Characterization of post-transcriptional regulation of gene expression in heart diseases
Final Report Abstract
The aim of his research project was to identify mechanisms of post-transcriptional gene regulation utilizing a transriptome-wide approach on RNA-binding proteins and miRNAs. These questions were addressed with a combination biochemical and RNA deep-sequencing methods, first and foremost an enhanced CLIP-seq protocol that makes use of photoactivatable nucleosides (PAR-CLIP) and a multiplexed small RNA-sequencing protocol which allows for an absolute quantification of miRNA expression. We profiled myocardial and circulating miRNAs in more than 150 samples from patients with advanced heart failure, healthy controls and fetal hearts. Comparing non-failing postnatal and failing postnatal left ventricular myocardium we identified a characteristic miRNA heart failure signature that was in part reflected in fetal heart tissue, but lacked critical changes promoting cell proliferation. This signature was consistent across all co-transcribed members of a miRNA precursor cluster, and had an inverse correlation to the expression on their target mRNAs in a seed-sequence dependent manner. The total RNA amount in the blood circulation was about 10000-fold lower than in cardiac tissue. The predominant RNA species captured with our protocol were 5’ tRNA and YRNA fragments as well as miRNAs. The muscle-typic miRNAs contributed less than 1 % of all circulating miRNAs, the majority consisted of miRNAs highly expressed, in part specifically, in hematopoetic and endothelial cells. The circulating miRNA profile had pronounced changes in patients with advanced heart failure and cardiogenic shock. The muscle- and heart-enriched miRNAs as well as other highly expressed miRNAs in the heart were strongly elevated in the circulation of these patients, but approach normal levels as early as 3 months after the initiation of mechanical unloading (ventricular assist device therapy). In contrast to this, there were very few changes in the circulating miRNA profile of patients with a highly reduced ventricular function but in stable condition, revealing important limitations in the use of circulating miRNAs as biomarkers in heart failure. The PAR-CLIP experiments with the three RNA-binding proteins CELF1, MBNL1, and RBM20 identified mRNA targets and target sites at nucleotide resolution. Ongoing structural and functional studies are currently done to elucidate the functions of these proteins in the myocardium. Together, the results and generated data offer a valuable framework to direct future research in drug and biomarker assay development.
