title: Sequence-specific cleavage of dsRNA by Mini-III RNase.
abstract: Ribonucleases (RNases) play a critical role in RNA processing and degradation by hydrolyzing phosphodiester bonds (exo- or endonucleolytically). Many RNases that cut RNA internally exhibit substrate specificity, but their target sites are usually limited to one or a few specific nucleotides in single-stranded RNA and often in a context of a particular three-dimensional structure of the substrate. Thus far, no RNase counterparts of restriction enzymes have been identified which could cleave double-stranded RNA (dsRNA) in a sequence-specific manner. Here, we present evidence for a sequence-dependent cleavage of long dsRNA by RNase Mini-III from Bacillus subtilis (BsMiniIII). Analysis of the sites cleaved by this enzyme in limited digest of bacteriophage Φ6 dsRNA led to the identification of a consensus target sequence. We defined nucleotide residues within the preferred cleavage site that affected the efficiency of the cleavage and were essential for the discrimination of cleavable versus non-cleavable dsRNA sequences. We have also determined that the loop α5b-α6, a distinctive structural element in Mini-III RNases, is crucial for the specific cleavage, but not for dsRNA binding. Our results suggest that BsMiniIII may serve as a prototype of a sequence-specific dsRNase that could possibly be used for targeted cleavage of dsRNA.
authors: Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology in Warsaw, ul. Ks. Trojdena 4, 02-109 Warsaw, Poland.
authors: Dawid
authors: D
authors: Głów
authors: Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology in Warsaw, ul. Ks. Trojdena 4, 02-109 Warsaw, Poland.
authors: Dariusz
authors: D
authors: Pianka
authors: Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology in Warsaw, ul. Ks. Trojdena 4, 02-109 Warsaw, Poland.
authors: Agata A
authors: AA
authors: Sulej
authors: Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology in Warsaw, ul. Ks. Trojdena 4, 02-109 Warsaw, Poland.
authors: Łukasz P
authors: ŁP
authors: Kozłowski
authors: Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology in Warsaw, ul. Ks. Trojdena 4, 02-109 Warsaw, Poland.
authors: Justyna
authors: J
authors: Czarnecka
authors: Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology in Warsaw, ul. Ks. Trojdena 4, 02-109 Warsaw, Poland.
authors: Grzegorz
authors: G
authors: Chojnowski
authors: Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology in Warsaw, ul. Ks. Trojdena 4, 02-109 Warsaw, Poland.
authors: Krzysztof J
authors: KJ
authors: Skowronek
authors: Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology in Warsaw, ul. Ks. Trojdena 4, 02-109 Warsaw, Poland iamb@genesilico.pl.
authors: Janusz M
authors: http://orcid.org/0000-0002-6633-165X
authors: JM
authors: Bujnicki
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012330:RNA, Double-Stranded / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D043244:Ribonuclease III / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D012330:RNA, Double-Stranded; D043244:Ribonuclease III
references: Nature. 1991 Aug 8;352(6335):497-505
references: 1865905
references: Acta Crystallogr D Biol Crystallogr. 2010 Apr;66(Pt 4):486-501
references: 20383002
references: Proc Natl Acad Sci U S A. 1989 Sep;86(17):6513-7
references: 2771938
references: Structure. 2006 May;14(5):869-80
references: 16698548
references: RNA. 2010 Sep;16(9):1692-724
references: 20675404
references: Genome Biol. 2010;11(8):R86
references: 20738864
references: J Appl Crystallogr. 2007 Aug 1;40(Pt 4):658-674
references: 19461840
references: J Biol Chem. 2004 Apr 30;279(18):18425-33
references: 14973123
references: Nucleic Acids Res. 2011 May;39(10):4007-22
references: 21300639
references: J Virol. 1998 Feb;72(2):1504-15
references: 9445054
references: Biochemistry. 1993 Jul 27;32(29):7549-58
references: 8338852
references: Mol Microbiol. 2008 Jun;68(5):1096-106
references: 18363798
references: Nucleic Acids Res. 1993 Apr 25;21(8):1919-25
references: 8493105
references: Mol Microbiol. 2008 Jan;67(1):143-54
references: 18047582
references: Infect Immun. 1998 Aug;66(8):3952-8
references: 9673285
references: Nucleic Acids Res. 2009 Jul;37(Web Server issue):W202-8
references: 19458158
references: Biochemistry. 2000 May 30;39(21):6380-9
references: 10828952
references: Biochemistry. 2004 Mar 16;43(10):2854-62
references: 15005620
references: Nucleic Acids Res. 2006;34(5):1381-92
references: 16522648
references: Acta Crystallogr D Biol Crystallogr. 1997 May 1;53(Pt 3):240-55
references: 15299926
references: Nucleic Acids Res. 2001 Sep 15;29(18):3705-27
references: 11557805
references: Fold Des. 1996;1(4):R78-88
references: 9079386
references: FEMS Microbiol Lett. 2011 Feb;315(1):30-7
references: 21133991
references: Nat Commun. 2012;3:1147
references: 23093184
references: EMBO J. 2000 Mar 1;19(5):997-1009
references: 10698941
references: Cell. 2010 Oct 15;143(2):225-37
references: 20946981
references: Science. 2004 Dec 17;306(5704):2068-72
references: 15604402
references: Methods Mol Biol. 1999;118:73-91
references: 10549516
references: Proc Natl Acad Sci U S A. 1997 Dec 9;94(25):13437-41
references: 9391043
references: Nucleic Acids Res. 2003 Jun 1;31(11):2751-8
references: 12771201
references: Cell. 2006 Jan 27;124(2):355-66
references: 16439209
references: Bioinformatics. 2011 Jun 15;27(12):1653-9
references: 21543442
references: Nat Methods. 2012 Apr;9(4):357-9
references: 22388286
references: Proc Natl Acad Sci U S A. 2004 Jun 1;101(22):8307-12
references: 15150409
references: Nucleic Acids Res. 2008 Feb;36(3):963-9
references: 18086702
references: Cell Mol Life Sci. 2013 Jun;70(11):1875-95
references: 22918483
references: Biotechniques. 2001 Mar;30(3):574-80, 582
references: 11252793
references: J Biol Chem. 1990 Feb 5;265(4):2216-22
references: 2298746
references: Mol Microbiol. 2009 Mar;71(5):1145-54
references: 19154332
references: Mol Microbiol. 2008 Jun;68(5):1073-6
references: 18430137
references: Acta Crystallogr D Biol Crystallogr. 2010 Feb;66(Pt 2):125-32
references: 20124692