Long Non-Coding RNA NEAT1 Associates with SRp40 to Temporally Regulate PPARγ2 Splicing during Adipogenesis in 3T3-L1 Cells.
Long Non-Coding RNA NEAT1 Associates with SRp40 to Temporally Regulate PPARγ2 Splicing during Adipogenesis in 3T3-L1 Cells.
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DOI：
10.3390/genes5041050
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发表时间：
2014-11-27
期刊：
影响因子：
3.5
通讯作者：
Patel NA
中科院分区：
文献类型：
--
作者：
Cooper DR;Carter G;Li P;Patel R;Watson JE;Patel NA
长非编码 (lnc) RNA 在细胞中发挥多种功能。 NEAT1 RNA 是细胞核中高度丰富的 4 kb lncRNA，与副斑点（控制副斑点蛋白隔离的核结构域）一致。我们检测了 3T3-L1 细胞分化为脂肪细胞过程中的 NEAT1 RNA 水平及其功能。通过 RT-PCR 测量，NEAT1 转录物水平在分化过程中出现短暂波动，这表明其在 PPARγ mRNA（驱动脂肪形成的主要转录因子）的选择性剪接中发挥作用。当细胞在第 0 天被胰岛素、地塞米松和异丁基甲黄嘌呤 (IBMX) 混合培养基诱导分化时，NEAT1 水平在第 4 天下降，此时 PPARγ2 变体被剪接，并且当发生终末分化时，PPARγ2 的出现与 PPARγ1 变体协调驱动脂肪细胞的分化。用于耗尽 NEAT1 的 SiRNA 导致细胞无法磷酸化富含丝氨酸/精氨酸的剪接蛋白 SRp40。 SRp40 的 siRNA 处理导致 PPARγ1 的失调，主要是 PPARγ2 mRNA 水平的失调。如第 0 天和第 8 天的 RNA-IP 所示，SRp40 与 NEAT1 相关，但在第 4 天下降，并且浓度比 IgG 对照有所增加。 SRp40 的过表达会增加 PPARγ2，但不会增加 γ1。尽管 lncRNA MALAT1 已在 SR 蛋白功能中进行了研究，但 NEAT1 尚未被证明可以结合 SR 蛋白进行磷酸化，从而产生选择性剪接结果。细胞增加 PPARγ2 剪接磷酸化 SR 蛋白的能力表明，脂肪生成过程中 NEAT1 水平的变化调节选择性剪接事件。
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.
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