lncRNA的定位，功能

听了一个发育讲坛以后做的关于lncRNA的笔记

lncRNA的定位，功能

• Q:whether processing of lncRNA orthologs is conserved?
  • 从这四点来观察保守性，如果sequence相同，保守性一般也相同
    • conservation of long noncoding RNAs(lncRNAs) occures at multiple levels
    • expressed lncRNAs in mESCs(R1) are more frequently localized to nuclei than those in hESCs (H9)
      • 在人的干细胞中，一半在细胞核，一半在细胞浆
    • sequence-conserved lncRNAs exhibit stronger cytoplasmic localization patterns in H9 cells than R1 cells
    • subcellular distribution of positionally conserved lncRNAs is more cytoplasmic in H9 cells and more nuclear in RI cells
      • 分成三类：分散的，集中的，相同的
        • 在人中，lncRNA更多的趋向于细胞浆
        • 在小鼠中，lncRNA更多的趋向于细胞核
    • the localization difference of both sequence-conserved and positionally conserved lncRNAs between lncRNAs between H9 and RI cells is significantly higher than that of conserved mRNAs
      • 检查了两者的promoter是否有共同的
      • 将两种lncRNA取出来与相对应的mRNA进行比较，发现两种lncRNA都要比mRNA差别要大
    • different pluripotent states of ECSs had little effect on the distinct localization patterns of lncRNA orthologs
      • 来进行RNA FISH
        • 红色：细胞质 蓝色：细胞核
• Q:are there any functional consequences of these distinctly localized lncRNA orthologs in the context of pluripotency?
  • 进行敲除或者敲低实验
    • crisper/cas9-KO nuclear lncRNAs in R1 cells
    • shRNA-KD cytoplasmic lncRNA in H9 cells
  • FAST( FOXD3 Antisense transcript 1) is a conserved lncRNA that is specifically expressed in ESCs
    • 比较人和小鼠的 FAST
      • 先找相关资料：与之相对的DNA位置，长度，人和老鼠的对比
      • 为何说它是个保守的lncRNA
      • 通过胶图来看RNA的长度
      • qRT-PCR
        • • 提示与干细胞功能有关系
    • hFAST is required for puripotency of all examined hESCs(H1 H9 CT1)
      • hFAST by NB
      • colony formation
      • AP staining for pluripotency
      • confirme the effect of hFAST on pluripotency via crisper/cas9 KO
    • loss of hFAST led to altered expression of stem cell maintenance-related genes in H9 cells
      • RNA-seq
      • WB
      • ectopic expression of hFAST rescued OCT4 and NANOG expression in hFAST KD H9 cell
  • FAST定位在细胞浆的lncRNA能不能编码小肽
    • to test hFAST coding potential RNA fish
      • Flag-tag KI three continous nucleotides downstream of the predicted ORF,followed by WB
        • 子主题
  • in hESCs,hFAST is likely involved in Wnr signaling
    • wnt signaling 通路模式
    • • Q：FAST是否可以结合destruction complex
        • in hESCs,hFAST is likely invovled in Wnt signaling by binding to the E3 ubiquitin ligase β-TrCP
      • Q：确实有结合，怎么作用？
• Q:How does hFAST work in hESCs?
  • hFAST is a multivalent β-TrCP binding platform via five individual loop regions
  • hFAST is specifically binds to the WD40 domains of FLAG-β-TrCP
    • β-TrCP belongs to the Fbw(F-box/WD40 repeat-containing) protein family,with an F-box motif at the N terminus and seven WD40 repeat at the C terminus
    • β-TrCP 的结构
    • 将β-TrCP进行各种变异后，查看结合情况
    • 有很多种都有WD40 domain，来看结合情况
      • in vitro binding assays
      • RIP assays
  • Q: is hFAST sufficiently abundant to affect levels of β-TrCP involved in WNT signaling in hESCs?
    • 绝对定量
      • β-TrCP并不是特别abundant的protein
      • 子主题
    • 理论上，>22%的细胞质β-TrCP可以由hFAST来结合
  • mFAST（1,377nt）is not required for puripotency in mESCs,nor interact with β-TrCP
    • FISH
    • RT-PCR
    • WB
    • in vitro binding assays
    • black lines indicate conserved sequence between hFAST and mFAST
• Q: what factors regulate the distinct processing of lncRNA orthologs in m/hESCs?
  • ectopic expression of hFAST and mFAST:both were localized to the cytoplasm in H9 cells,whereas both were more nuclear in R1 cells
    • comparable levels
    • not cis-elements,but some(differentially expressed) trans-factors,contribute primarily to such distinct localizations
• Q:what factors determine the distinct processing of lncRNA orthologs in m/hESCs?
  • Q:which trans-factor(s) might regulate distinct FAST processinng and export in H9 and R1 cells?
    • 
    • PPIE:belongs to the peptidyl-prolyl cis-trans isomerase (PPIase) family and is highly conserved
      • 
      • 
      • 
    • PPIE KD resulted in an increased mFast export(reduced N/C),accompanying by increased splicing and export of mFast
      • RNA FISH
        • 
      • WB
        • 
      • RT-PCR
        • 
    • PPIE KD also reduced the N/C distribution of other positionally conserved lncRNAs in R1 cells,which can be largely rescued by re-introducing Ppie into Ppie KD cells
      • 
      • 
    • 表达的不同剪接抑制子PPIE对lncRNA亚群的不同定位有贡献
• 总结
  • 1
    • subcellular localization of conserved lncRNAs is different in hESCs and mESC
    • cytoplasmic hFAST but not nuclear mFAST promotes WNT signaling in hESCs pluripotency
    • 老鼠的保守的lncRNA在细胞浆和细胞质的定位和人的lncRNA是很不一样的，这种不一样性可能对于干细胞的多能性潜能有一定影响。
  • 2
    • PPIE regulate distinct FAST processing in hESCs and mESCs
  • 3
    • 保守的lncRNA,不保守的加工，“新的”生物学功能
• 背景
  • PWS region new lncRNAs：SPAs and sno-lncRNAs
  • 检测了保守性
    • 猴子
    • 老鼠
    • 可变剪接AS导致sno-lncRNA的非保守性表达
  • SNORD II16del mice could not fully recapitulate the clinical features of human PWS patients
    • • lncRNAs evolve more rapidly than mRNAs
      • the comparison is not comparable (between mRNA and lncRNA)
• 文章
  • Distinct processing of lncRNAs contributes to non-conserved functions in stem cells
• 主题
• 发现lncRNA更多的跟人的干细胞有关