Abstract
BackgroundRecent 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.ResultsWe 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.ConclusionThese 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.
Highlights
Recent work has identified that many long mRNA-like noncoding RNAs are expressed in the developing nervous system
Though conventional sequence alignment searches did not identify any nonmammalian orthologues of Retinal Non-coding RNA 2 (RNCR2) [19], a syntenybased search identified putative RNCR2 orthologues from chick and Xenopus tropicalis
RNCR2 expression declines after the first few days postnatal, and by P8 is confined to a subset of cells in the inner nuclear layer and ganglion cell layer of the peripheral retina (Figure 2C)
Summary
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. Several thousand mammalian ncRNAs have been identified that span multiple kilobases in length [2]. Some of these ncRNAs show extensive conservation at the nucleotide level, despite lacking evolutionarily conserved open reading frames (ORFs). Other long ncRNAs such as Air and H19 have been implicated in genomic imprinting [5,6] In both of these cases, ncRNAs act locally, coating the nearby genomic loci and inducing the formation of heterochromatin and repression of gene expression [7]
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