Heterodimerization of the human RNase P/MRP subunits Rpp20 and Rpp25 is a prerequisite for interaction with the P3 arm of RNase MRP RNA

Rpp20 and Rpp25 are two key subunits of the human endoribonucleases RNase P and MRP. Formation of an Rpp20–Rpp25 complex is critical for enzyme function and sub-cellular localization. We present the first detailed in vitro analysis of their conformational properties, and a biochemical and biophysical characterization of their mutual interaction and RNA recognition. This study specifically examines the role of the Rpp20/Rpp25 association in the formation of the ribonucleoprotein complex. The interaction of the individual subunits with the P3 arm of the RNase MRP RNA is revealed to be negligible whereas the 1:1 Rpp20:Rpp25 complex binds to the same target with an affinity of the order of nM. These results unambiguously demonstrate that Rpp20 and Rpp25 interact with the P3 RNA as a heterodimer, which is formed prior to RNA binding. This creates a platform for the design of future experiments aimed at a better understanding of the function and organization of RNase P and MRP. Finally, analyses of interactions with deletion mutant proteins constructed with successively shorter N- and C-terminal sequences indicate that the Alba-type core domain of both Rpp20 and Rpp25 contains most of the determinants for mutual association and P3 RNA recognition

EGR1 controls divergent cellular responses of distinctive nucleus pulposus cell types

Background Immediate early genes (IEGs) encode transcription factors which serve as first line response modules to altered conditions and mediate appropriate cell responses. The immediate early response gene EGR1 is involved in physiological adaptation of numerous different cell types. We have previously shown a role for EGR1 in controlling processes supporting chondrogenic differentiation. We recently established a unique set of phenotypically distinct cell lines from the human nucleus pulposus (NP). Extensive characterization showed that these NP cellular subtypes represented progenitor-like cell types and more functionally mature cells. Methods To further understanding of cellular heterogeneity in the NP, we analyzed the response of these cell subtypes to anabolic and catabolic factors. Here, we test the hypothesis that physiological responses of distinct NP cell types are mediated by EGR1 and reflect specification of cell function using an RNA interference-based experimental approach. Results We show that distinct NP cell types rapidly induce EGR1 exposure to either growth factors or inflammatory cytokines. In addition, we show that mRNA profiles induced in response to anabolic or catabolic conditions are cell type specific: the more mature NP cell type produced a strong and more specialized transcriptional response to IL-1β than the NP progenitor cells and aspects of this response were controlled by EGR1. Conclusions Our current findings provide important substantiation of differential functionality among NP cellular subtypes. Additionally, the data shows that early transcriptional programming initiated by EGR1 is essentially restrained by the cells’ epigenome as it was determined during development and differentiation. These studies begin to define functional distinctions among cells of the NP and will ultimately contribute to defining functional phenotypes within the adult intervertebral disc.info:eu-repo/semantics/publishedVersio

Current practice in bicistronic ires reporter use: A systematic review

Bicistronic reporter assays have been instrumental for transgene expression, understanding of internal ribosomal entry site (IRES) translation, and identification of novel cap-independent translational elements (CITE). We observed a large methodological variability in the use of bicistronic reporter assays and data presentation or normalization procedures. Therefore, we systematically searched the literature for bicistronic IRES reporter studies and analyzed methodological details, data visualization, and normalization procedures. Two hundred fifty-seven publications were identified using our search strategy (published 1994–2020). Experimental studies on eukaryotic adherent cell systems and the cell-free translation assay were included for further analysis. We evaluated the following methodological details for 176 full text articles: the bicistronic reporter design, the cell line or type, transfection methods, and time point of analyses post-transfection. For the cell-free translation assay, we focused on methods of in vitro transcription, type of translation lysate, and incubation times and assay temperature. Data can be presented in multiple ways: raw data from individual cistrons, a ratio of the two, or fold changes thereof. In addition, many different control experiments have been suggested when studying IRES-mediated translation. In addition, many different normalization and control experiments have been suggested when studying IRES-mediated translation. Therefore, we also categorized and summarized their use. Our unbiased analyses provide a representative overview of bicistronic IRES reporter use. We identified parameters that were reported inconsistently or incompletely, which could hamper data reproduction and interpretation. On the basis of our analyses, we encourage adhering to a number of practices that should improve transparency of bicistronic reporter data presentation and improve methodological descriptions to facilitate data replication

Gene expression signatures of synovial fluid multipotential stromal cells (MSCs) in advanced knee osteoarthritis and following knee joint distraction

Osteoarthritis (OA) is the most common musculoskeletal disorder. Although joint replacement remains the standard of care for knee OA patients, knee joint distraction (KJD), which works by temporarily off-loading the joint for 6–8 weeks, is becoming a novel joint-sparing alternative for younger OA sufferers. The biological mechanisms behind KJD structural improvements remain poorly understood but likely involve joint-resident regenerative cells including multipotent stromal cells (MSCs). In this study, we hypothesized that KJD leads to beneficial cartilage-anabolic and anti-catabolic changes in joint-resident MSCs and investigated gene expression profiles of synovial fluid (SF) MSCs following KJD as compared with baseline. To obtain further insights into the effects of local biomechanics on MSCs present in late OA joints, SF MSC gene expression was studied in a separate OA arthroplasty cohort and compared with subchondral bone (SB) MSCs from medial (more loaded) and lateral (less loaded) femoral condyles from the same joints. In OA arthroplasty cohort (n = 12 patients), SF MSCs expressed lower levels of ossification- and hypotrophy-related genes [bone sialoprotein (IBSP), parathyroid hormone 1 receptor (PTH1R), and runt-related transcription factor 2 (RUNX2)] than did SB MSCs. Interestingly, SF MSCs expressed 5- to 50-fold higher levels of transcripts for classical extracellular matrix turnover molecules matrix metalloproteinase 1 (MMP1), a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), and tissue inhibitor of metalloproteinase-3 (TIMP3), all (p < 0.05) potentially indicating greater cartilage remodeling ability of OA SF MSCs, compared with SB MSCs. In KJD cohort (n = 9 patients), joint off-loading resulted in sustained, significant increase in SF MSC colonies’ sizes and densities and a notable transcript upregulation of key cartilage core protein aggrecan (ACAN) (weeks 3 and 6), as well as reduction in pro-inflammatory C–C motif chemokine ligand 2 (CCL2) expression (weeks 3 and 6). Additionally, early KJD changes (week 3) were marked by significant increases in MSC chondrogenic commitment markers gremlin 1 (GREM1) and growth differentiation factor 5 (GDF5). In combination, our results reveal distinct transcriptomes on joint-resident MSCs from different biomechanical environments and show that 6-week joint off-loading leads to transcriptional changes in SF MSCs that may be beneficial for cartilage regeneration. Biomechanical factors should be certainly considered in the development of novel MSC-based therapies for OA

The Longitudinal Assessment of Osteomyelitis Development by Molecular Imaging in a Rabbit Model

Introduction. Osteomyelitis is a severe orthopaedic complication which is difficult to diagnose and treat. Previous experimental studies mainly focussed on evaluating osteomyelitis in the presence of an implant or used a sclerosing agent to promote infection onset. In contrast, we focused on the longitudinal assessment of a nonimplant related osteomyelitis. Methods. An intramedullary tibial infection with S. aureus was established in NZW rabbits. Clinical and haematological infection status was evaluated weekly, combined with X-ray radiographs, biweekly injections of calcium binding fluorophores, and postmortem micro-CT. The development of the infection was assessed by micro-PET at consecutive time points using 18F-FDG as an infection tracer. Results. The intramedullary contamination of the rabbit tibia resulted in an osteomyelitis. Haematological parameters confirmed infection in mainly the first postoperative weeks (CRP at the first 5 postoperative weeks, leucocyte differentiation at the second and sixth postoperative weeks, and ESR on the second postoperative week only), while micro-PET was able to detect the infection from the first post-operative week onward until the end of the study. Conclusions. This study shows that osteomyelitis in the rabbit can be induced without use of an implant or sclerosing agent. The sequential follow-up indicates that the diagnostic value of each infection parameter is time point dependant. Furthermore, from all parameters used, the diagnostic value of  18F-FDG micro-PET is the most versatile to assess the presence of an orthopaedic infection in this model

Viperin mRNA is a novel target for the human RNase MRP/RNase P endoribonuclease

RNase MRP is a conserved endoribonuclease, in humans consisting of a 267-nucleotide RNA associated with 7–10 proteins. Mutations in its RNA component lead to several autosomal recessive skeletal dysplasias, including cartilage-hair hypoplasia (CHH). Because the known substrates of mammalian RNase MRP, pre-ribosomal RNA, and RNA involved in mitochondrial DNA replication are not likely involved in CHH, we analyzed the effects of RNase MRP (and the structurally related RNase P) depletion on mRNAs using DNA microarrays. We confirmed the upregulation of the interferon-inducible viperin mRNA by RNAi experiments and this appeared to be independent of the interferon response. We detected two cleavage sites for RNase MRP/RNase P in the coding sequence of viperin mRNA. This is the first study providing direct evidence for the cleavage of a mRNA by RNase MRP/RNase P in human cells. Implications for the involvement in the pathophysiology of CHH are discussed