Trafficking of Mature miRNA-122 into the Nucleus of Live Liver Cells

Abstract

The binding of superquencher molecular beacon (SQMB) probes to human single-stranded cellular miRNA-122 targets was detected in various single live cells with femtosecond laser microscopy. For delivery of the SQMB-probes, 3D-nanoprocessing of single cells with sub-15 femtosecond 85 MHz near-infrared laser pulses was applied. Transient nanopores were formed by focusing the laser beam for some milliseconds on the membrane of a single cell in order to import of SQMB-probes into the cells. In single cells of the human liver cell lines Huh-7D12 and IHH that expressed miRNA-122, we measured target binding in the cytoplasm by two-photon fluorescence imaging. We found increased fluorescence with time in a nonlinear manner up to the point where steady state saturation was reached. We also studied the intracellular distribution of target SQMB and provide for the first time strong experimental evidence that cytoplasmic miRNA travels into the cell nucleus. To interpret nonlinear binding, a number of individual miRNA-122 positive cells (Huh-7D12 and IHH) and negative control cells, human VA13 fibroblasts and Caco-2 cells were analyzed. Our experimental data are consistent with the cytoplasmic assembly of nuclear miRNA and provide further mechanistic insight in the regulatory function of miRNAs in cellular physiology. An open issue in the regulation of gene expression by miRNA is whether miRNA can activate gene expression in addition to the well-known inhibitory effect. A first step for such a regulatory role could be the travelling of miRNA-RISC into the nucleus.

Nuclear and cytoplasmic localization of neural stem cell microRNAs

“…A number of reports have described the occurrence of particular miRNAs in animal cell nuclei and nucleoli (Hwang et al 2007a;Kim et al 2008;Marcon et al 2008;Ohrt et al 2008;Place et al 2008;Foldes-Papp et al 2009;Politz et al 2009;Liao et al 2010). In this article, we wished to evaluate the generality of miRNA nuclear localization by assaying a nontumor, minimally passaged, progenitor cell line.…”

“…While miRNAs generally function in the cytoplasm, mature miRNAs have been found in the nucleus, and, in some cases, shown to alter promoter activity (Hwang et al 2007a;Kim et al 2008;Marcon et al 2008;Ohrt et al 2008;Place et al 2008;Foldes-Papp et al 2009). Other miRNAs have been localized to the nucleolus (Politz et al 2009;Liao et al 2010) and, notably, the nuclear and nucleolar abundances of some miRNAs have been reported to be greater than their cytoplasmic levels.…”

Importin 8 Regulates the Transport of Mature MicroRNAs into the Cell Nucleus

“…MicroRNAs (miRNAs), 5 a class of 19 -24-nucleotide noncoding RNAs (ncRNAs), are widely involved in post-transcriptional gene regulation (1). According to the canonical model, miRNAs recognize target mRNAs and exert their roles predominantly in the cytoplasm.…”

“…Accumulating evidences, however, have demonstrated that certain mature miRNAs can reenter the nucleus (2-4). Földes-Papp et al (5) were the first to report the expression of miR-122 in liver cells and show unequivocal proof for the relocation of cytoplasm-assembled single-stranded miR-122 into the cell nucleus. Hwang et al (4) demonstrated that miR-29b could be imported into the cell nucleus.…”

Functional Roles of microRNAs in Cancer: microRNomes and oncomiRs Connection

“…Recently, it has been evidenced that mature microRNAs may also be localized in nucleus, through a specific hexanucleotide (AGUGUU) sequence which acts as a transferable nuclear localization element [24]. An example of this is the positive regulatory effect on transcriptional level of mir-122 in liver cells [25]. Moreover, it has been shown that vesicles of endocytic origin known as exosomes may contain both mRNA and microRNAs, which can be delivered to adjacent cells, and can be functional therein.…”