The pancancer overexpressed and cell cycle regulated NFYC antisense 1 controls tumor E2F/MYC proliferation programs through in cis and in trans modes of action

Antisense RNAs (asRNAs) represent a new layer of gene expression regulation, generally acting in cis on sense gene due to sequence complementarity or by their act of transcription. Besides this, evidence is accumulating regarding the existence of in trans-acting asRNAs, which leave their site of transcription to act on different molecular targets, both in the nucleus and in the cytoplasm. In this context, we studied NFYC Antisense 1 (NFYC-AS1), an asRNA transcribed head-to-head to NFYC, the subunit C of the trimeric proliferation-associated NF-Y transcription factor. We showed that NFYC-AS1, but not its sense gene NFYC, is overexpressed pancancer with preferential association with RB1 mutation. NFYC-AS1 transcript characterization revealed that it is a nuclear asRNA with 3 isoforms, of which the longest is the dominant, which peaks in the early G1 phase of cell cycle. Knockdown by gapmers in lung squamous cell carcinoma and small cell lung cancer cells impairs cell growth, a phenotype recapitulated also by CRISPR-deletion of NFYC-AS1 transcription start site. Surprisingly, expression of sense gene is affected only when endogenous transcription of NFYC-AS1 is manipulated, suggesting that regulation of cell proliferation is at least in part independent of the in cis transcription-mediated effect on NFYC and is possibly mediated by RNA-dependent in trans effects, which converge on the regulation of E2F1/MYC-transcriptional programs. Among NFYC-AS1 putative targets, interesting candidates for further validation as therapeutic hits in different cancer types, including the very aggressive RB1-mutated tumors, emerged, ultimately supporting a role for NFYC-AS1 role in promoting cancer cell proliferation.