Abstract
In the nervous system, control of gene expression by microRNAs (miRNAs) has been investigated in fundamental processes, such as development and adaptation to ambient demands. The action of these short nucleotide sequences on specific genes depends on intracellular concentration, which in turn reflects the balance of biosynthesis and degradation. Whereas mechanisms underlying miRNA biogenesis has been investigated in recent studies, little is known about miRNA-stability related proteins. We first detected two genes in the retina that have been associated to miRNA stability, XRN2 and PAPD4. These genes are highly expressed during retinal development, however with distinct subcellular localization. We investigated whether these proteins are regulated during specific phases of the cell cycle. Combined analyses of nuclei position in neuroblastic layer and labeling using anti-cyclin D1 revealed that both proteins do not accumulate in S or M phases of the cell cycle, being poorly expressed in progenitor cells. Indeed, XRN2 and PAPD4 were observed mainly after neuronal differentiation, since low expression was also observed in astrocytes, endothelial and microglial cells. XRN2 and PAPD4 are expressed in a wide variety of neurons, including horizontal, amacrine and ganglion cells. To evaluate the functional role of both genes, we carried out experiments addressed to the retinal adaptation in response to different ambient light conditions. PAPD4 is upregulated after 3 and 24 hours of dark- adaptation, revealing that accumulation of this protein is governed by ambient light levels. Indeed, the fast and functional regulation of PAPD4 was not related to changes in gene expression, disclosing that control of protein levels occurs by post-transcriptional mechanisms. Furthermore, we were able to quantify changes in PAPD4 in specific amacrine cells after dark -adaptation, suggesting for circuitry-related roles in visual perception. In summary, in this study we first described the ontogenesis and functional expression of these two miRNA-stability related proteins in the retina.
Highlights
In addition to the expression in the adult retina, we observed that XRN2 and PAPD4 transcripts are present throughout all analyzed developmental ages, with distinct gene expression profiles (n = 6)
Our results indicated that XRN2 has higher expression in Embryonic day 19 (E19) retinas (2ˆ0.86 = 1.82 fold-expression, P,0.01) and P10 (2ˆ1.40 = 2.64 fold-expression, P,0.01) when compared to P60
Width of specific miRNA activity on mRNA targets depends on intracellular concentrations, which obviously relies on the balance of the synthesis and degradation processes [16]
Summary
MiRNA comprises a distinct class of 20– 24 nucleotide base pair single-stranded noncoding RNA which post-transcriptionally regulates mRNA copy levels and translation efficiency through complementary binding of small stretches of base pairs, typically in the 39 untranslated region [9,10]. The action of these short nucleotide sequences on specific genes depends on intracellular concentration [11], which in turn reflects the balance of biosynthesis and degradation. Functional expression of XRN2 and PAPD4 in the retina was assessed after adaptation to different ambient light conditions
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