Research Article: Dynamic Localisation of Mature MicroRNAs in Human Nucleoli is Influenced by Exogenous Genetic Materials

Date Published: August 6, 2013

Publisher: Public Library of Science

Author(s): Zhou Fang Li, Yi Min Liang, Pui Ngan Lau, Wei Shen, Dai Kui Wang, Wing Tai Cheung, Chun Jason Xue, Lit Man Poon, Yun Wah Lam, Sebastien Pfeffer.


Although microRNAs are commonly known to function as a component of RNA-induced silencing complexes in the cytoplasm, they have been detected in other organelles, notably the nucleus and the nucleolus, of mammalian cells. We have conducted a systematic search for miRNAs in HeLa cell nucleoli, and identified 11 abundant miRNAs with a high level of nucleolar accumulation. Through in situ hybridisation, we have localised these miRNAs, including miR-191 and miR-484, in the nucleolus of a diversity of human and rodent cell lines. The nucleolar association of these miRNAs is resistant to various cellular stresses, but highly sensitive to the presence of exogenous nucleic acids. Introduction of both single- and double-stranded DNA as well as double stranded RNA rapidly induce the redistribution of nucleolar miRNAs to the cytoplasm. A similar change in subcellular distribution is also observed in cells infected with the influenza A virus. The partition of miRNAs between the nucleolus and the cytoplasm is affected by Leptomycin B, suggesting a role of Exportin-1 in the intracellular shuttling of miRNAs. This study reveals a previously unknown aspect of miRNA biology, and suggests a possible link between these small noncoding RNAs and the cellular management of foreign genetic materials.

Partial Text

MicroRNAs (miRNAs) have emerged as ubiquitous regulators of gene expression. The latest version of miRbase reveals 1600 unique human miRNAs (Release 19: August 2012); many of them are highly cell type- or disease-specific [1]. Biogenesis of miRNAs requires multiple enzymatic complexes located in different subcellular compartments. Canonically, miRNAs are transcribed and processed in the nucleus, and matured in the cytoplasm, where they are incorporated into an RNA-induced silencing complex (RISC). Engaged RISCs affect the stability and/or translational potential of mRNA targets [2]–[3]. Bioinformatic estimations suggest that up to one-third of all protein-coding genes in the human genome are under the regulation of at least one miRNA [4].

The protocol for animal work was approved by the Animal Experimentation Ethics Committee of the Chinese University of Hong Kong (Permit Number: 09/050/MIS).

In this study, 11 abundant miRNAs are identified in the nucleolus of HeLa. It is the first work on human cells showing the nucleolar localization of miRNAs. The presence of miRNAs in the nucleolus has been reported in other mammals [17]–[18]. The Pederson Lab identified more than 50 miRNAs that are highly enriched in the nucleolus of rat myoblast cells [18]. Three out of the 11 nucleolar miRNAs identified in our study, including miR-191 and miR-484, are also on their list (Table S1). This is a remarkable level of overlapping, given the cell type-specificity of mammalian miRNAs [38]. We have demonstrated here the nucleolar localisation of mature miRNAs in a wide diversity of human, mouse and canine cell lines, regardless of cell types and transformation status. This suggests that the localisation of miRNAs in nucleoli is a general phenomenon in mammalian cells, rather than a unique property of myogenic cells. The nucleolus is a very dynamic structure [21], [39]. Activities of a wide range of proteins, including MDM2 [40], ADAR2 [41], HAND1 [42] and RelA [43], are controlled by their nucleolar localisation, possibly through a mechanism mediated by a noncoding RNA encoded in the ribosomal intergenic region [44]. Moreover, the nucleolar structure can be reversibly disrupted in response to cellular stress, which allows sequestered nucleolar factors to disperse out [34], [45]–[46]. In this report, we show for the first time that the nucleolar location of mature miRNAs is also highly dynamic. Although nucleolar miRNAs were not affected by metabolic perturbations known to disrupt the nucleolar structure, a single dose of exogenous nucleic acid was sufficient to disperse these miRNAs into the nucleoplasm and cytoplasm. Future studies will include a detailed investigation on the possible roles of the type and sequence of nucleic acids in this phenomenon. The functional significance of this redistribution is still not clear. The need to manage foreign genomes and their degradation products may saturate the same molecular machineries used for the subcellular partitioning of miRNAs. Interestingly, we showed that miR-484, a nucleolar miRNA, is much less efficient in the binding to RISC when compared to miR-20a, a cytoplasmic miRNA. This suggests that the displacement of miRNAs from the nucleolus to the cytoplasm makes them accessible to RISCs and the sites of translational suppression.