Prokaryotic homologs of Argonaute proteins are predicted to function as key components of a novel system of defense against mobile genetic elements.
BACKGROUND
In eukaryotes, RNA interference (RNAi) is a major mechanism of defense against viruses and transposable elements as well of regulating translation of endogenous mRNAs. The RNAi systems recognize the target RNA molecules via small guide RNAs that are completely or partially complementary to a region of the target. Key components of the RNAi systems are proteins of the Argonaute-PIWI family some of which function as slicers, the nucleases that cleave the target RNA that is base-paired to a guide RNA. Numerous prokaryotes possess the CRISPR-associated system (CASS) of defense against phages and plasmids that is, in part, mechanistically analogous but not homologous to eukaryotic RNAi systems. Many prokaryotes also encode homologs of Argonaute-PIWI proteins but their functions remain unknown.


RESULTS
We present a detailed analysis of Argonaute-PIWI protein sequences and the genomic neighborhoods of the respective genes in prokaryotes. Whereas eukaryotic Ago/PIWI proteins always contain PAZ (oligonucleotide binding) and PIWI (active or inactivated nuclease) domains, the prokaryotic Argonaute homologs (pAgos) fall into two major groups in which the PAZ domain is either present or absent. The monophyly of each group is supported by a phylogenetic analysis of the conserved PIWI-domains. Almost all pAgos that lack a PAZ domain appear to be inactivated, and the respective genes are associated with a variety of predicted nucleases in putative operons. An additional, uncharacterized domain that is fused to various nucleases appears to be a unique signature of operons encoding the short (lacking PAZ) pAgo form. By contrast, almost all PAZ-domain containing pAgos are predicted to be active nucleases. Some proteins of this group (e.g., that from Aquifex aeolicus) have been experimentally shown to possess nuclease activity, and are not typically associated with genes for other (putative) nucleases. Given these observations, the apparent extensive horizontal transfer of pAgo genes, and their common, statistically significant over-representation in genomic neighborhoods enriched in genes encoding proteins involved in the defense against phages and/or plasmids, we hypothesize that pAgos are key components of a novel class of defense systems. The PAZ-domain containing pAgos are predicted to directly destroy virus or plasmid nucleic acids via their nuclease activity, whereas the apparently inactivated, PAZ-lacking pAgos could be structural subunits of protein complexes that contain, as active moieties, the putative nucleases that we predict to be co-expressed with these pAgos. All these nucleases are predicted to be DNA endonucleases, so it seems most probable that the putative novel phage/plasmid-defense system targets phage DNA rather than mRNAs. Given that in eukaryotic RNAi systems, the PAZ domain binds a guide RNA and positions it on the complementary region of the target, we further speculate that pAgos function on a similar principle (the guide being either DNA or RNA), and that the uncharacterized domain found in putative operons with the short forms of pAgos is a functional substitute for the PAZ domain.


CONCLUSIONS
The hypothesis that pAgos are key components of a novel prokaryotic immune system that employs guide RNA or DNA molecules to degrade nucleic acids of invading mobile elements implies a functional analogy with the prokaryotic CASS and a direct evolutionary connection with eukaryotic RNAi. The predictions of the hypothesis including both the activities of pAgos and those of the associated endonucleases are readily amenable to experimental tests.
Makarova
Kira S
KS
National Center for Biotechnology Information, NLM, National Institutes of Health, Bethesda, Maryland 20894, USA. makarova@ncbi.nlm.nih.gov
Wolf
Yuri I
YI
van der Oost
John
J
Koonin
Eugene V
EV
D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000235:genetics; D003851:Deoxyribonucleases / Q000378:metabolism; D039642:Eukaryotic Initiation Factors / Q000737:chemistry* / Q000276:immunology*; D022761:Gene Transfer, Horizontal / Q000235:genetics; D016678:Genome / Q000235:genetics; D007107:Immune System; D020071:Interspersed Repetitive Sequences / Q000235:genetics* / Q000276:immunology*; D008969:Molecular Sequence Data; D010802:Phylogeny; D011387:Prokaryotic Cells / Q000276:immunology*; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D039642:Eukaryotic Initiation Factors; D003851:Deoxyribonucleases
Nucleic Acids Res. 1999 Dec 1;27(23):4658-70
10556324
Bioinformatics. 2000 Apr;16(4):404-5
10869041
Science. 2008 Dec 19;322(5909):1843-5
19095942
Trends Biochem Sci. 2003 Apr;28(4):196-201
12713903
Nature. 2008 Dec 18;456(7224):921-6
19092929
Cell Metab. 2008 Feb;7(2):104-12
18249170
Biol Direct. 2007 Nov 27;2:33
18042280
FEMS Microbiol Rev. 2005 Aug;29(3):435-63
16125007
Bioinformatics. 1998;14(1):48-54
9520501
Cell. 2009 Feb 20;136(4):642-55
19239886
Trends Genet. 2005 Jul;21(7):399-404
15913835
Structure. 2009 Jun 10;17(6):904-12
19523907
Curr Opin Microbiol. 2007 Apr;10(2):117-24
17376733
Trends Biochem Sci. 1999 Feb;24(2):47-53
10098397
Nature. 2005 Mar 31;434(7033):663-6
15800628
Proc Natl Acad Sci U S A. 2002 May 28;99(11):7536-41
12032318
EMBO J. 2004 Dec 8;23(24):4727-37
15565169
Cell. 2009 Feb 20;136(4):615-28
19239884
Nucleic Acids Res. 2007 Jan;35(Database issue):D61-5
17130148
Science. 2004 Sep 3;305(5689):1434-7
15284453
Trends Biochem Sci. 2003 May;28(5):226-9
12765832
Trends Biochem Sci. 2005 Feb;30(2):106-14
15691656
Nucleic Acids Res. 2005 Jul 1;33(Web Server issue):W244-8
15980461
Cell Death Differ. 2002 Apr;9(4):394-404
11965492
Biol Direct. 2006 Sep 19;1:29
16984643
Genome Biol. 2004;5(5):224
15128440
Nature. 2009 Jan 22;457(7228):421-5
19158788
BMC Struct Biol. 2007 Jun 20;7:40
17584917
Genome Res. 2000 Aug;10(8):1074-7
10958625
Methods Enzymol. 1996;266:418-27
8743697
Nat Struct Biol. 2001 Jul;8(7):621-5
11427894
Biochem Soc Trans. 2007 Dec;35(Pt 6):1437-44
18031241
Genes Dev. 2009 May 15;23(10):1151-64
19451215
Genome Res. 2000 Aug;10(8):1204-10
10958638
Nat Chem Biol. 2007 Jan;3(1):36-43
17173028
Nature. 2009 Jan 22;457(7228):413-20
19158787
Nat Rev Microbiol. 2008 Mar;6(3):181-6
18157154
Nat Rev Genet. 2009 Feb;10(2):94-108
19148191
Biochemistry. 2006 Jun 20;45(24):7511-21
16768447
Nat Biotechnol. 2000 Jun;18(6):609-13
10835597
Cold Spring Harb Symp Quant Biol. 2000;65:297-302
12760043
Mol Microbiol. 2003 Nov;50(4):1111-24
14622403
Curr Opin Hematol. 2008 Jul;15(4):326-31
18536570
Trends Ecol Evol. 2008 Oct;23(10):578-87
18715673
Nucleic Acids Res. 2007 Jan;35(Database issue):D269-70
17202163
Nature. 2005 Mar 31;434(7033):666-70
15800629
PLoS Genet. 2008 Aug 22;4(8):e1000163
18725932
Nature. 2009 Jan 22;457(7228):396-404
19158785
Nat Rev Mol Cell Biol. 2005 Feb;6(2):127-38
15654322
Nature. 2002 Jul 11;418(6894):244-51
12110901
Science. 2005 Sep 2;309(5740):1519-24
16141061
Handb Exp Pharmacol. 2008;(183):21-50
18071653
Biol Direct. 2006 Mar 16;1:7
16545108
Crit Rev Biochem Mol Biol. 2005 Mar-Apr;40(2):93-113
15814430
J Mol Biol. 2004 Mar 26;337(3):731-41
15019790
Cold Spring Harb Symp Quant Biol. 2006;71:67-72
17381282
Science. 2008 Aug 15;321(5891):960-4
18703739
Mol Biotechnol. 2003 Mar;23(3):225-43
12665693
Cell. 2005 Jul 15;122(1):17-20
16009129
J Bacteriol. 2008 Feb;190(4):1276-83
18083809
Nucleic Acids Res. 2002 Jan 15;30(2):482-96
11788711
Nature. 2008 Nov 13;456(7219):209-13
18754009
Nucleic Acids Res. 1997 Sep 1;25(17):3389-402
9254694
Genes Dev. 2005 Dec 1;19(23):2837-48
16287716
Nucleic Acids Res. 2005 Jun 22;33(11):3598-605
15972856
J Biol Chem. 2007 May 4;282(18):13824-32
17130125
Nature. 2009 Jan 22;457(7228):405-12
19158786
Curr Opin Microbiol. 2005 Aug;8(4):473-9
15979388
Biol Direct. 2009 Jun 03;4:19
19493340
Trends Biochem Sci. 2009 Aug;34(8):401-7
19646880
Structure. 1995 Feb 15;3(2):131-4
7735828
Sci STKE. 2007 Aug 28;2007(401):pe46
17726177
Mol Cell. 2005 Aug 5;19(3):405-19
16061186
Nucleic Acids Res. 2004 Mar 19;32(5):1792-7
15034147
Nucleic Acids Res. 2004 Oct 05;32(17):5260-79
15466593
Nucleic Acids Res. 2008 Apr;36(7):2295-300
18287115