Limits...
Human nuclear Dicer restricts the deleterious accumulation of endogenous double-stranded RNA.

White E, Schlackow M, Kamieniarz-Gdula K, Proudfoot NJ, Gullerova M - Nat. Struct. Mol. Biol. (2014)

Bottom Line: Dicer interacts with RNA polymerase II (Pol II) at actively transcribed gene loci.Our results suggest that Pol II-associated Dicer restricts endogenous dsRNA formation from overlapping noncoding-RNA transcription units.Failure to do so has catastrophic effects on cell function.

View Article: PubMed Central - PubMed

Affiliation: Sir William Dunn School of Pathology, University of Oxford, Oxford, UK.

ABSTRACT
Dicer is a central enzymatic player in RNA-interference pathways that acts to regulate gene expression in nearly all eukaryotes. Although the cytoplasmic function of Dicer is well documented in mammals, its nuclear function remains obscure. Here we show that Dicer is present in both the nucleus and cytoplasm, and its nuclear levels are tightly regulated. Dicer interacts with RNA polymerase II (Pol II) at actively transcribed gene loci. Loss of Dicer causes the appearance of endogenous double-stranded RNA (dsRNA), which in turn leads to induction of the interferon-response pathway and consequent cell death. Our results suggest that Pol II-associated Dicer restricts endogenous dsRNA formation from overlapping noncoding-RNA transcription units. Failure to do so has catastrophic effects on cell function.

Show MeSH
Dicer localizes in both nucleus and cytoplasm of HEK293 cellsa) Immunofluorescence analyses of HEK293 cells with an integrated inducible Dicer shRNA expression cassette, using DAPI staining (blue) and anti-Dicer antibody (green). Top panels: Uninduced cells (normal),showing cytoplasmic and nuclear localization of Dicer Bottom panels: Induction of the shRNA cassette (Dicer knockdown), Dicer signals are absent (lower panels).b) Expression of GFP-Dicer or GFP alone (green) in HEK293 cells; nucleus stained by DAPI (blue). Uninduced cells (normal) show no colocalization of GFP-Dicer signals with DAPI staining. In contrast GFP colocalizes with DAPI (top two rows). Following Dicer knockdown (bottom panels), GFP-Dicer, like GFP, localizes in both cytoplasm and nucleus. Although GFP-Dicer is ultimately down-regulated by Dicer shRNA expression, it is expressed at detectable levels during experimental time frame.c) FRAP analysis measuring GFP-Dicer nuclear dynamics in Dicer knockdown cells transfected with Dicer-GFP. Top, fluorescence microscopy shows GFP-Dicer in green. Left, the cell before bleaching. Middle, the red circle corresponds to bleached area; fluorescent recovery was measured in the green circle; fluorescence background values were measured in the blue circle. Right, the cell after recovery. Bottom left, absolute levels of fluorescence; bottom right, relative levels of fluorescence (subtracting background fluorescence). All experiments described in Fig. 1a-c were independently replicated three times.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4129937&req=5

Figure 1: Dicer localizes in both nucleus and cytoplasm of HEK293 cellsa) Immunofluorescence analyses of HEK293 cells with an integrated inducible Dicer shRNA expression cassette, using DAPI staining (blue) and anti-Dicer antibody (green). Top panels: Uninduced cells (normal),showing cytoplasmic and nuclear localization of Dicer Bottom panels: Induction of the shRNA cassette (Dicer knockdown), Dicer signals are absent (lower panels).b) Expression of GFP-Dicer or GFP alone (green) in HEK293 cells; nucleus stained by DAPI (blue). Uninduced cells (normal) show no colocalization of GFP-Dicer signals with DAPI staining. In contrast GFP colocalizes with DAPI (top two rows). Following Dicer knockdown (bottom panels), GFP-Dicer, like GFP, localizes in both cytoplasm and nucleus. Although GFP-Dicer is ultimately down-regulated by Dicer shRNA expression, it is expressed at detectable levels during experimental time frame.c) FRAP analysis measuring GFP-Dicer nuclear dynamics in Dicer knockdown cells transfected with Dicer-GFP. Top, fluorescence microscopy shows GFP-Dicer in green. Left, the cell before bleaching. Middle, the red circle corresponds to bleached area; fluorescent recovery was measured in the green circle; fluorescence background values were measured in the blue circle. Right, the cell after recovery. Bottom left, absolute levels of fluorescence; bottom right, relative levels of fluorescence (subtracting background fluorescence). All experiments described in Fig. 1a-c were independently replicated three times.

Mentions: We next carried out immunofluorescence analysis on these HEK293 cells using confocal microscopy. As shown in representative cell images (Fig. 1a), strong Dicer signals were detected especially around the cytoplasmic side of the nuclear periphery, where pre-microRNAs are processed into mature microRNAs2. However, a clear punctuate pattern was also visible throughout the nucleoplasm; these signals were absent following anti-Dicer shRNA induction.


Human nuclear Dicer restricts the deleterious accumulation of endogenous double-stranded RNA.

White E, Schlackow M, Kamieniarz-Gdula K, Proudfoot NJ, Gullerova M - Nat. Struct. Mol. Biol. (2014)

Dicer localizes in both nucleus and cytoplasm of HEK293 cellsa) Immunofluorescence analyses of HEK293 cells with an integrated inducible Dicer shRNA expression cassette, using DAPI staining (blue) and anti-Dicer antibody (green). Top panels: Uninduced cells (normal),showing cytoplasmic and nuclear localization of Dicer Bottom panels: Induction of the shRNA cassette (Dicer knockdown), Dicer signals are absent (lower panels).b) Expression of GFP-Dicer or GFP alone (green) in HEK293 cells; nucleus stained by DAPI (blue). Uninduced cells (normal) show no colocalization of GFP-Dicer signals with DAPI staining. In contrast GFP colocalizes with DAPI (top two rows). Following Dicer knockdown (bottom panels), GFP-Dicer, like GFP, localizes in both cytoplasm and nucleus. Although GFP-Dicer is ultimately down-regulated by Dicer shRNA expression, it is expressed at detectable levels during experimental time frame.c) FRAP analysis measuring GFP-Dicer nuclear dynamics in Dicer knockdown cells transfected with Dicer-GFP. Top, fluorescence microscopy shows GFP-Dicer in green. Left, the cell before bleaching. Middle, the red circle corresponds to bleached area; fluorescent recovery was measured in the green circle; fluorescence background values were measured in the blue circle. Right, the cell after recovery. Bottom left, absolute levels of fluorescence; bottom right, relative levels of fluorescence (subtracting background fluorescence). All experiments described in Fig. 1a-c were independently replicated three times.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4129937&req=5

Figure 1: Dicer localizes in both nucleus and cytoplasm of HEK293 cellsa) Immunofluorescence analyses of HEK293 cells with an integrated inducible Dicer shRNA expression cassette, using DAPI staining (blue) and anti-Dicer antibody (green). Top panels: Uninduced cells (normal),showing cytoplasmic and nuclear localization of Dicer Bottom panels: Induction of the shRNA cassette (Dicer knockdown), Dicer signals are absent (lower panels).b) Expression of GFP-Dicer or GFP alone (green) in HEK293 cells; nucleus stained by DAPI (blue). Uninduced cells (normal) show no colocalization of GFP-Dicer signals with DAPI staining. In contrast GFP colocalizes with DAPI (top two rows). Following Dicer knockdown (bottom panels), GFP-Dicer, like GFP, localizes in both cytoplasm and nucleus. Although GFP-Dicer is ultimately down-regulated by Dicer shRNA expression, it is expressed at detectable levels during experimental time frame.c) FRAP analysis measuring GFP-Dicer nuclear dynamics in Dicer knockdown cells transfected with Dicer-GFP. Top, fluorescence microscopy shows GFP-Dicer in green. Left, the cell before bleaching. Middle, the red circle corresponds to bleached area; fluorescent recovery was measured in the green circle; fluorescence background values were measured in the blue circle. Right, the cell after recovery. Bottom left, absolute levels of fluorescence; bottom right, relative levels of fluorescence (subtracting background fluorescence). All experiments described in Fig. 1a-c were independently replicated three times.
Mentions: We next carried out immunofluorescence analysis on these HEK293 cells using confocal microscopy. As shown in representative cell images (Fig. 1a), strong Dicer signals were detected especially around the cytoplasmic side of the nuclear periphery, where pre-microRNAs are processed into mature microRNAs2. However, a clear punctuate pattern was also visible throughout the nucleoplasm; these signals were absent following anti-Dicer shRNA induction.

Bottom Line: Dicer interacts with RNA polymerase II (Pol II) at actively transcribed gene loci.Our results suggest that Pol II-associated Dicer restricts endogenous dsRNA formation from overlapping noncoding-RNA transcription units.Failure to do so has catastrophic effects on cell function.

View Article: PubMed Central - PubMed

Affiliation: Sir William Dunn School of Pathology, University of Oxford, Oxford, UK.

ABSTRACT
Dicer is a central enzymatic player in RNA-interference pathways that acts to regulate gene expression in nearly all eukaryotes. Although the cytoplasmic function of Dicer is well documented in mammals, its nuclear function remains obscure. Here we show that Dicer is present in both the nucleus and cytoplasm, and its nuclear levels are tightly regulated. Dicer interacts with RNA polymerase II (Pol II) at actively transcribed gene loci. Loss of Dicer causes the appearance of endogenous double-stranded RNA (dsRNA), which in turn leads to induction of the interferon-response pathway and consequent cell death. Our results suggest that Pol II-associated Dicer restricts endogenous dsRNA formation from overlapping noncoding-RNA transcription units. Failure to do so has catastrophic effects on cell function.

Show MeSH