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Ribosome profiling (Ribo-Seq) is a highly sensitive method to quantify ribosome occupancies along individual mRNAs on a genome-wide scale. Hereby, ribosome-protected fragments (= footprints) are generated by nuclease digestion, isolated, and sequenced together with the corresponding randomly fragmented input samples, to determine ribosome densities (RD). For library preparation, equal amounts of total RNA are used. Subsequently, all transcript fragments are subjected to linker ligation, cDNA synthesis, and PCR amplification. Importantly, the number of reads obtained for every transcript in input and footprint samples during sequencing depends on sequencing depth and library size, as well as the relative abundance of the transcript in the sample. However, the information pertaining to the absolute amount of input and footprint sequences is lost during sample preparation, hence ruling out any conclusion whether translation is generally suppressed or activated in one condition over the other. Therefore, the RD fold-changes determined for individual genes do not reflect absolute regulation, but have to be interpreted as relative to bulk mRNA translation. Here, we modified the original ribosome profiling protocol that was first established by Ingolia et al. (2009), by adding small amounts of yeast lysate to the mammalian lysates of interest as a spike-in. This allows us to not only detect changes in the RD of specific transcripts relative to each other, but also to simultaneously measure global differences in RD (normalized ribosome density values) between samples. Graphic abstract: Global changes in translation efficiency can be detected with polysome profiling, where the proportion of polysomal ribosomes is interpreted as a proxy for ribosome density (RD) on bulk mRNA. Ribo-Seq measures changes in RD of specific mRNAs relative to bulk mRNA. The addition of a yeast-lysate, as a spike-in for normalization of read counts, allows for an absolute measurement of changes in RD., Competing Interests: Competing interestsThe authors declare no competing interests., (Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC.)

In general, mRNAs are assumed to be loaded with ribosomes instantly upon entry into the cytoplasm. To measure ribosome density (RD) on nascent mRNA, we developed nascent Ribo-Seq by combining Ribo-Seq with progressive 4-thiouridine labeling. In mouse macrophages, we determined experimentally the lag between the appearance of nascent mRNA and its association with ribosomes, which was calculated to be 20-22 min for bulk mRNA. In mouse embryonic stem cells, nRibo-Seq revealed an even stronger lag of 35-38 min in ribosome loading. After stimulation of macrophages with lipopolysaccharide, the lag between cytoplasmic and translated mRNA leads to uncoupling between input and ribosome-protected fragments, which gives rise to distorted RD measurements under conditions where mRNA amounts are far from steady-state expression. As a result, we demonstrate that transcriptional changes affect RD in a passive way., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The Dnmt2 enzyme utilizes the catalytic mechanism of eukaryotic DNA methyltransferases to methylate several tRNAs at cytosine 38. Dnmt2 mutant mice, flies, and plants were reported to be viable and fertile, and the biological function of Dnmt2 has remained elusive. Here, we show that endochondral ossification is delayed in newborn Dnmt2-deficient mice, which is accompanied by a reduction of the haematopoietic stem and progenitor cell population and a cell-autonomous defect in their differentiation. RNA bisulfite sequencing revealed that Dnmt2 methylates C38 of tRNA Asp(GTC), Gly(GCC), and Val(AAC), thus preventing tRNA fragmentation. Proteomic analyses from primary bone marrow cells uncovered systematic differences in protein expression that are due to specific codon mistranslation by tRNAs lacking Dnmt2-dependent methylation. Our observations demonstrate that Dnmt2 plays an important role in haematopoiesis and define a novel function of C38 tRNA methylation in the discrimination of near-cognate codons, thereby ensuring accurate polypeptide synthesis., (© 2015 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

For a rapid induction and efficient resolution of the inflammatory response, gene expression in cells of the immune system is tightly regulated at the transcriptional and post-transcriptional level. The control of mRNA translation has emerged as an important determinant of protein levels, yet its role in macrophage activation is not well understood. We systematically analyzed the contribution of translational regulation to the early phase of the macrophage response by polysome fractionation from mouse macrophages stimulated with lipopolysaccharide (LPS). Individual mRNAs whose translation is specifically regulated during macrophage activation were identified by microarray analysis. Stimulation with LPS for 1 h caused translational activation of many feedback inhibitors of the inflammatory response including NF-κB inhibitors (Nfkbid, Nfkbiz, Nr4a1, Ier3), a p38 MAPK antagonist (Dusp1) and post-transcriptional suppressors of cytokine expression (Zfp36 and Zc3h12a). Our analysis showed that their translation is repressed in resting and de-repressed in activated macrophages. Quantification of mRNA levels at a high temporal resolution by RNASeq allowed us to define groups with different expression patterns. Thereby, we were able to distinguish mRNAs whose translation is actively regulated from mRNAs whose polysomal shifts are due to changes in mRNA levels. Active up-regulation of translation was associated with a higher content in AU-rich elements (AREs). For one example, Ier3 mRNA, we show that repression in resting cells as well as de-repression after stimulation depends on the ARE. Bone-marrow derived macrophages from Ier3 knockout mice showed reduced survival upon activation, indicating that IER3 induction protects macrophages from LPS-induced cell death. Taken together, our analysis reveals that translational control during macrophage activation is important for cellular survival as well as the expression of anti-inflammatory feedback inhibitors that promote the resolution of inflammation.

NR4A1 (Nur77) and NR4A3 (Nor-1) function as tumor suppressor genes as demonstrated by the rapid development of acute myeloid leukemia in the NR4A1 and NR4A3 knockout mouse. The aim of our study was to investigate NR4A1 and NR4A3 expression and function in lymphoid malignancies. We found a vastly reduced expression of NR4A1 and NR4A3 in chronic lymphocytic B-cell leukemia (71%), in follicular lymphoma (FL, 70%), and in diffuse large B-cell lymphoma (DLBCL, 74%). In aggressive lymphomas (DLBCL and FL grade 3), low NR4A1 expression was significantly associated with a non-germinal center B-cell subtype and with poor overall survival. To investigate the function of NR4A1 in lymphomas, we overexpressed NR4A1 in several lymphoma cell lines. Overexpression of NR4A1 led to a higher proportion of lymphoma cells undergoing apoptosis. To test the tumor suppressor function of NR4A1 in vivo, the stable lentiviral-transduced SuDHL4 lymphoma cell line harboring an inducible NR4A1 construct was further investigated in xenografts. Induction of NR4A1 abrogated tumor growth in the NSG mice, in contrast to vector controls, which formed massive tumors. Our data suggest that NR4A1 has proapoptotic functions in aggressive lymphoma cells and define NR4A1 as a novel gene with tumor suppressor properties involved in lymphomagenesis.

Tumor necrosis factor-α (TNF-α) is the most potent proinflammatory cytokine in mammals. The degradation of TNF-α mRNA is critical for restricting TNF-α synthesis and involves a constitutive decay element (CDE) in the 3' UTR of the mRNA. Here, we demonstrate that the CDE folds into an RNA stem-loop motif that is specifically recognized by Roquin and Roquin2. Binding of Roquin initiates degradation of TNF-α mRNA and limits TNF-α production in macrophages. Roquin proteins promote mRNA degradation by recruiting the Ccr4-Caf1-Not deadenylase complex. CDE sequences are highly conserved and are found in more than 50 vertebrate mRNAs, many of which encode regulators of development and inflammation. In macrophages, CDE-containing mRNAs were identified as the primary targets of Roquin on a transcriptome-wide scale. Thus, Roquin proteins act broadly as mediators of mRNA deadenylation by recognizing a conserved class of stem-loop RNA degradation motifs., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: Histiocytic sarcoma (HS) is a rare but highly aggressive disease. The cancer-specific survival of patients with HS is short and only limited response to conventional chemotherapy or radiation therapy is seen. Some data from single case reports have suggested efficacy for high-dose chemotherapy and autologous/allogeneic stem cell transplantation., Case Report: We report on 4 cases of HS, and demonstrate that different druggable receptors are expressed on HS. Using immunohistochemistry, we detected the expression of platelet-derived growth factor receptor, vascular endothelial growth factor receptor and epidermal growth factor receptor, which are all well-known targets for novel targeted agents. Based on the marker profile, different novel targeted therapies including imatinib, sorafenib and bevacizumab were applied to the patients. We observed a varying clinical course for each patient., Conclusion: In our case series, we demonstrated that different receptors, which represent potential targets for novel drugs, are expressed on HS tumor cells. For a definitive assessment of the efficacy of these agents a prospective case study of a larger number of patients should be performed., (Copyright © 2012 S. Karger AG, Basel.)

In patients with haemophilia A knowledge of the pathogenetic mutation is important i) as basis for carrier diagnosis and ii) for risk estimation of inhibitor formation. The pathogenetic mutations were identified by testing inversions in intron 1 and 22 (IVS22 and IVS1) and sequencing part of the promoter, the coding region and the exon/intron boundaries in a cohort of Austrian haemophilia A patients. A total of 239 patients from nine participating centres, who had consented to genetic testing and of whom clinical information was available were included in the study. First, IVS22 and IVS1 were tested; in case of absence of either inversion patients were subjected to sequencing. Mutations within the FVIII gene were identified in 234 patients. Notably, 53 mutations had not previously been described in HAMSTeRS. Of our patient cohort, 72.5 % had either an IVS22 or a missense mutation. Interestingly, in three brothers with severe haemophilia, we found a double mutation in exon 14 (missense + small deletion). The spectrum of mutations in Austrian haemophilia A patients was comparable to that found in the German and Italian population; however, it differed from the spectrum reported in the UK. In conclusion, 53 not previously published mutations were identified in Austrian haemophilia A patients. The occurrence of double mutations in the factor VIII gene could be confirmed and their low frequency was corroborated. We speculate that the differences between mutations in Austria and other European countries are due to ethnic diversity. Detailed investigations of the association of ethnicity and the mutation spectrum are planned.

The treatment of haemophilia requires continuous development of knowledge related to various aspects of diagnosis and therapy. It is, therefore, essential to collect valid and representative data, which are comparable on an international level. The Austrian Haemophilia Registry was set up by the Scientific Advisory Panel of the Austrian Haemophilia Society and by the patient organisation. For the design, it was decided to divide the registry into three sections, two concerning quality control and a third concerning scientific questions, the latter requiring written informed consent. A web-based software is used to collect data. Transfer and storage of data are secured and the server is situated in a computer center with video and access control. Data entry was initiated early 2008. Currently, only preliminary data are available. Our further focus is on continued data entry, which will further enable us to provide information concerning the characteristics of the haemophilia patient population in Austria and the actual treatment modalities used.

Patient registries are databases essential for identifying and tracking individuals with a particular disease and for collecting epidemiological information. The data obtained are necessary for quality control and quality assurance in treatment and for studying the impact of new developments on prevention, diagnosis and treatment. The Austrian Hemophilia Registry is a joint initiative between Austrian hemophilia treaters, represented by the Austrian Hemophilia Society's scientific advisory board, and the Austrian Hemophilia Society (OHG). The registry's main objective is to record information on patients with hemophilia, such as severity of disease, types of treatment and general health status. The registry also aims to improve the planning of supply of factor concentrates and to provide an instrument for early detection of side effects, such as an increase in inhibitor development or certain infections. The registry consists of three parts: the first contains basic information on quality control, the second contains extended data for quality control collected annually, and the third, "study part", covers scientific data and is also updated annually. For the latter, written informed consent of each patient is a prerequisite. Data are stored centrally on a server of an independent, public institution (University for Health Sciences, Medical Informatics and Technology: UMIT). The server is situated in a data-processing center with sophisticated security measures including physical control of access. Participating institutions comprise the main hemophilia care centers in Austria. Statistical analysis is carried out on anonymized data only. The project was financed by a public-private partnership with financial resources provided by the Austrian Ministry of Health (BMGFJ) and the pharmaceutical industry. The entire project, which is planned to be long term, will be monitored, evaluated and adjusted by the scientific advisory board accordingly.