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Silencing by small RNAs is linked to endosomal trafficking.

Lee YS, Pressman S, Andress AP, Kim K, White JL, Cassidy JJ, Li X, Lubell K, Lim do H, Cho IS, Nakahara K, Preall JB, Bellare P, Sontheimer EJ, Carthew RW - Nat. Cell Biol. (2009)

Bottom Line: Here, we show that GW-bodies are associated with late endosomes (multivesicular bodies, MVBs).These results indicate that active RISCs are physically and functionally coupled to MVBs.We suggest that the recycling of RISCs is promoted by MVBs, resulting in RISCs more effectively engaging with small RNA effectors and possibly target RNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60201, USA. ys-lee@korea.ac.kr

ABSTRACT
Small RNAs direct RNA-induced silencing complexes (RISCs) to regulate stability and translation of mRNAs. RISCs associated with target mRNAs often accumulate in discrete cytoplasmic foci known as GW-bodies. However, RISC proteins can associate with membrane compartments such as the Golgi and endoplasmic reticulum. Here, we show that GW-bodies are associated with late endosomes (multivesicular bodies, MVBs). Blocking the maturation of MVBs into lysosomes by loss of the tethering factor HPS4 (ref. 5) enhances short interfering RNA (siRNA)- and micro RNA (miRNA)-mediated silencing in Drosophila melanogaster and humans. It also triggers over-accumulation of GW-bodies. Blocking MVB formation by ESCRT (endosomal sorting complex required for transport) depletion results in impaired miRNA silencing and loss of GW-bodies. These results indicate that active RISCs are physically and functionally coupled to MVBs. We further show that MVBs promote the competence of RISCs in loading small RNAs. We suggest that the recycling of RISCs is promoted by MVBs, resulting in RISCs more effectively engaging with small RNA effectors and possibly target RNAs. It may provide a means to enhance the dynamics of RNA silencing in the cytoplasm.

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MVBs are sites of miRNA-mediated silencinga–c, Expression of the GFP::Brd reporter of miRNA silencing in pupal eyes that contain clones of hrsD28 (a,b), and vps25A3 (c) mutant cells. Presumed clone boundaries are marked in yellow. Two hrs clones are shown at different z-planes to highlight GFP up-regulation in cone cells (a) and pigment cells (b). d–e, Expression of a lacZ::E(spl)m8 reporter of miRNA silencing in larval eye discs that contain vps25A3 clones. (d) Whole eye discs that were stained with X-Gal, with the left disc lacking any clones and the right disc containing vps25A3 clones. (e) A region of an eye disc with vps25A3 clones stained for the lacZ::E(spl)m8 reporter protein (green) and Ago1 protein (red). Note the correlation between enhanced lacZ::E(spl)m8 expression and Ago1 concentration in the presumed clones. f–i, Larval eye discs were stained for Ago1 protein (red) to detect miRISC distribution. Magnified views of wildtype (f) and dHPS4W515X mutant (g) eye discs counterstained for nuclei (green). Perinuclear Ago1 localization in wildtype cells is highlighted with arrowheads. This localization is lacking in the mutant cells. Eye discs containing clones of vps25A3 (h), and myopicT612 (i) mutant cells. In these genetically mosaic eyes, mutant cells are visualized by lack of expression of a marker gene (green). Yellow lines mark boundaries of clones. Mutant cells exhibit concentrated Ago1 around large vesicles (arrows). j–k, S2 cells showing Lamp1-RFP (red) that marks MVBs and lysosomes. Cells have been transfected with YFP-GW182 (j) or GFP-Me31b (k) to highlight GW-bodies (green). Note selected examples (arrows) of bodies juxtaposed with Lamp1-positive membranes. l, Distributions of Ago2-GFP cytoplasmic bodies per cell in HeLa cell populations that underwent different siRNA treatments. Indicated in each plot are the siRNAs that were incubated with cells prior to counting GW-body numbers. Inset images are single HeLa cells from selected treatments showing Ago2-GFP bodies.
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Figure 3: MVBs are sites of miRNA-mediated silencinga–c, Expression of the GFP::Brd reporter of miRNA silencing in pupal eyes that contain clones of hrsD28 (a,b), and vps25A3 (c) mutant cells. Presumed clone boundaries are marked in yellow. Two hrs clones are shown at different z-planes to highlight GFP up-regulation in cone cells (a) and pigment cells (b). d–e, Expression of a lacZ::E(spl)m8 reporter of miRNA silencing in larval eye discs that contain vps25A3 clones. (d) Whole eye discs that were stained with X-Gal, with the left disc lacking any clones and the right disc containing vps25A3 clones. (e) A region of an eye disc with vps25A3 clones stained for the lacZ::E(spl)m8 reporter protein (green) and Ago1 protein (red). Note the correlation between enhanced lacZ::E(spl)m8 expression and Ago1 concentration in the presumed clones. f–i, Larval eye discs were stained for Ago1 protein (red) to detect miRISC distribution. Magnified views of wildtype (f) and dHPS4W515X mutant (g) eye discs counterstained for nuclei (green). Perinuclear Ago1 localization in wildtype cells is highlighted with arrowheads. This localization is lacking in the mutant cells. Eye discs containing clones of vps25A3 (h), and myopicT612 (i) mutant cells. In these genetically mosaic eyes, mutant cells are visualized by lack of expression of a marker gene (green). Yellow lines mark boundaries of clones. Mutant cells exhibit concentrated Ago1 around large vesicles (arrows). j–k, S2 cells showing Lamp1-RFP (red) that marks MVBs and lysosomes. Cells have been transfected with YFP-GW182 (j) or GFP-Me31b (k) to highlight GW-bodies (green). Note selected examples (arrows) of bodies juxtaposed with Lamp1-positive membranes. l, Distributions of Ago2-GFP cytoplasmic bodies per cell in HeLa cell populations that underwent different siRNA treatments. Indicated in each plot are the siRNAs that were incubated with cells prior to counting GW-body numbers. Inset images are single HeLa cells from selected treatments showing Ago2-GFP bodies.

Mentions: Does HPS4 affect RNAi because of its role in endosome trafficking? To explore this issue, we disrupted other steps in the endosome trafficking pathway. Normally, cargo from early endosomes arrives at the late endosome en route to the lysosome6. In this transition, regions of the endosomal limiting membrane invaginate into the interior and form lumenal vesicles, generating a late endosome known as the multivesicular body (MVB). The mature MVB packed with vesicles fuses with a lysosome to deliver its cargo. It is this turnover step where HPS4 acts. Upstream of this step, the formation of MVBs requires a number of ESCRT proteins to direct the process14. We eliminated ESCRT factors using mutants, and observed the effects on RNA-mediated silencing. The ESCRT genes hrs and vps25 are required for maturation of early endosomes in Drosophila15–17. We observed impaired miRNA-mediated silencing of the GFP::Brd reporter in hrs and vps25 mutant cells (Fig. 3a–c). A different reporter for miRNA-mediated silencing was also de-repressed in vps25 mutant clones (Fig. 3d–e). Altogether, these data indicate that blocking MVB formation (with ESCRT mutants) inhibits silencing while blocking MVB turnover (with HPS4 mutants) stimulates silencing. These results implicate MVBs as important compartments for RNA silencing.


Silencing by small RNAs is linked to endosomal trafficking.

Lee YS, Pressman S, Andress AP, Kim K, White JL, Cassidy JJ, Li X, Lubell K, Lim do H, Cho IS, Nakahara K, Preall JB, Bellare P, Sontheimer EJ, Carthew RW - Nat. Cell Biol. (2009)

MVBs are sites of miRNA-mediated silencinga–c, Expression of the GFP::Brd reporter of miRNA silencing in pupal eyes that contain clones of hrsD28 (a,b), and vps25A3 (c) mutant cells. Presumed clone boundaries are marked in yellow. Two hrs clones are shown at different z-planes to highlight GFP up-regulation in cone cells (a) and pigment cells (b). d–e, Expression of a lacZ::E(spl)m8 reporter of miRNA silencing in larval eye discs that contain vps25A3 clones. (d) Whole eye discs that were stained with X-Gal, with the left disc lacking any clones and the right disc containing vps25A3 clones. (e) A region of an eye disc with vps25A3 clones stained for the lacZ::E(spl)m8 reporter protein (green) and Ago1 protein (red). Note the correlation between enhanced lacZ::E(spl)m8 expression and Ago1 concentration in the presumed clones. f–i, Larval eye discs were stained for Ago1 protein (red) to detect miRISC distribution. Magnified views of wildtype (f) and dHPS4W515X mutant (g) eye discs counterstained for nuclei (green). Perinuclear Ago1 localization in wildtype cells is highlighted with arrowheads. This localization is lacking in the mutant cells. Eye discs containing clones of vps25A3 (h), and myopicT612 (i) mutant cells. In these genetically mosaic eyes, mutant cells are visualized by lack of expression of a marker gene (green). Yellow lines mark boundaries of clones. Mutant cells exhibit concentrated Ago1 around large vesicles (arrows). j–k, S2 cells showing Lamp1-RFP (red) that marks MVBs and lysosomes. Cells have been transfected with YFP-GW182 (j) or GFP-Me31b (k) to highlight GW-bodies (green). Note selected examples (arrows) of bodies juxtaposed with Lamp1-positive membranes. l, Distributions of Ago2-GFP cytoplasmic bodies per cell in HeLa cell populations that underwent different siRNA treatments. Indicated in each plot are the siRNAs that were incubated with cells prior to counting GW-body numbers. Inset images are single HeLa cells from selected treatments showing Ago2-GFP bodies.
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Figure 3: MVBs are sites of miRNA-mediated silencinga–c, Expression of the GFP::Brd reporter of miRNA silencing in pupal eyes that contain clones of hrsD28 (a,b), and vps25A3 (c) mutant cells. Presumed clone boundaries are marked in yellow. Two hrs clones are shown at different z-planes to highlight GFP up-regulation in cone cells (a) and pigment cells (b). d–e, Expression of a lacZ::E(spl)m8 reporter of miRNA silencing in larval eye discs that contain vps25A3 clones. (d) Whole eye discs that were stained with X-Gal, with the left disc lacking any clones and the right disc containing vps25A3 clones. (e) A region of an eye disc with vps25A3 clones stained for the lacZ::E(spl)m8 reporter protein (green) and Ago1 protein (red). Note the correlation between enhanced lacZ::E(spl)m8 expression and Ago1 concentration in the presumed clones. f–i, Larval eye discs were stained for Ago1 protein (red) to detect miRISC distribution. Magnified views of wildtype (f) and dHPS4W515X mutant (g) eye discs counterstained for nuclei (green). Perinuclear Ago1 localization in wildtype cells is highlighted with arrowheads. This localization is lacking in the mutant cells. Eye discs containing clones of vps25A3 (h), and myopicT612 (i) mutant cells. In these genetically mosaic eyes, mutant cells are visualized by lack of expression of a marker gene (green). Yellow lines mark boundaries of clones. Mutant cells exhibit concentrated Ago1 around large vesicles (arrows). j–k, S2 cells showing Lamp1-RFP (red) that marks MVBs and lysosomes. Cells have been transfected with YFP-GW182 (j) or GFP-Me31b (k) to highlight GW-bodies (green). Note selected examples (arrows) of bodies juxtaposed with Lamp1-positive membranes. l, Distributions of Ago2-GFP cytoplasmic bodies per cell in HeLa cell populations that underwent different siRNA treatments. Indicated in each plot are the siRNAs that were incubated with cells prior to counting GW-body numbers. Inset images are single HeLa cells from selected treatments showing Ago2-GFP bodies.
Mentions: Does HPS4 affect RNAi because of its role in endosome trafficking? To explore this issue, we disrupted other steps in the endosome trafficking pathway. Normally, cargo from early endosomes arrives at the late endosome en route to the lysosome6. In this transition, regions of the endosomal limiting membrane invaginate into the interior and form lumenal vesicles, generating a late endosome known as the multivesicular body (MVB). The mature MVB packed with vesicles fuses with a lysosome to deliver its cargo. It is this turnover step where HPS4 acts. Upstream of this step, the formation of MVBs requires a number of ESCRT proteins to direct the process14. We eliminated ESCRT factors using mutants, and observed the effects on RNA-mediated silencing. The ESCRT genes hrs and vps25 are required for maturation of early endosomes in Drosophila15–17. We observed impaired miRNA-mediated silencing of the GFP::Brd reporter in hrs and vps25 mutant cells (Fig. 3a–c). A different reporter for miRNA-mediated silencing was also de-repressed in vps25 mutant clones (Fig. 3d–e). Altogether, these data indicate that blocking MVB formation (with ESCRT mutants) inhibits silencing while blocking MVB turnover (with HPS4 mutants) stimulates silencing. These results implicate MVBs as important compartments for RNA silencing.

Bottom Line: Here, we show that GW-bodies are associated with late endosomes (multivesicular bodies, MVBs).These results indicate that active RISCs are physically and functionally coupled to MVBs.We suggest that the recycling of RISCs is promoted by MVBs, resulting in RISCs more effectively engaging with small RNA effectors and possibly target RNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60201, USA. ys-lee@korea.ac.kr

ABSTRACT
Small RNAs direct RNA-induced silencing complexes (RISCs) to regulate stability and translation of mRNAs. RISCs associated with target mRNAs often accumulate in discrete cytoplasmic foci known as GW-bodies. However, RISC proteins can associate with membrane compartments such as the Golgi and endoplasmic reticulum. Here, we show that GW-bodies are associated with late endosomes (multivesicular bodies, MVBs). Blocking the maturation of MVBs into lysosomes by loss of the tethering factor HPS4 (ref. 5) enhances short interfering RNA (siRNA)- and micro RNA (miRNA)-mediated silencing in Drosophila melanogaster and humans. It also triggers over-accumulation of GW-bodies. Blocking MVB formation by ESCRT (endosomal sorting complex required for transport) depletion results in impaired miRNA silencing and loss of GW-bodies. These results indicate that active RISCs are physically and functionally coupled to MVBs. We further show that MVBs promote the competence of RISCs in loading small RNAs. We suggest that the recycling of RISCs is promoted by MVBs, resulting in RISCs more effectively engaging with small RNA effectors and possibly target RNAs. It may provide a means to enhance the dynamics of RNA silencing in the cytoplasm.

Show MeSH
Related in: MedlinePlus