DNA-guided DNA interference by a prokaryotic Argonaute

Abstract

RNA interference is widely distributed in eukaryotes and has a variety of functions, including antiviral defence and gene regulation1,2. All RNA interference pathways use small single-stranded RNA (ssRNA) molecules that guide proteins of the Argonaute (Ago) family to complementary ssRNA targets: RNA-guided RNA interference1,2. The role of prokaryotic Ago variants has remained elusive, although bioinformatics analysis has suggested their involvement in host defence3. Here we demonstrate that Ago of the bacterium Thermus thermophilus (TtAgo) acts as a barrier for the uptake and propagation of foreign DNA. In vivo, TtAgo is loaded with 5′-phosphorylated DNA guides, 13–25 nucleotides in length, that are mostly plasmid derived and have a strong bias for a 5′-end deoxycytidine. These small interfering DNAs guide TtAgo to cleave complementary DNA strands. Hence, despite structural homology to its eukaryotic counterparts, TtAgo functions in host defence by DNA-guided DNA interference.

A bacterial Argonaute with noncanonical guide RNA specificity

“…S1D). However, attempts to purify native protein using immunoprecipitation were not successful, consistent with previous reports of low Ago concentrations in bacteria and archaea (6)(7)(8).…”

“…Recent studies suggested that DNA-guided bacterial and archaeal Argonaute proteins are directly involved in host defense by cleaving foreign DNA elements, such as DNA viruses and plasmids (6,7). In addition, a catalytically inactive Argonaute protein in Rhodobacter sphaeroides (RsAgo) was demonstrated to use RNA guides and possibly recruits an associated nuclease for subsequent target cleavage (8).…”

DNA interference: DNA-induced gene silencing in the appendicularian Oikopleura dioica

“…The molecular pathway that mediates DNAi in O. dioica will be the focus of future investigations. As a host defence against invading DNA, it has recently been reported that Argonaute protein of T. thermophilus (TtAgo) mediates small single-stranded DNA-induced DNAi [10]. Similarly to TtAgo, Argonaute of O. dioica may mediate DNAi, inhibiting transcription and degrading mRNA.…”

“…Furthermore, a recent study of the archaean Thermus thermophilus has suggested that it may have a system for DNA-induced DNAi [10]. Argonaute protein in T. thermophilus has shown to cleave exogenously supplied DNA strands in a sequence-specific manner [10]. While RNAi is a highly conserved genesilencing system in a wide range of eukaryotes, DNAi has not been explored in any multicellular animals studied to date.…”

“…In Paramecium, a ciliate, plasmid DNA carrying a protein-encoding region induces posttranscriptional gene silencing of the corresponding endogenous gene [9]. Furthermore, a recent study of the archaean Thermus thermophilus has suggested that it may have a system for DNA-induced DNAi [10]. Argonaute protein in T. thermophilus has shown to cleave exogenously supplied DNA strands in a sequence-specific manner [10].…”