In situ fluorescence analysis demonstrates active siRNA exclusion from the nucleus by Exportin 5

Institute for Biophysics, BIOTEC, Dresden University of Technology, Tatzberg 47-51, 01307 Dresden, Germany.
Nucleic Acids Research (Impact Factor: 9.11). 02/2006; 34(5):1369-80. DOI: 10.1093/nar/gkl001
Source: PubMed


Two types of short double-stranded RNA molecules, namely microRNAs (miRNAs) and short interfering RNAs (siRNAs), have emerged
recently as important regulators of gene expression. Although these molecules show similar sizes and structural features,
the mechanisms of action underlying their respective target silencing activities appear to differ: siRNAs act primarily through
mRNA degradation, whereas most miRNAs appear to act primarily through translational inhibition. Our understanding of how these
overlapping pathways are differentially regulated within the cell remains incomplete. In the present work, quantitative fluorescence
microscopy was used to study how siRNAs are processed within human cells. We found that siRNAs are excluded from non-nucleolar
areas of the nucleus in an Exportin-5 dependent process that specifically recognizes key structural features shared by these
and other small RNAs such as miRNAs. We further established that the Exportin-5-based exclusion of siRNAs from the nucleus
can, when Exp5 itself is inhibited, become a rate-limiting step for siRNA-induced silencing activity. Exportin 5 therefore
represents a key point of intersection between the siRNA and miRNA pathways, and, as such, is of fundamental importance for
the design and interpretation of RNA interference experimentation.

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Available from: Petra Schwille, Oct 08, 2014
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    • "Drosha is responsible for recognizing and polishing primiRNAs by cleaving it with special structure. This binding is called a conserved structural functionality since no conserved sequences are found on pre-miRNA for Drosha (Ohrt et al. 2006). Furthermore, Drosha binds and cleaves various primiRNAs in different efficiencies and specificities. "
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    • "This implied that one or a few components of the RNAi pathway, which are engaged by si, sh and miRNAs, are rate-limiting and thus titratable (4,13–15). To narrow down these factors, we and others inhibited or overexpressed different proteins involved in RNAi and characterized the effects on co-delivered si or shRNAs in vitro and in vivo (3,10,11,16–20). One of the hits was Exportin-5, the karyopherin shuttling mi and shRNA precursors from the nucleus to the cytoplasm (3,18–21). "
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    • "Depletion of CLIMP-63 protein levels in 293T-REx cells was not associated with a concomitant decrease in the level of let-7, miR-17 and miR-21 microRNAs (our unpublished data), suggesting that the effects on RNA silencing may be related to changes in the function, rather than the level, of endogenous microRNAs. However, the caveats have to be taken into account that: (i) the cells that were the most depleted in CLIMP-63, and thus most likely to exhibit a phenotype, may not have survived and been included in our analyses; (ii) changes in the level, activity or function of protein and RNA components of the microRNA pathway other than Exportin-5, which is considered as the rate-limiting step in microRNA biogenesis in human cells (33), need to fall below that threshold to be detected, a situation that may not be achieved upon incomplete CLIMP-63 depletion and (iii) the pool of Dicer proteins affected upon knockdown of CLIMP-63 may not be involved in microRNA biogenesis and may function instead as part of the microRNA effector complex, independently of microRNA levels. "
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