SRA

The major cardiac cell types composing the adult heart arise from common multipotent precursor cells. Cardiac lineage decisions are guided by extrinsic and cell-autonomous factors, including recently discovered long noncoding RNAs (lncRNAs). The CARMEN locus, which is known to dictate specification towards the cardiomyocyte (CM) and the smooth muscle cell (SMC) fates, generates a diversity of alternatively spliced lncRNAs. Here, we identify one of these isoforms, CARMEN-201, to be crucial for SMC commitment. CARMEN-201 activity is encoded within an alternatively-spliced exon containing a MIRc short interspersed nuclear element. This element binds the transcriptional repressor REST (RE1 Silencing Transcription Factor), targets it to cardiogenic loci, including ISL1, IRX1, IRX5, and SFRP1, and thereby blocks the CM gene program. In turn, genes regulating SMC differentiation are induced. These data show how a critical physiological switch is wired by alternative splicing and functional transposable elements in a long noncoding RNA. Overall design: Human fetal cardiac precursor cells (CPCs) were infected with lentivirus expressing CARMEN7 Exon2 bearing either a native WT MIRc (D1FCPCLV and D7PFCPCLV) or a scrambled (D1FCPCMV and D7FCPCMV) MIRc, with conservation of base composition. The cells were cultured in differentiation consitions and total cellular RNA was extracted 1 day (D1) or 7 days (D7) after initiation of differentaition. Uninfected fetal human CPCs maintained under proliferation conditions (PFCPC) were included as controls. Additionally, uninfected cells were also also subjected to differentiation and harvested 1 day (D1FCPC) or 7 days (D7FCPC) post initiation of differentiation. Triplicate samples were used for each biological condition. RNA samples were subjected to RNASeq analysis. For RNA immunoprescipitation, adult control CPCs (DIFFCTRL and PROCTRL) and CPCs lacking CARMEN C201 (DKOC201 and PKOC201) were grown under differentiation (DIFFCTRL and DKOC201) or proliferation (PROCTRL and PKOC201) conditions, then RIP was performed using anti-REST IgG. REST-associated transcripts were purified from differentiated adult CPCs using the RNeasy isolation kit (Qiagen). Sequencing libraries were prepared from 3 samples for each condition according to Illumina RNA Seq library kit instructions with Poly(A) selection. Libraries were sequenced with the Illumina HiSeq4000.