Long Non-coding RNA Lnc-Fbxl10 And FBXL10-LF Promotes Cortical Neurogenesis

The mammalian cerebral cortex is the most complex region of the nervous system,performing the most sophisticated cognitive and perceptual functions.The adult neocortex is a six-layered structure comprising a plethora of projection neurons,interneurons and glial cells.The projection neurons(PNs)are the main functional units,whose generation and migration are precisely spatiotemporally regulated,but the mechanisms still remain unknown.Many recent studies point to long non-coding RNAs(lncRNAs)as essential regulators in biological process such as cell proliferation and differentiation,apoptosis,immune response and embryonic development.LncRNAs in the brain,exerting high conservation and special expression patterns,may play important roles in development.Here,we identified Fbxl10-LF and its divergent non-coding RNALnc-Fbxl10(also known as lncKdm2b)by transcriptome analysis,with similar expression patterns in mice cortex.Knocking down of Lnc-Fbxl10 by sh RNAs or ASOs in mice primary neural precursor cells(NPCs)or Neuro-2a neuroblastoma cells respectively significantly downregulated the m RNA levels of Fbxl10-LF,but not Fbxl10-SF.Similar regulation could be found in Lnc-Fbxl10 poly A knock-in m ESCs.Importantly,nuclear run-on assay revealed that depletion of Lnc-Fbxl10 attenuated the transcription rate of Fbxl10-LF,suggesting a role of Lnc-Fbxl10 in cis promoting the basal transcription of Fbxl10-LF.In order to test whether Lnc-Fbxl10 displays intrinsic ability to promote gene expression,we performed the Gal4-?N/BoxB system assay,showing the evolutionarily conserved 5'part of Lnc-Fbxl10 has a intrinsic-activating function.RNA pull-down experiments followed by mass spectrometry identified Lnc-Fbxl10 binding protein heterogeneous nuclear ribonucleoprotein A/B(hnRNPAB),depletion of which significantly decreased Fbxl10-LF's expression,suggesting Lnc-Fbxl10 transcripts may activate the transcriontion of Fbxl10-LF by interactiong with hnRNPAB.Fbxl10-LF is transiently expressed in differentiating freshly born PNs.To further validate the fate of Fbxl10-LF-expressing cells during cortical neurogenesis,we performed lineage tracing experiments by Fbxl10-LF^{Cre ERT2} knock-in mouse line,indicating the progenies of Fbxl10-LF-expressing cells are largely projection neurons.Then we analysed the roles of FBXL10 in cortical neurogenesis in vivo.Fbxl10-LF^{Cre ERT2/Cre ERT2} mice develop normally probably resulting from FBXL10 is reduced to approximately half of wide-type levels.Cortex of conditional knock-out(Fbxl10 cKO)postnatal pups are slightly thinner and the migration of SATB2⁺and CTIP2⁺neurons are slightly impaired compared to the wide-type.Lnc-Fbxl10 is transiently expressed in freshly born projection neurons and Lnc-Fbxl10 cis-activates Fbxl10-LF expression,we expected that Lnc-Fbxl10 and Fbxl10-LF may have a similar function on cortical neuronal differentiation.To this end,depletion the expression of Lnc-Fbxl10 or Fbxl10-LF in vitro or in vivo enhanced self-renewal but decreased neuronal differentiation of NPCs.Most importantly,overexpressing Fbxl10-LF can mostly rescue the phenotypes caused by Lnc-Fbxl10knockdown.Moreover,hnRNPAB can also regulates neuronal differentiation in vivo similarly to FBXL10-LF,verifying Lnc-Fbxl10 may promote cortical neuronal differentiation via FBXL10-LF.Rescue experiments in vivo indicated that the LRR domain is indispensable for FBXL10's role in cortical development.Then we identified the proteins interacting with LRR domain—MCM complex(components of pre-replication complex),by immunoprecipitation and MS in N2a cell lines.Decreasing the expression of MCM2(a subunit of MCM complex)in vivo impaired self-renewal of NPCs,in contrast with the phenotype of FBXL10-LF,suggesting FBXL10-LF may affect the cell cycles of NPCs in neurogenesis by interacting with and antagonizing MCM complex.To elucidate downstream signaling mediated by FBXL10 in cortical neurogenesis,we used the CRISPR/Cas9 technique to generate Fbxl10 mutant mice(Fbxl10^-/-).Fbxl10^-/-embryos display prominent growth retardation and neural tube closure defects at E9.5 and eventually die around E11.RNA-seq transcriptome analyses using E9.5telencephalon tissues from wild-type and Fbxl10^-/-embryos showed significant downregulation of oxidative phosphorylation related genes,and significant upregulation of glycolysis and mitochondrion organization related genes in Fbxl10^-/-embryos.More importantly,FBXL10-LF may regulate morphological dynamics of mitochondria dependent of LRR domain,and depletion of Lnc-Fbxl10 or Fbxl10-LF significantly decreased cells'capability of oxidative phosphorylation.Thus,Lnc-Fbxl10 and Fbxl10-LF may regulate neurogenesis in a way of affecting morphological dynamics and oxidative phosphorylation.Overall,we explored the roles and mechanisms of lncRNALnc-Fbxl10 and its neighbour gene Fbxl10-LF in cortical neurogenesis,providing a new theoretical basis for neurogenesis.