Untersuchung 3D miRNA Netzwerken in Alterungsprozessen.
Biochemie
Zellbiologie
Zusammenfassung der Projektergebnisse
The results of this project constitute clear evidence of a miRNA locus engaging in spatial regulatory interactions with a protein-encoding gene, in this case PERP, which encodes a p53-associated effector critical for cellular senescence entry by human primary lung fibroblasts. To sum up, first, we used profiling of chromatin- and “transcription factory”-enriched miRNAs to find that miR-3145 is specific to nuclei of proliferating cells, and becomes suppressed upon senescence, when PERP is strongly induced. Second, co-regulation of the two transcripts was validated via miRNA-inhibition and PERP- CRISPRa experiments. Third, native i4C and intronic RNA FISH in proliferating and senescent cells from different donors showed that the two loci interact spatially in proliferating cells, and that abolishing this interaction promotes PERP activation that coincides with senescence entry. Last, a combination of “transcription factory” RNA-IP and ChIP allowed us to identify the Drosha protein in complex with the precursor miRNA-3145 as the key effectors in this coordinated interaction, and the PERP promoter as they main target region. The case study of human miRNA-3145 and PERP interaction in the context of cellular senescence using cutting edge methodology provides new insights into the molecular mechanisms behind miRNA networks. Based on this study we are able to propose a novel regulatory axis that exploits the three-dimensional folding of human chromosomes to coordinate targeting of gene loci by nuclear miRNAs via Drosha in a co-transcriptional manner.
