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PMID: 17237357 Published · ppublish English Journal Article Research Support, N.I.H., Extramural

Comparison of siRNA-induced off-target RNA and protein effects.

RNA (New York, N.Y.) ·Vol. 13 ·No. 3 ·2007-03-00 ·Pages 385-95

Alemán LM, Doench J, Sharp PA

Abstract

The downregulation of many mRNAs has been observed through bioinformatic analysis of microarray results following transfection of short interfering RNAs (siRNAs). Many of these mRNA changes are due to the interaction of the siRNA guide strand with partially complementary sites and thus are considered "off-target" effects. To examine the mRNA:siRNA interactions important for off-target effects, we generated a panel of mRNA:siRNA combinations containing single and double mismatches, bulges, and noncanonical base-pairing interactions in the 9th, 10th, and 11th positions of two siRNA binding sites located in the 3' UTR of an integrated reporter gene. Approximately half of the mRNA:siRNA combinations containing mismatches in positions 9-11 result in a twofold or more mRNA decrease with varying degrees of protein knockdown. However, mRNA and protein analysis of the various mRNA:siRNA combinations reveals instances in which mRNA and protein levels do not correlate. Analysis of the resulting degradation products recovered from an imperfectly complementary siRNA interaction with an endogenous gene reveals a small fraction of products that map to the canonical siRNA cleavage site. Furthermore, downregulation of ARGONAUTE 2 (AGO2), the only AGO family protein known to catalyze canonical siRNA-mediated cleavage, did not significantly affect the degree of mRNA knockdown observed for one of the stably expressed reporters after transfection of an imperfectly complementary siRNA. These results indicate that although some degree of canonical siRNA cleavage can take place between a siRNA and an off-target transcript, most off-target mRNA reductions are likely attributable to AGO2-independent degradation processes.

MeSH Terms
Animals Argonaute Proteins Base Sequence Eukaryotic Initiation Factor-2 Gene Expression Regulation Genes, Reporter Humans Luciferases, Renilla/analysis,genetics Molecular Sequence Data Peptide Initiation Factors/antagonists & inhibitors,genetics,metabolism Protein Biosynthesis/genetics RNA Stability RNA, Messenger/antagonists & inhibitors,metabolism RNA, Small Interfering/metabolism Transcription, Genetic
Chemicals
AGO2 protein, human Argonaute Proteins Eukaryotic Initiation Factor-2 Peptide Initiation Factors RNA, Messenger RNA, Small Interfering Luciferases, Renilla
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Alemán Lourdes M
Center for Cancer Research, Massachusetts Institute of Technology, Cambridge 02139, USA,
Doench John
Sharp Phillip A
References (43)
43 references, click to expand
  1. Kinetic analysis of the RNAi enzyme complex.
    Nat Struct Mol Biol. 2004 Jul;11(7):599-606 PMID: 15170178
  2. AtXRN4 degrades mRNA in Arabidopsis and its substrates include selected miRNA targets.
    Mol Cell. 2004 Jul 23;15(2):173-83 PMID: 15260969
  3. Crystal structure of Argonaute and its implications for RISC slicer activity.
    Science. 2004 Sep 3;305(5689):1434-7 PMID: 15284453
  4. Argonaute2 is the catalytic engine of mammalian RNAi.
    Science. 2004 Sep 3;305(5689):1437-41 PMID: 15284456
  5. Chemiluminescent and bioluminescent reporter gene assays.
    Anal Biochem. 1994 Jun;219(2):169-81 PMID: 8080073
  6. A link between mRNA turnover and RNA interference in Arabidopsis.
    Science. 2004 Nov 5;306(5698):1046-8 PMID: 15528448
  7. Substrate requirements for let-7 function in the developing zebrafish embryo.
    Nucleic Acids Res. 2004;32(21):6284-91 PMID: 15585662
  8. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets.
    Cell. 2005 Jan 14;120(1):15-20 PMID: 15652477
  9. Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs.
    Nature. 2005 Feb 17;433(7027):769-73 PMID: 15685193
  10. Involvement of microRNA in AU-rich element-mediated mRNA instability.
    Cell. 2005 Mar 11;120(5):623-34 PMID: 15766526
  11. Decay of mRNAs targeted by RISC requires XRN1, the Ski complex, and the exosome.
    RNA. 2005 Apr;11(4):459-69 PMID: 15703439
  12. Principles of microRNA-target recognition.
    PLoS Biol. 2005 Mar;3(3):e85 PMID: 15723116
  13. Argonaute 2/RISC resides in sites of mammalian mRNA decay known as cytoplasmic bodies.
    Nat Cell Biol. 2005 Jun;7(6):633-6 PMID: 15908945
  14. siRNA-mediated off-target gene silencing triggered by a 7 nt complementation.
    Nucleic Acids Res. 2005;33(14):4527-35 PMID: 16091630
  15. Tolerated wobble mutations in siRNAs decrease specificity, but can enhance activity in vivo.
    Nucleic Acids Res. 2005;33(15):4704-10 PMID: 16113241
  16. Regulation by let-7 and lin-4 miRNAs results in target mRNA degradation.
    Cell. 2005 Aug 26;122(4):553-63 PMID: 16122423
  17. Micro-RNA regulation of the mammalian lin-28 gene during neuronal differentiation of embryonal carcinoma cells.
    Mol Cell Biol. 2005 Nov;25(21):9198-208 PMID: 16227573
  18. Disruption of GW bodies impairs mammalian RNA interference.
    Nat Cell Biol. 2005 Dec;7(12):1267-74 PMID: 16284622
  19. A role for the P-body component GW182 in microRNA function.
    Nat Cell Biol. 2005 Dec;7(12):1261-6 PMID: 16284623
  20. Short RNAs repress translation after initiation in mammalian cells.
    Mol Cell. 2006 Feb 17;21(4):533-42 PMID: 16483934
  21. 3' UTR seed matches, but not overall identity, are associated with RNAi off-targets.
    Nat Methods. 2006 Mar;3(3):199-204 PMID: 16489337
  22. MicroRNAs direct rapid deadenylation of mRNA.
    Proc Natl Acad Sci U S A. 2006 Mar 14;103(11):4034-9 PMID: 16495412
  23. Genome-wide analysis of mRNAs regulated by Drosha and Argonaute proteins in Drosophila melanogaster.
    Mol Cell Biol. 2006 Apr;26(8):2965-75 PMID: 16581772
  24. Zebrafish MiR-430 promotes deadenylation and clearance of maternal mRNAs.
    Science. 2006 Apr 7;312(5770):75-9 PMID: 16484454
  25. Widespread siRNA "off-target" transcript silencing mediated by seed region sequence complementarity.
    RNA. 2006 Jul;12(7):1179-87 PMID: 16682560
  26. Off-target effects by siRNA can induce toxic phenotype.
    RNA. 2006 Jul;12(7):1188-96 PMID: 16682561
  27. mRNA degradation by miRNAs and GW182 requires both CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes.
    Genes Dev. 2006 Jul 15;20(14):1885-98 PMID: 16815998
  28. Identification of miRNA targets with stable isotope labeling by amino acids in cell culture.
    Nucleic Acids Res. 2006;34(16):e107 PMID: 16945957
  29. Effects of Dicer and Argonaute down-regulation on mRNA levels in human HEK293 cells.
    Nucleic Acids Res. 2006;34(17):4801-15 PMID: 16971455
  30. Designing siRNA that distinguish between genes that differ by a single nucleotide.
    PLoS Genet. 2006 Sep 8;2(9):e140 PMID: 16965178
  31. Single-stranded antisense siRNAs guide target RNA cleavage in RNAi.
    Cell. 2002 Sep 6;110(5):563-74 PMID: 12230974
  32. P1/HC-Pro, a viral suppressor of RNA silencing, interferes with Arabidopsis development and miRNA unction.
    Dev Cell. 2003 Feb;4(2):205-17 PMID: 12586064
  33. siRNAs can function as miRNAs.
    Genes Dev. 2003 Feb 15;17(4):438-42 PMID: 12600936
  34. bantam encodes a developmentally regulated microRNA that controls cell proliferation and regulates the proapoptotic gene hid in Drosophila.
    Cell. 2003 Apr 4;113(1):25-36 PMID: 12679032
  35. Genomewide view of gene silencing by small interfering RNAs.
    Proc Natl Acad Sci U S A. 2003 May 27;100(11):6343-6 PMID: 12730368
  36. Specificity of short interfering RNA determined through gene expression signatures.
    Proc Natl Acad Sci U S A. 2003 May 27;100(11):6347-52 PMID: 12746500
  37. Expression profiling reveals off-target gene regulation by RNAi.
    Nat Biotechnol. 2003 Jun;21(6):635-7 PMID: 12754523
  38. Selective silencing by RNAi of a dominant allele that causes amyotrophic lateral sclerosis.
    Aging Cell. 2003 Aug;2(4):209-17 PMID: 12934714
  39. Small RNAs with imperfect match to endogenous mRNA repress translation. Implications for off-target activity of small inhibitory RNA in mammalian cells.
    J Biol Chem. 2003 Nov 7;278(45):44312-9 PMID: 12952966
  40. Nonspecific, concentration-dependent stimulation and repression of mammalian gene expression by small interfering RNAs (siRNAs).
    RNA. 2004 Jan;10(1):12-8 PMID: 14681580
  41. Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells.
    Proc Natl Acad Sci U S A. 2004 Feb 17;101(7):1892-7 PMID: 14769924
  42. Specificity of microRNA target selection in translational repression.
    Genes Dev. 2004 Mar 1;18(5):504-11 PMID: 15014042
  43. RISC is a 5' phosphomonoester-producing RNA endonuclease.
    Genes Dev. 2004 May 1;18(9):975-80 PMID: 15105377
Article Info
Journal
RNA (New York, N.Y.)
Abbr.
RNA
ISSN
1355-8382
DOI
10.1261/rna.352507
Published
2007-03-00
Epub
2007-00-19
Pages
385-95
Language
English
Region
United States
NLM ID
9509184
PMCID
PMC1800510
Subset
IM
Grants
NIGMS NIH HHS · 5F31GM068983-03 · United States
NCI NIH HHS · P30 CA014051 · United States
NCI NIH HHS · P30-CA14051 · United States
NIAID NIH HHS · U19 AI056900 · United States
NIGMS NIH HHS · R01 GM034277 · United States
NIGMS NIH HHS · F31 GM068983 · United States
NIGMS NIH HHS · R01-GM34277 · United States
External Links
PubMed source DOI: 10.1261/rna.352507 PMC Full Text View journal details
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