DICER1 deficit induces Alu RNA toxicity in age-related macular degeneration

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Abstract

Geographic atrophy (GA), an untreatable advanced form of age-related macular degeneration, results from retinal pigmented epithelium (RPE) cell death. Here we show that the microRNA (miRNA)-processing enzyme DICER1 is reduced in the RPE of humans with GA, and that conditional ablation of Dicer1, but not seven other miRNA-processing enzymes, induces RPE degeneration in mice. DICER1 knockdown induces accumulation of Alu RNA in human RPE cells and Alu-like B1 and B2 RNAs in mouse RPE. Alu RNA is increased in the RPE of humans with GA, and this pathogenic RNA induces human RPE cytotoxicity and RPE degeneration in mice. Antisense oligonucleotides targeting Alu/B1/B2 RNAs prevent DICER1 depletion-induced RPE degeneration despite global miRNA downregulation. DICER1 degrades Alu RNA, and this digested Alu RNA cannot induce RPE degeneration in mice. These findings reveal a miRNA-independent cell survival function for DICER1 involving retrotransposon transcript degradation, show that Alu RNA can directly cause human pathology, and identify new targets for a major cause of blindness.

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Most cited references 40

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Argonaute2 is the catalytic engine of mammalian RNAi.

Jidong Liu, Michelle A Carmell, Fabiola V Rivas … (2004)

Gene silencing through RNA interference (RNAi) is carried out by RISC, the RNA-induced silencing complex. RISC contains two signature components, small interfering RNAs (siRNAs) and Argonaute family proteins. Here, we show that the multiple Argonaute proteins present in mammals are both biologically and biochemically distinct, with a single mammalian family member, Argonaute2, being responsible for messenger RNA cleavage activity. This protein is essential for mouse development, and cells lacking Argonaute2 are unable to mount an experimental response to siRNAs. Mutations within a cryptic ribonuclease H domain within Argonaute2, as identified by comparison with the structure of an archeal Argonaute protein, inactivate RISC. Thus, our evidence supports a model in which Argonaute contributes "Slicer" activity to RISC, providing the catalytic engine for RNAi.

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Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs.

Gunter Meister, Markus Landthaler, Agnieszka Patkaniowska … (2004)

Argonaute proteins associate with small RNAs that guide mRNA degradation, translational repression, or a combination of both. The human Argonaute family has eight members, four of which (Ago1 through Ago4) are closely related and coexpressed in many cell types. To understand the biological function of the different Ago proteins, we set out to determine if Ago1 through Ago4 are associated with miRNAs as well as RISC activity in human cell lines. Our results suggest that miRNAs are incorporated indiscriminately of their sequence into Ago1 through Ago4 containing microRNPs (miRNPs). Purification of the FLAG/HA-epitope-tagged Ago containing complexes from different human cell lines revealed that endonuclease activity is exclusively associated with Ago2. Exogenously introduced siRNAs also associate with Ago2 for guiding target RNA cleavage. The specific role of Ago2 in guiding target RNA cleavage was confirmed independently by siRNA-based depletion of individual Ago members in combination with a sensitive positive-readout reporter assay.

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Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes.

Toshiaki Watanabe, Yasushi Totoki, Atsushi Toyoda … (2008)

RNA interference (RNAi) is a mechanism by which double-stranded RNAs (dsRNAs) suppress specific transcripts in a sequence-dependent manner. dsRNAs are processed by Dicer to 21-24-nucleotide small interfering RNAs (siRNAs) and then incorporated into the argonaute (Ago) proteins. Gene regulation by endogenous siRNAs has been observed only in organisms possessing RNA-dependent RNA polymerase (RdRP). In mammals, where no RdRP activity has been found, biogenesis and function of endogenous siRNAs remain largely unknown. Here we show, using mouse oocytes, that endogenous siRNAs are derived from naturally occurring dsRNAs and have roles in the regulation of gene expression. By means of deep sequencing, we identify a large number of both approximately 25-27-nucleotide Piwi-interacting RNAs (piRNAs) and approximately 21-nucleotide siRNAs corresponding to messenger RNAs or retrotransposons in growing oocytes. piRNAs are bound to Mili and have a role in the regulation of retrotransposons. siRNAs are exclusively mapped to retrotransposons or other genomic regions that produce transcripts capable of forming dsRNA structures. Inverted repeat structures, bidirectional transcription and antisense transcripts from various loci are sources of the dsRNAs. Some precursor transcripts of siRNAs are derived from expressed pseudogenes, indicating that one role of pseudogenes is to adjust the level of the founding source mRNA through RNAi. Loss of Dicer or Ago2 results in decreased levels of siRNAs and increased levels of retrotransposon and protein-coding transcripts complementary to the siRNAs. Thus, the RNAi pathway regulates both protein-coding transcripts and retrotransposons in mouse oocytes. Our results reveal a role for endogenous siRNAs in mammalian oocytes and show that organisms lacking RdRP activity can produce functional endogenous siRNAs from naturally occurring dsRNAs.

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Author and article information

Journal

Journal ID (nlm-journal-id): 0410462

Journal ID (pubmed-jr-id): 6011

Journal ID (nlm-ta): Nature

Journal ID (iso-abbrev): Nature

Title: Nature

ISSN (Print): 0028-0836

ISSN (Electronic): 1476-4687

Publication date Nihms-submitted: 20 January 2011

Publication date (Electronic): 06 February 2011

Publication date (Print): 17 March 2011

Publication date PMC-release: 17 September 2011

Volume: 471

Issue: 7338

Pages: 325-330

Affiliations

[1 ]Department of Ophthalmology & Visual Sciences, University of Kentucky, Lexington, Kentucky 40506, USA

[2 ]Department of Physiology, University of Kentucky, Lexington, Kentucky 40506, USA

[3 ]Department of Internal Medicine, University of Kentucky, Lexington, Kentucky 40506, USA

[4 ]Department of Anatomy, Yonsei University Wonju College of Medicine, Wonju City 220-701, Korea

[5 ]Department of Chemistry and Biochemistry, University of Colorado at Boulder, Boulder, Colorado 80309, USA

[6 ]Department of Ophthalmology, University of Florida, Gainesville, Florida 32610, USA

[7 ]Department of Neurosurgery, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA

[8 ]Global Research Laboratory for RNAi Medicine & BK21 School of Chemical Materials Science and Department of Chemistry, Sungkyunkwan University, Suwon 440-746, Korea

[9 ]F.M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA

[10 ]Department of Microbiology and Immunology, Meharry Medical College, Nashville, TN 37208, USA

[11 ]The Jackson Laboratory, Bar Harbor, ME 04609, USA

[12 ]The Arnold and Mabel Beckman Macular Research Center at the Doheny Eye Institute, University of Southern California, Los Angeles, California 90033, USA

[13 ]Departments of Ophthalmology & Pathology, Emory University Atlanta, Georgia 30322, USA

[14 ]ARC Centre of Excellence in Vision Science and Research School of Biology & ANU Medical School, The Australian National University, Canberra, ACT 0200, Australia

[15 ]School of Optometry and Vision Science, The University of NSW, Kensington, NSW 2033 & and Save Sight Institute, The University of Sydney, Sydney, NSW 2001, Australia

[16 ]Howard Hughes Medical Institute, Laboratory of Mammalian Cell Biology and Development, The Rockefeller University, New York, New York 10065, USA

[17 ]Howard Hughes Medical Institute, The Kimmel Center for Biology and Medicine of the Skirball Institute, New York University School of Medicine, New York, New York 10016, USA

[18 ]Department of Ophthalmology and Visual Sciences, Moran Eye Center, University of Utah School of Medicine, Salt Lake City, Utah 84132, USA

[19 ]Department of Ophthalmology, Veterans Affairs Salt Lake City Healthcare System, Salt Lake City, Utah 84148, USA

[20 ]Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Johns Hopkins University, Baltimore, Maryland, 21231, USA

[21 ]The Walter and Eliza Hall Institute, Autoimmunity and Transplantation Division, Parkville, VIC 3052, Australia

[22 ]CHUL Research Center/CHUQ and Faculty of Medicine, Université Laval, Quebec, QC G1K 7P4, Canada

Author notes

Correspondence and requests for materials should be addressed to J.A. ( jamba2@ 123456email.uky.edu )

[*]

These authors contributed equally to this work.

Article

Manuscript ID: NIHMS265989

DOI: 10.1038/nature09830

PMC ID: 3077055

PubMed ID: 21297615

SO-VID: 44d30993-b80b-48a1-9bb5-432cc76ebcdd

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