THE LONG NON-CODING RNA DAGAR IS REGU- LATED BY M6 A MODIFICATION AND BOTH PATHWAYS ARE IMPORTANT FOR SMOOTH MUSCLE CELL PLAS- TICITY

DE LA CRUZ-THEA, BENJAMÍN; HO, XUAN; PEINADO, VICTOR; PAI, BALAGOPAL; BRUCKMANN, ASTRID; COLL-BONFILL, NURIA; NATALI, LAUTARO; MEISTER, GUNTER; MUSRI, MELINA M.

Congreso; Reunión conjunta de SAIC. SAI. AAFE. NANOMED; 2021

SAIC. SAI. AAFE. NANOMED

Adult vascular smooth muscle cells (SMCs) change between a con-tractile-differentiated and a proliferative-dedifferentiated phenotypein response to environmental cues constituting a key factor in theonset of cardiovascular diseases. Long non-coding RNAs (lncR-NAs) and mRNA modifications, in particular N-6-methyladenosine(m6A), regulate several physiological and pathological processes,but little is known about these molecules in SMC biology. Our aimwas to study both the role of lncRNAs and the m6A machineryduring SMC differentiation. By using an in vitro model of primaryhuman pulmonary artery SMCs phenotypic modulation and RNAdeep sequencing we found several regulated lncRNAs. RT-qPCRand Northern blot confirmed the dramatically increase of a candi-date during differentiation, which we refer to as Differentiation AndGrowth Arrest Related (DAGAR). DAGAR was downregulated inSMCs during tumor necrosis factor (TNF)-induced de-differentiationand in total RNA from pulmonary arteries of patients with COPDcompared to non-smoker patients. DAGAR Knockdown by siPoolsled to differentiation defects and increased SMC proliferation.RNA-Protein affinity purification identified the m6A machinery bound to DAGAR. Interestingly, DAGAR was enriched in m6A-RNA immunoprecipitation. Accordingly, we found a marked downregulation of YTHDF proteins during SMC differentiation, which is consistent withDAGAR increase. YTHDF2 immunoprecipitation followed by RNAdeep sequencing displayed an enrichment of mRNAs associatedto SMC fundamental processes, including smooth muscle myosinheavy chain (MYH11), the most specific SMC marker, and membersof the TGFb, PDGF and VEGF pathways. Remarkably, knockdownof YTHDF2 by siPools induced a significant increase of DAGAR andSMC marker gene expression. We conclude that the lncRNA DA-GAR and the m6A reader YTHDF2 contribute to the regulation ofSMC plasticity and differentiation programs.