mRNA translation regulation in Diamond Blackfan Anaemia

Diamond Blackfan Anemia is a bone marrow failure syndrome in which patients lack erythroid precursor cells in the bone marrow. Therefore, it is also designated as pure red cell aplasia, as other hematological indices are at normal levels. Around half of DBA patients bear an autosomal dominant mutation in one of the ribosomal protein genes which leads to decreased cellular ribosomal content. How mutations in such general proteins can result in an erythroid restricted phenotype is as yet unknown. In this thesis we aimed to elucidate the pathological mechanisms of erythroid failure in DBA. We hypothesized, that decreased ribosomal content in erythroid precursor cells could cause a decrease in the translation of important transcripts. For this purpose, we set up shRNA mediated knockdown of ribosomal proteins in proerythroblasts followed by translational profiling. After we have confirmed that translational deregulation of erythroid-specific transcripts occurs in DBA patients too, we further investigated their function. We aimed to characterize erythroid in vitro phenotype of Bag1 knockout mice and investigate the molecular role of Bag1 in the unfolded protein response. Further, we set out to delineate the function of Csde1 in translational regulation during erythropoiesis. In summary, we identified novel genes that are subject to selective translation and important for erythropoiesis. Reduced translation of the encoded transcripts may contribute to the pathophysiological mechanism by which mutations in ribosomal proteins in Diamond Blackfan Anemia patients cause the decrease in erythroid expansion and anemia

Grsf1-induced translation of the SNARE protein use1 is required for expansion of the erythroid compartment

Induction of cell proliferation requires a concomitant increase in the synthesis of glycosylated lipids and membrane proteins, which is dependent on ER-Golgi protein transport by CopII-coated vesicles. In this process, retrograde transport of ER resident proteins from the Golgi is crucial to maintain ER integrity, and allows for anterograde transport to continue. We previously showed that expression of the CopI specific SNARE protein Use1 (Unusual SNARE in the ER 1) is tightly regulated by eIF4E-dependent translation initiation of Use1 mRNA. Here we investigate the mechanism that controls Use1 mRNA translation. The 5'UTR of mouse Use1 contains a 156 nt alternatively spliced intron. The non-spliced form is the predominantly translated mRNA. The alternatively spliced sequence contains G-repeats that bind the RNA-binding protein G-rich sequence binding factor 1 (Grsf1) in RNA band shift assays. The presence of these G-repeats rendered translation of reporter constructs dependent on the Grsf1 concentration. Down regulation of either Grsf1 or Use1 abrogated expansion of erythroblasts. The 5'UTR of human Use1 lacks the splice donor site, but contains an additional upstream open reading frame in close proximity of the translation start site. Similar to mouse Use1, also the human 5'UTR contains G-repeats in front of the start codon. In conclusion, Grsf1 controls translation of the SNARE protein Use1, possibly by positioning the 40S ribosomal subunit and associated translation factors in front of the translation start site

Ribosomal deficiencies in Diamond-Blackfan anemia impair translation of transcripts essential for differentiation of murine and human erythroblasts

Diamond-Blackfan anemia (DBA) is associated with developmental defects and profound anemia. Mutations in genes encoding a ribosomal protein of the small (eg, RPS19) or large (eg, RPL11) ribosomal subunit are found in more than half of these patients. The mutations cause ribosomal haploinsufficiency, which reduces overall translation efficiency of cellular mRNAs. We reduced the expression of Rps19 or Rpl11 in mouse erythroblasts and investigated mRNA polyribosome association, which revealed deregulated translation initiation of specific transcripts. Among these were Bag1, encoding a Hsp70 cochaperone, and Csde1, encoding an RNA-binding protein, and both were expressed at increased levels in erythroblasts. Their translation initiation is cap independent and starts from an internal ribosomal entry site, which appeared sensitive to knockdown of Rps19 or Rpl11. Mouse embryos lacking Bag1 die at embryonic day 13.5, with reduced erythroid colony forming cells in the fetal liver, and low Bag1 expression impairs erythroid differentiation in vitro. Reduced expression of Csde1 impairs the proliferation and differentiation of erythroid blasts. Protein but not mRNA expression of BAG1 and CSDE1 was reduced in erythroblasts cultured from DBA patients. Our data suggest that impaired internal ribosomal entry site-mediated translation of mRNAs expressed at increased levels in erythroblasts contributes to the erythroid phenotype of DBA. (Blood. 2012; 119(1): 262-272

miR-Blood – a small RNA atlas of human blood components

Abstract miR-Blood is a high-quality, small RNA expression atlas for the major components of human peripheral blood (plasma, erythrocytes, thrombocytes, monocytes, neutrophils, eosinophils, basophils, natural killer cells, CD4+ T cells, CD8+ T cells, and B cells). Based on the purified blood components from 52 individuals, the dataset provides a comprehensive repository for the expression of 4971 small RNAs from eight non-coding RNA classes

A blood-based miRNA signature with prognostic value for overall survival in advanced stage non-small cell lung cancer treated with immunotherapy

Abstract Immunotherapies have recently gained traction as highly effective therapies in a subset of late-stage cancers. Unfortunately, only a minority of patients experience the remarkable benefits of immunotherapies, whilst others fail to respond or even come to harm through immune-related adverse events. For immunotherapies within the PD-1/PD-L1 inhibitor class, patient stratification is currently performed using tumor (tissue-based) PD-L1 expression. However, PD-L1 is an accurate predictor of response in only ~30% of cases. There is pressing need for more accurate biomarkers for immunotherapy response prediction. We sought to identify peripheral blood biomarkers, predictive of response to immunotherapies against lung cancer, based on whole blood microRNA profiling. Using three well-characterized cohorts consisting of a total of 334 stage IV NSCLC patients, we have defined a 5 microRNA risk score (miRisk) that is predictive of overall survival following immunotherapy in training and independent validation (HR 2.40, 95% CI 1.37–4.19; P < 0.01) cohorts. We have traced the signature to a myeloid origin and performed miRNA target prediction to make a direct mechanistic link to the PD-L1 signaling pathway and PD-L1 itself. The miRisk score offers a potential blood-based companion diagnostic for immunotherapy that outperforms tissue-based PD-L1 staining

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field