Abstract
Long non-coding (lnc) RNAs serve a multitude of functions in cells. NEAT1 RNA is a highly abundant 4 kb lncRNA in nuclei, and coincides with paraspeckles, nuclear domains that control sequestration of paraspeckle proteins. We examined NEAT1 RNA levels and its function in 3T3-L1 cells during differentiation to adipocytes. Levels of NEAT1 transcript, measured by RT-PCR, fluctuated in a temporal manner over the course of differentiation that suggested its role in alternative splicing of PPARγ mRNA, the major transcription factor driving adipogenesis. When cells were induced to differentiate by a media cocktail of insulin, dexamethasone, and isobutylmethyxanthine (IBMX) on Day 0, NEAT1 levels dropped on Day 4, when the PPARγ2 variant was spliced and when terminal differentiation occurs The appearance of PPARγ2 coordinates with the PPARγ1 variant to drive differentiation of adipocytes. SiRNA used to deplete NEAT1 resulted in the inability of cells to phosphorylate the serine/arginine-rich splicing protein, SRp40. SiRNA treatment for SRp40 resulted in dysregulation of PPARγ1 and, primarily, PPARγ2 mRNA levels. SRp40 associated with NEAT1, as shown by RNA-IP on days 0 and 8, but decreased on day 4, and concentrations increased over that of IgG control. Overexpression of SRp40 increased PPARγ2, but not γ1. Although lncRNA MALAT1 has been investigated in SR protein function, NEAT1 has not been shown to bind SR proteins for phosphorylation such that alternative splicing results. The ability of cells to increase phosphorylated SR proteins for PPARγ2 splicing suggests that fluxes in NEAT1 levels during adipogenesis regulate alternative splicing events.
Highlights
Alternative splicing of pre-mRNA during adipogenesis is a means of ushering preadipocytes towards fully differentiated adipocytes by expanding the diversity of the proteome that accompanies maturation of the cells
Two Peroxisome proliferator activated receptor (PPAR) splice variants, PPAR1 and PPAR2 have been cloned from mouse and human [26,27]
When cells are placed in a cocktail of insulin, dexamethasone and IBMX to initiate differentiation, PPAR1 is the first splice variant to be detected
Summary
Alternative splicing of pre-mRNA during adipogenesis is a means of ushering preadipocytes towards fully differentiated adipocytes by expanding the diversity of the proteome that accompanies maturation of the cells. LncRNAs can act as components of nuclear bodies by providing a scaffolding to organize nuclear structures In this role, they regulate gene expression by controlling pre-mRNA processing and export [9]. NEAT1 and MALAT1 (metastasisassociated lung adenocarcinoma transcript 1) appear to serve as structural docking sites for accumulating specific splicing factors such as SR proteins [15] This class of lncRNA is retained within the nucleus and localizes to specific nuclear bodies. We further hypothesize that NEAT1 associates with the serine-arginine rich “SR” protein, SRp40, so that Clk kinase phosphorylates it for regulating alternative splicing of pre-mRNA during adipocyte differentiation. This study increases our knowledge of how varying levels of NEAT1 can function in the timing of alternative splicing of primary transcripts regulating adipogenesis
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