Sirna in Human Cells Selectively Localizes to Target RNA Sites

siRNA in human cells selectively localizes to target RNA sites Svitlana Y. Berezhna*†, Lubica Supekova†‡, Frantisek Supek§, Peter G. Schultz‡¶, and Ashok A. Deniz*¶ Departments of *Molecular Biology (MB 19) and ‡Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037; and §Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, CA 92121 Contributed by Peter G. Schultz, March 28, 2006 Recent observations of RNA interference (RNAi) in the nuclei of the cytoplasm and nuclei of HeLa cells. Given these results, human cells raise key questions about the extent to which nuclear different mechanisms can be envisioned by which nuclear- and cytoplasmic RNAi pathways are shared. By directly visualizing programmed RISCs are formed and localize to the nucleus. One the localization of small interfering RNA (siRNA) in live human cells, possibility is that the guide (antisense) siRNA strand forms we show here that siRNA either selectively localizes in the cyto- RISC in the cytoplasm, and this assembled and active RISC is plasm or translocates into the nucleus, depending on where the then transported to the nucleus. This mechanism could represent silencing target RNA resides. Two siRNAs that target the small a common RISC formation pathway for both cytoplasmic and nuclear 7SK and U6 RNAs localize into the nucleus as duplexes. In nuclear targets, with subsequent cytoplasmic and nuclear par- contrast, an siRNA targeting the cytoplasmic hepatitis C virus titioning of variously programmed RISC complexes. Alternately, replicon RNA dissociates, and only antisense strand distributes in siRNA could first enter the nucleus as duplexes, followed by the cytoplasm of the cells harboring the target RNA, whereas sense active RISC assembly in the nucleus. A third possibility is that strand gets degraded. At the same time, both strands of the latter RISC complexes can be formed both in the cytoplasm and in the siRNA are distributed throughout the cytoplasm and nucleus in nucleus, with or without later redistribution in the cell. In a more cells lacking the silencing target RNA. These results suggest the general context, it is not known whether, after transfection, existence of a mechanism by which the RNAi machinery orches- siRNA is randomly distributed and can form RISC complexes trates a target-determined localization of the siRNA and the throughout the cell, or a more selective cytoplasmic versus corresponding RNAi activity, and also provide evidence for forma- nuclear distribution and localized activity is achieved through tion of nuclear-programmed active RNA induced silencing com- passive or active transport. plexes directly in the nucleus. Results and Discussion confocal imaging ͉ nuclear͞cytoplasmic localization ͉ RNA-induced To investigate these mechanistic possibilities, we used confocal silencing complex ͉ RNA interference mechanism ͉ small interfering RNA imaging to directly visualize the localization of fluorescently labeled siRNA after transfection in human Huh-7 hepatoma cells harboring the subgenomic hepatitis C virus (HCV) repli- early a decade after the discovery that double-stranded con. HCV replication occurs at the virus-induced specialized RNA can trigger an RNAi response that inhibits gene N membrane structures derived from the endoplasmic reticulum, expression in a sequence-specific manner (1), the complexity of which are dispersed in the cytoplasm (14). In the imaging the mechanisms by which small RNAs regulate gene expression experiments, we separately examined the intracellular locations continues to unfold (2–11). RNA interference (RNAi) has of two siRNA duplexes, 7SK siRNA targeting the small nuclear generally been defined as a cellular pathway that mediates 7SK RNA (6, 15–17) and NS3 siRNA that targets the cytoplas- posttranscriptional gene silencing either by sequence-specific mic HCV replicon RNA (18). Each siRNA duplex was labeled degradation of targeted RNAs or via sequence-specific inhibi- with Alexa Fluor 488 and Cy5 fluorophores at the 3Ј ends of tion of translation. Thus, RNAi studies in mammalian cells have sense and antisense strands, respectively, to visualize the local- mainly focused on the cytoplasm, where mature mRNA is izations of the two strands in the cells. All siRNAs were translated and key proteins of RNA-induced silencing complexes determined to be active, and labeled and unlabeled siRNAs had (RISCs) were thought to localize and function. These RISCs, by identical activities within error (Figs. 6 and 7, which are pub- which the RNAi machinery implements silencing of gene ex- lished as supporting information on the PNAS web site). pression, are composed of several proteins (including Ago1 and Two hours after transfection, both sense and antisense strands Ago2) and one strand of small interfering RNA (siRNA) (12, ͞ of 7SK siRNA clearly accumulate in the nuclei of Huh-7 HCV 13). During the course of RISC assembly, the siRNA cells (Fig. 1). Antisense and sense siRNA strands were visualized microRNA duplex dissociates, and the guide strand enters active by using their fluorescent labels, depicted in red (Fig. 1a) and RISCs, allowing binding and degradation of the complementary green (Fig. 1b), respectively, and the nuclei of the cells were target mRNA. stained with DAPI (blue, Fig. 1c). In sharp contrast to these Target specificity in RNAi is achieved through RNA–RNA observations, Huh-7 cells identically transfected with NS3 sequence recognition and base pairing. Because RNA can also siRNA exhibited a completely different localization behavior recognize and form duplexes with DNA, RNAi should be (Fig. 2). In the latter case, no visible accumulation of NS3 capable of affecting gene function at the level of genomic DNA, siRNAs was seen in the nucleus. Furthermore, significant strand- extending the realm of RNAi function into the nucleus. Indeed, separation of NS3 siRNA was observed. The antisense NS3 recent demonstrations of siRNA-induced transcriptional gene silencing through involvement of DNA methylation (2, 3) in various human cell types, siRNA-dependent knock-down of Conflict of interest statement: No conflicts declared. nucleus-restricted transcripts (4, 5), and a direct documentation Abbreviations: RISC, RNA-induced silencing complex; RNAi, RNA interference; HCV, hepa- of potent and specific down-regulation of 7SK and U6 small titis C virus; siRNA, small interfering RNA. nuclear RNAs (6) have uncovered such nuclear RNAi pathways †S.Y.B. and L.S. contributed equally to the work. in human cells. In the latter work, immunoblot analysis and ¶To whom correspondence may be addressed. E-mail: [email protected] or schultz@ activity assays confirmed the presence of the key RISC proteins scripps.edu. Ago1 and Ago2, as well as functional RISC complexes, in both © 2006 by The National Academy of Sciences of the USA 7682–7687 ͉ PNAS ͉ May 16, 2006 ͉ vol. 103 ͉ no. 20 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0600148103 Downloaded by guest on October 1, 2021 CELL BIOLOGY Fig. 1. 7SK siRNA accumulates in the nucleus in Huh-7 cells 2 h after transfection. (a–c) 7SK siRNA antisense (red) and sense (green) strands colocalize primarily in the nucleus (blue), seen in the cytoplasm only in small amounts. (Scale bar, 20 ␮m.) (d) Overlay of red, green, and blue. (e) Two cells. (Scale bar, 10 ␮m.) (f) Intensity profiles along the selected line (red arrow in d) across cells show that both siRNA strands enter the nucleus, where they apparently get separated, because primarily the antisense strand is visible in the nucleoli (marked by arrows, DAPI-depleted dark spots against a blue background in c). siRNA strand was observed to create perinuclear ring-like fection, whereas cytoplasmically targeted NS3 siRNA remains patterns of clustered dots (seen in red in Fig. 2d around nuclei distributed largely in the cytoplasm 4 h after transfection, in blue), whereas the sense strand nearly disappeared and was showing no nuclear accumulation. In addition, we also carried only sparsely visible as distinct dots (Fig. 2b). out 7SK siRNA transfection for HeLa cells, and observed similar Colocalization of the three signal profiles (DAPI, Alexa Fluor behavior. 488, and Cy5) along multiple lines through the cells (examples RISC assembly involves incorporation of the antisense siRNA are given in Figs. 1f and 2f) provided additional information strand into the complex, and therefore requires duplex siRNA about the distributions of these siRNAs. In the case of 7SK strand separation (12, 13). Recent experiments have shown that siRNA, a minor amount of the RNA could be found as a diffuse Ago2 protein likely cleaves the sense strand after siRNA binding, background in the cytoplasm but clusters were typically not resulting in the sense strand dissociation and only antisense observed to concentrate in the perinuclear area. Within the strand incorporation into active RISC (10, 11). Therefore, RISC nuclear boundaries, the two strands showed different levels of formation will be correlated with siRNA duplex strand separa- accumulation in the nucleoli (Fig. 1f, arrows), which could tion in our imaging experiments. Furthermore, because the signify partial strand separation. In the case of NS3 siRNA, sharp incorporated siRNA single strand is known to be tightly bound peaks of the red signal at the edges of nuclear boundaries verify to Ago proteins in RISC (19), the localization of RISC is that the two strands are separated, and the antisense strand tends expected to parallel the observed siRNA

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