Rapicavoli Nicole A, Poth Erin M, Blackshaw Seth
Department of Neuroscience, Neurology and Ophthalmology, Center for High-Throughput Biology and Institute for Cell Engineering, Johns Hopkins University School of Medicine, 733 N, Broadway Avenue, Baltimore, MD, USA.
BMC Dev Biol. 2010 May 11;10:49. doi: 10.1186/1471-213X-10-49.
Recent work has identified that many long mRNA-like noncoding RNAs (lncRNAs) are expressed in the developing nervous system. Despite their abundance, the function of these ncRNAs has remained largely unexplored. We have investigated the highly abundant lncRNA RNCR2 in regulation of mouse retinal cell differentiation.
We find that the RNCR2 is selectively expressed in a subset of both mitotic progenitors and postmitotic retinal precursor cells. ShRNA-mediated knockdown of RNCR2 results in an increase of both amacrine cells and Müller glia, indicating a role for this lncRNA in regulating retinal cell fate specification. We further report that RNCR2 RNA, which is normally nuclear-retained, can be exported from the nucleus when fused to an IRES-GFP sequence. Overexpression of RNCR2-IRES-GFP phenocopies the effects of shRNA-mediated knockdown of RNCR2, implying that forced mislocalization of RNCR2 induces a dominant-negative phenotype. Finally, we use the IRES-GFP fusion approach to identify specific domains of RNCR2 that are required for repressing both amacrine and Müller glial differentiation.
These data demonstrate that the lncRNA RNCR2 plays a critical role in regulating mammalian retinal cell fate specification. Furthermore, we present a novel approach for generating dominant-negative constructs of lncRNAs, which may be generally useful in the functional analysis of this class of molecules.
最近的研究发现，许多长链mRNA样非编码RNA（lncRNA）在发育中的神经系统中表达。尽管它们数量众多，但这些非编码RNA的功能在很大程度上仍未被探索。我们研究了高度丰富的lncRNA RNCR2在小鼠视网膜细胞分化调控中的作用。
我们发现RNCR2在有丝分裂祖细胞和有丝分裂后视网膜前体细胞的一个子集中选择性表达。通过shRNA介导敲低RNCR2会导致无长突细胞和穆勒胶质细胞数量增加，表明这种lncRNA在调节视网膜细胞命运决定中发挥作用。我们进一步报道，通常保留在细胞核中的RNCR2 RNA，当与IRES-GFP序列融合时可以从细胞核输出。RNCR2-IRES-GFP的过表达模拟了shRNA介导敲低RNCR2的效果，这意味着RNCR2的强制错误定位会诱导显性负性表型。最后，我们使用IRES-GFP融合方法来鉴定RNCR2中抑制无长突细胞和穆勒胶质细胞分化所需的特定结构域。
这些数据表明lncRNA RNCR2在调节哺乳动物视网膜细胞命运决定中起关键作用。此外，我们提出了一种生成lncRNA显性负性构建体的新方法，这可能在这类分子的功能分析中普遍有用。
