Regulation and function of YTHDF2 in a human pulmonary artery smooth muscle differentiation model

NATALI, LAUTARO; DE LA CRUZ-THEA, BENJAMÍN; HO, XUAN; STOLZ, ALEXANDRA; MUSRI, MELINA MARA

Córdoba

Congreso; Reunión Anual de la Sociedad Argentina de Investigación en bioquímica y biología molecular; 2024

Sociedad Argentina de Investigación en Bioquímica (SAIB)

Smooth Muscle Cells (SMCs) are one of the maincomponents of the vascular wall, where they play a fundamental rol inmaintaining vascular function and integrity. Although a fully differentiatedcell type, they retain a high degree of phenotypic plasticity which allows themto switch between a contractile-quiescent and a synthetic-proliferative state. Understandingthe molecular mechanisms controlling SMCs phenotypic switch is fundamental todevelop therapeutics for the diseases in which it’s implicated, such as COPD,artherosclerosis, pulmonary hypertension, among others. The methylation of mRNAis a recently discovered layer of gene expression control that has beenimplicated in the regulation of many cellular processes, such as proliferation,apoptosis and cell differentiation. This regulatory mechanism relies on a complexmachinery of proteins to “write”, “erase” and “read” the modification, makingit reversible and dynamic. Given the importance of these regulatory pathways,we investigated the role of mRNA methylation machinery in SMC differentiationand proliferation. We employed a primary cell line of human pulmonary arterySMCs in combination with a contact-inhibition model of differentiation toassess changes in methylation machinery expression during the differentiation.Our study revealed that the protein levels of the YTHDF family are downregulatedduring differentiation of SMCs, while the mRNA levels remain unchanged, hintingat a posttranslational regulatory mechanism. This decrease in protein levelswas attributed to proteasomal degradation and autophagy, as shown by recoveryexperiments using MG132 and Bafilomycin A1, respectively. Employing RIP-seq, wedemonstrated that YTHDF2 is bound to several key transcripts involved in SMChomeostasis, including several mRNAs of the TGF beta pathway and related to SMCcontraction. Knockdown of YTHDF2 followed by RNA-seq and TMT-mass spectrometryin different time points of the differentiation process resulted in alteredpatterns of expression of several mRNAs and proteins, with a potentupregulation of the interferon pathway. We identified a set of potential novelYTHDF2 targets, among which FBLN5 was validated using knockdown of YTHDF2followed by western blot and qPCR. Lastly we used live cell imaging andimmunofluorescence to assess the impact of YTHDF protein knockdown in SMCdifferentiation and proliferation, which surprisingly resulted in an impairedproliferation upon the depletion of YTHDF3. In conclusion, this studydemonstrated that the expression of the YTHDF family of proteins is inverselycorrelated with SMC differentiation. Furthermore we characterized the alterationsto the transcriptome and proteome product of the absence of YTHDF2 during thedifferentiation, together with the identification of a novel target. This workwill serve as a stepping stone in the quickly developing field of methylatedRNA biology and its intersection with smooth muscle cell development anddisease.