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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.

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Loss of Dicer leads to a reduction of siRNA that co-localise with chromatin-associated Dicer sites and dsRNA sitesa) Northern Blot analysis of small RNA isolated from normal and Dicer knockdown HEK293 cells and hybridized to specific 32P-radiolabelled probes. Probes against GAPDH and U6 snRNA were used as negative and loading controls respectively. Signals were visualised by PhosphoImager. See Supplementary Fig. 9 for uncropped blot images.b) Snapshot of the three data sets in the UCSC genome browser; the examples show the intergenic region of KPNA2 and the terminator region of MTRNR2L6 (as in Fig. 5a). The last three panels show small RNAs isolated from whole cell, cytosolic and nuclear fractions of IMR90 cells respectively.c) Left: Distribution of Dicer binding sites that overlap with sites of dsRNA and siRNA based on small RNA RNA-seq data. Right: Division of loci where Dicer, dsRNA and siRNA co-localise, according to dsRNA levels increasing (blue) or remaining the same (red) upon Dicer depletion.
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Figure 6: Loss of Dicer leads to a reduction of siRNA that co-localise with chromatin-associated Dicer sites and dsRNA sitesa) Northern Blot analysis of small RNA isolated from normal and Dicer knockdown HEK293 cells and hybridized to specific 32P-radiolabelled probes. Probes against GAPDH and U6 snRNA were used as negative and loading controls respectively. Signals were visualised by PhosphoImager. See Supplementary Fig. 9 for uncropped blot images.b) Snapshot of the three data sets in the UCSC genome browser; the examples show the intergenic region of KPNA2 and the terminator region of MTRNR2L6 (as in Fig. 5a). The last three panels show small RNAs isolated from whole cell, cytosolic and nuclear fractions of IMR90 cells respectively.c) Left: Distribution of Dicer binding sites that overlap with sites of dsRNA and siRNA based on small RNA RNA-seq data. Right: Division of loci where Dicer, dsRNA and siRNA co-localise, according to dsRNA levels increasing (blue) or remaining the same (red) upon Dicer depletion.

Mentions: The co-occurrence of dsRNA and Dicer genome-wide, and the fact that dsRNA accumulates upon Dicer depletion, suggests that dsRNA is processed into siRNA. For three of the four Dicer-positive loci, tested in these studies (Fig. 2 and 3), siRNAs were detected above background levels using a chemical cross-linking Northern blot technique. Furthermore, these weak siRNA signals were reduced following Dicer knockdown (Fig. 6a). No RdRP activity has been observed in mammals, unlike in S. pombe and plants, where this RNA polymerase acts to amplify the levels of siRNA allowing their ready detection3,14. In spite of this, small 5′ monophosphate RNAs have been detected by genomic sequencing in various cell lines33. Examination of these small RNAs from IMR90 cells revealed the presence of small RNAs associated with the co-localising Dicer binding sites and dsRNA loci (Fig. 6b). We note that these small RNA clusters appear consistent between the various cell lines tested33. Bioinformatic analysis revealed that there is substantial overlap (97%) between Dicer binding sites, dsRNA loci and sites of small RNA detection (Fig. 6c) suggesting that these small RNAs are siRNAs. Furthermore, Dicer depletion leads to accumulation of dsRNA in 94% of cases, implying an RNAi associated mechanism (Fig. 6c).


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)

Loss of Dicer leads to a reduction of siRNA that co-localise with chromatin-associated Dicer sites and dsRNA sitesa) Northern Blot analysis of small RNA isolated from normal and Dicer knockdown HEK293 cells and hybridized to specific 32P-radiolabelled probes. Probes against GAPDH and U6 snRNA were used as negative and loading controls respectively. Signals were visualised by PhosphoImager. See Supplementary Fig. 9 for uncropped blot images.b) Snapshot of the three data sets in the UCSC genome browser; the examples show the intergenic region of KPNA2 and the terminator region of MTRNR2L6 (as in Fig. 5a). The last three panels show small RNAs isolated from whole cell, cytosolic and nuclear fractions of IMR90 cells respectively.c) Left: Distribution of Dicer binding sites that overlap with sites of dsRNA and siRNA based on small RNA RNA-seq data. Right: Division of loci where Dicer, dsRNA and siRNA co-localise, according to dsRNA levels increasing (blue) or remaining the same (red) upon Dicer depletion.
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Figure 6: Loss of Dicer leads to a reduction of siRNA that co-localise with chromatin-associated Dicer sites and dsRNA sitesa) Northern Blot analysis of small RNA isolated from normal and Dicer knockdown HEK293 cells and hybridized to specific 32P-radiolabelled probes. Probes against GAPDH and U6 snRNA were used as negative and loading controls respectively. Signals were visualised by PhosphoImager. See Supplementary Fig. 9 for uncropped blot images.b) Snapshot of the three data sets in the UCSC genome browser; the examples show the intergenic region of KPNA2 and the terminator region of MTRNR2L6 (as in Fig. 5a). The last three panels show small RNAs isolated from whole cell, cytosolic and nuclear fractions of IMR90 cells respectively.c) Left: Distribution of Dicer binding sites that overlap with sites of dsRNA and siRNA based on small RNA RNA-seq data. Right: Division of loci where Dicer, dsRNA and siRNA co-localise, according to dsRNA levels increasing (blue) or remaining the same (red) upon Dicer depletion.
Mentions: The co-occurrence of dsRNA and Dicer genome-wide, and the fact that dsRNA accumulates upon Dicer depletion, suggests that dsRNA is processed into siRNA. For three of the four Dicer-positive loci, tested in these studies (Fig. 2 and 3), siRNAs were detected above background levels using a chemical cross-linking Northern blot technique. Furthermore, these weak siRNA signals were reduced following Dicer knockdown (Fig. 6a). No RdRP activity has been observed in mammals, unlike in S. pombe and plants, where this RNA polymerase acts to amplify the levels of siRNA allowing their ready detection3,14. In spite of this, small 5′ monophosphate RNAs have been detected by genomic sequencing in various cell lines33. Examination of these small RNAs from IMR90 cells revealed the presence of small RNAs associated with the co-localising Dicer binding sites and dsRNA loci (Fig. 6b). We note that these small RNA clusters appear consistent between the various cell lines tested33. Bioinformatic analysis revealed that there is substantial overlap (97%) between Dicer binding sites, dsRNA loci and sites of small RNA detection (Fig. 6c) suggesting that these small RNAs are siRNAs. Furthermore, Dicer depletion leads to accumulation of dsRNA in 94% of cases, implying an RNAi associated mechanism (Fig. 6c).

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