5 - 4.8]) and cyclophosphamide treatment (HR 3.6 [95% CI: 1.3 - 10.3]). eGFR decline was significantly faster in subjects with old age at baseline (HR 1.04 [95% CI: 1 - 1.1]). CONCLUSION: High serum-albumin levels, and treatment with cyclophosphamide are associated with faster proteinuria reduction and/or serum-albumin normalization. Old age constitutes a risk factor for eGFR decline in subjects with PMN.

Eculizumab as a treatment for C3 glomerulopathy: a single-center retrospective study.

BACKGROUND: C3 Glomerulopathy (C3G) is a rare glomerular disease caused by dysregulation of the complement pathway. Based on its pathophysiology, treatment with the monoclonal antibody eculizumab targeting complement C5 may be a therapeutic option. Due to the rarity of the disease, observational data on the clinical response to eculizumab treatment is scarce. METHODS: Fourteen patients (8 female, 57%) treated for C3 glomerulopathy at the medical center of the University of Freiburg between 2013 and 2022 were included. Subjects underwent biopsy before enrollment. Histopathology, clinical data, and response to eculizumab treatment were analyzed. Key parameters to determine the primary outcome were changes of estimated glomerular filtration rate (eGFR) over time. Positive outcome was defined as > 30% increase, stable outcome as ±30%, negative outcome as decrease > 30% of eGFR. RESULTS: Eleven patients (78.8%) were treated with eculizumab, three received standard of care (SoC, 27.2%). Median follow-up time was 68 months (IQR: 45-98 months). Median eculizumab treatment duration was 10 months (IQR 5-46 months). After eculizumab treatment, five patients showed a stable outcome, six patients showed a negative outcome. Among patients receiving SoC, one patient showed a stable outcome, two patients showed a negative outcome. CONCLUSIONS: The benefit of eculizumab in chronic progressive C3 glomerulopathy is limited.

An autoinhibitory intramolecular interaction proof-reads RNA recognition by the essential splicing factor U2AF2.

The recognition of cis-regulatory RNA motifs in human transcripts by RNA binding proteins (RBPs) is essential for gene regulation. The molecular features that determine RBP specificity are often poorly understood. Here, we combined NMR structural biology with high-throughput iCLIP approaches to identify a regulatory mechanism for U2AF2 RNA recognition. We found that the intrinsically disordered linker region connecting the two RNA recognition motif (RRM) domains of U2AF2 mediates autoinhibitory intramolecular interactions to reduce nonproductive binding to weak Py-tract RNAs. This proofreading favors binding of U2AF2 at stronger Py-tracts, as required to define 3' splice sites at early stages of spliceosome assembly. Mutations that impair the linker autoinhibition enhance the affinity for weak Py-tracts result in promiscuous binding of U2AF2 along mRNAs and impact on splicing fidelity. Our findings highlight an important role of intrinsically disordered linkers to modulate RNA interactions of multidomain RBPs.

Response to SARS-CoV-2 vaccines in patients receiving B-cell modulating antibodies for renal autoimmune disease.

BACKGROUND: Effective SARS-CoV-2 vaccination in patients receiving treatment with B-cell depleting agents is challenging. Information on vaccination responses in these patients are a valuable tool to develop efficient vaccination regimens. METHODS: In this single-center retrospective observational study, we report the humoral and cellular response in 34 patients receiving anti-CD20 antibody treatment for renal immune disease. RESULTS: After base immunization with SARS-CoV-2-vaccines, 92.0% developed a cellular, 32.4% a humoral response. Humoral immunity correlated with B-cell counts and the timespan between anti-CD20 antibody treatment and vaccination. All patients with > 21/µl B-cells, or > 197 days after treatment showed humoral response. CONCLUSIONS: Adequate timing of SARS-CoV-2-vaccinations after anti-CD20 antibody treatment and CD19 measurements are crucial to generate immunity. Awaiting partial B-cell recovery by postponing regularly scheduled anti-CD20 treatment should be considered in patients with stable immune disease. TRIAL REGISTRATION: This study has been retrospectively registered in the German Clinical Trials Register (DRKS00027049) on 29/10/2021.

Noncompetitive binding of PpiD and YidC to the SecYEG translocon expands the global view on the SecYEG interactome in Escherichia coli.

The SecYEG translocon constitutes the major protein transport channel in bacteria and transfers an enormous variety of different secretory and inner-membrane proteins. The minimal core of the SecYEG translocon consists of three inner-membrane proteins, SecY, SecE, and SecG, which, together with appropriate targeting factors, are sufficient for protein transport in vitro However, in vivo the SecYEG translocon has been shown to associate with multiple partner proteins, likely allowing the SecYEG translocon to process its diverse substrates. To obtain a global view on SecYEG plasticity in Escherichia coli, here we performed a quantitative interaction proteomic analysis, which identified several known SecYEG-interacting proteins, verified the interaction of SecYEG with quality-control proteins, and revealed several previously unknown putative SecYEG-interacting proteins. Surprisingly, we found that the chaperone complex PpiD/YfgM is the most prominent interaction partner of SecYEG. Detailed analyses of the PpiD-SecY interaction by site-directed cross-linking revealed that PpiD and the established SecY partner protein YidC use almost completely-overlapping binding sites on SecY. Both PpiD and YidC contacted the lateral gate, the plug domain, and the periplasmic cavity of SecY. However, quantitative MS and cross-linking analyses revealed that despite having almost identical binding sites, their binding to SecY is noncompetitive. This observation suggests that the SecYEG translocon forms different substrate-independent subassemblies in which SecYEG either associates with YidC or with the PpiD/YfgM complex. In summary, the results of this study indicate that the PpiD/YfgM chaperone complex is a primary interaction partner of the SecYEG translocon.

Structural and Kinetic Profiling of Allosteric Modulation of Duplex DNA Induced by DNA-Binding Polyamide Analogues.

A combined structural and quantitative biophysical profile of the DNA binding affinity, kinetics and sequence-selectivity of hairpin polyamide analogues is described. DNA duplexes containing either target polyamide binding sites or mismatch sequences are immobilized on a microelectrode surface. Quantitation of the DNA binding profile of polyamides containing N-terminal 1-alkylimidazole (Im) units exhibit picomolar binding affinities for their target sequences, whereas 5-alkylthiazole (Nt) units are an order of magnitude lower (low nanomolar). Comparative NMR structural analyses of the polyamide series shows that the steric bulk distal to the DNA-binding face of the hairpin iPr-Nt polyamide plays an influential role in the allosteric modulation of the overall DNA duplex structure. This combined kinetic and structural study provides a foundation to develop next-generation hairpin designs where the DNA-binding profile of polyamides is reconciled with their physicochemical properties.