LncRNA-PM Maintains Transcription Of Cbln1 And Synapse Integrity

LncRNAs are a class of regulatory transcripts longer than 200 nucleotides that do not have traditional open reading frames and play biological functions in the form of RNA.With the development of RNA sequencing technology and the in-depth study of lncRNA related functional mechanisms,thousands of lncRNAs have been discovered and proved to play necessary roles in a variety of physiological and pathological conditions.Due to the diversity of species and the complexity of functional mechanisms,there are still a lot of problems to be explored in the field of lncRNAs.Recent studies have shown that lncRNAs are critical regulators in the central nervous system.However,their roles in the cerebellum are currently unclear.Cbln1 is a synaptic organizer protein mainly expressed in cerebellar granule cells and plays an essential role in cerebellar synaptic integrity and plasticity.Despite its essential functions and therapeutic potential for cerebellar ataxic disorders,the gene expression regulation of Cbln1 is yet-to-be elucidated.In this work,we tested the function of the lncRNA Gm2694 isoforms and found that among the five examined splicing isoforms,only Gm2694-204(Designated as lncRNA-Promoting Methylation,lncRNA-PM)positively regulated the expression of Cblnl mRNA.FISH experiments found that lncRNA-PM is highly expressed in the nucleus of cerebellar granule cells.RT-qPCR detection of different brain regions and cerebellum at four key postnatal stages showed lncRNA-PM exhibits similar spatiotemporal expression pattern as Cbln1.We performed RT-qPCR after knockdown or overexpression,dual-luciferase reporter assays,subcellular fractionation assays,FISH,immunofluorescence,and ChIP to confirm that lncRNA-PM activates the transcription of Cblnl through Pax6-Mll1 complex-mediated H3K4me3.In mouse cerebellum,UV-RIP,re-ChIP and ChIRP experiments suggested an association of lncRNA-PM and Pax6-Mll1 complex with the promoter regulatory regions of Cbln1.Virus injection induced cerebellar lncRNA-PM-knockdown mice were validated to have deficiencies in Cblnl expression,cerebellar synaptic integrity and motor function by FISH,immunofluorescence,electron microscopy and motor behavioral experiments.Together,our work reveals the mechanism of Cblnl transcriptional activation via the Pax6-Mll1-H3K4me3 pathway mediated by cell-type-specifically expressed lncRNA splicing isoforms,and provides novel insights of the essential roles of lncRNA in the cerebellum.