Bacterial Argonaute samples the transcriptome to identify
foreign DNA
Ivan Olovnikov
1,2
,
Ken Chan
1
,
Ravi Sachidanandam
3
,
Dianne K. Newman
1,4
, and
Alexei A.
Aravin
1,&
1
California Institute of Technology, Division of Biology, 147-75, 1200E California Blvd. Pasadena,
CA 91125, USA
2
Institute of Molecular Genetics, Russian Academy of Sciences, Kurchatov sq. 2, Moscow,
123182, Russia
3
Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York, NY 10029, USA
4
Howard Hughes Medical Institute, Division of Biology, 147-75 1200E California Blvd. Pasadena,
CA 91125, USA
summary
Eukaryotic Argonautes bind small RNAs and use them as guides to find complementary RNA
targets and induce gene silencing. Though homologs of eukaryotic Argonautes are present in
many bacteria and archaea their small RNA partners and functions are unknown. We found that
the Argonaute of
Rhodobacter sphaeroides
(RsAgo) associates with 15-19 nt RNAs that
correspond to the majority of transcripts. RsAgo also binds single-stranded 22-24 nt DNA
molecules that are complementary to the small RNAs and enriched in sequences derived from
exogenous plasmids as well as genome-encoded foreign nucleic acids such as transposons and
phage genes. Expression of RsAgo in the heterologous
E. coli
system leads to formation of
plasmid– derived small RNA and DNA and plasmid degradation. In a
R. sphaeroides
mutant
lacking RsAgo, expression of plasmid-encoded genes is elevated. Our results indicate that RNAi-
related processes found in eukaryotes are also conserved in bacteria and target foreign nucleic
acids.
Introduction
Argonaute proteins are key players in RNA interference (RNAi) and related gene silencing
phenomena in diverse eukaryotic species. Argonautes form tight complexes with small (19
to 31 nt) RNA partners and use them as guides to recognize target RNA molecules by
Watson-Crick base pairing between the small and target RNAs (Meister, 2013). Argonaute
proteins harbor an RNaseH-like Piwi domain that is capable of endonucleolytic cleavage of
target RNA leading to its subsequent degradation and post-transcriptional silencing.
Endonucleolytic activity of Argonautes requires the DEDH tetrad that forms the catalytic
center of the Piwi domain (Liu et al., 2004; Nakanishi et al., 2012). In some Argonautes
these residues are mutated which correlates with their lack of endonuclease activity.
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Published in final edited form as:
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. 2013 September 12; 51(5): . doi:10.1016/j.molcel.2013.08.014.
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Argonautes that are deficient in endonuclease activity can nevertheless induce silencing of
complementary RNA targets through recruitment of additional proteins. Particularly,
Argonaute cleavage is not required for silencing of the majority of miRNA targets in
mammals and Drosophila (Cheloufi et al., 2010; Liu et al., 2004).
Beyond post-transcriptional silencing, Argonaute proteins associated with small RNA
partners are able to induce transcriptional gene repression through establishing repressive
chromatin structure on cognate genomic loci (Castel and Martienssen, 2013; Olovnikov et
al., 2012). In
S. pombe
Argonaute/small RNA complexes directly interact with nascent RNA
transcripts followed by recruitment of silencing complexes to chromatin (Verdel et al.,
2004). Similarly, the requirement for target transcription was shown or postulated in other
cases of small RNA-induced transcriptional repression in plants (Herr et al., 2005) and
Metazoa (Le Thomas et al., 2013; Shpiz et al., 2011; Sienski et al., 2012) suggesting that
even when Argonaute induce transcriptional repression it binds target RNA and not DNA
molecules.
In eukaryotes, several classes of Argonaute-associated small RNA have been described that
differ in both their mechanism of biogenesis and cellular functions. Two prominent classes,
siRNA and miRNA, are processed by RNaseIII-type enzymes from double-stranded RNA or
single-stranded molecules with hairpin structures, respectively. Other types of small RNA,
particularly piRNA in Metazoa, are processed from single-stranded precursors that lack
recognizable secondary structure without the involvement of RNaseIII-type enzymes (Vagin
et al., 2006). Overall, eukaryotes demonstrate a great diversity in small RNA biogenesis
mechanisms with some pathways requiring distinct secondary structures of the precursor
RNA molecules while others generate small RNA by sampling specific portions of or even
the entire transcriptome.
Studies of diverse small RNA pathways in eukaryotic species showed that they play two
major roles in cells: regulation of expression of host genes and protection against foreign
genetic material. In Metazoa and plants, miRNA and some siRNA target messages of
multiple genes to regulate their expression and fine-tune output of gene networks (Baek et
al., 2008; Tam et al., 2008). In many species siRNAs repress expression of double-stranded
RNA viruses (Li et al., 2002; Lu et al., 2005). siRNAs in plants and piRNAs in Metazoa are
involved in repressing endogenous transposable elements that, though they are not
infectious, are considered to be ‘selfish’ genomic elements capable of both multiplication
and damaging the host genome (Haag and Pikaard, 2011; Herr et al., 2005; Luteijn and
Ketting, 2013; Vagin et al., 2006).
Although studies of natural small RNA pathways have been restricted to eukaryotes,
Argonaute proteins are also present in many bacterial and archaeal species (Makarova et al.,
2009). Furthermore, the structure of several bacterial and archaeal Argonautes was solved
and used for modeling the structure of their eukaryotic orthologs (Ma et al., 2005; Parker et
al., 2005; Song et al., 2004; Wang et al., 2008b; Yuan et al., 2005).
In vitro
the Argonaute
protein of the eubacterium
Aquifex aeolicus
has the highest affinity to single-stranded DNA
oligonucleotides while binding affinities to single- and double-stranded RNA are
significantly lower (Ma et al., 2005). Similarly, Argonaute from the archaeon
Archaeoglobus fulgidus
binds single- and double-stranded short DNA and DNA-RNA
hybrids more efficiently than single- and double-stranded RNA (Yuan et al., 2005).
Furthermore,
in vitro
, bacterial members of the Argonaute family that have slicer activity are
able to use nucleic acid guides (preferentially DNA) for endonucleolytic cleavage of
complementary RNA targets (Wang et al., 2008a; Wang et al., 2008b; Yuan et al., 2005).
The bioinformatic analysis of Ago operons and their relationship with other genome defense
systems suggested that bacterial and archaeal Argonautes might play a role in protection of
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the host genome (Makarova et al., 2009), however, experimental analysis of Argonaute
functions and their native small RNA partners in either bacteria or archaea was lacking.
Here we identified the natural nucleic acid partners of Argonaute protein (RsAgo) from the
alphaproteobacterium
Rhodobacter sphaeroides
. Surprisingly, we found that in native cells
RsAgo associates with a complex population of both small RNA and small DNA species.
Analyses of nucleic acid partners of RsAgo suggest that associated small RNAs are
generated by broad sampling of the bacterial transcriptome, and formation of small DNA is
dependent on small RNAs. RsAgo-associated small DNAs are enriched in sequences
derived from foreign nucleic acids and RsAgo deficiency leads to increased expression of
plasmid-encoded genes implicating a role for bacterial Argonaute in the pathway that targets
foreign nucleic acids.
Results
Identification of nucleic acid partners of
Rhodobacter sphaeroides
Argonaute
To identify the natural nucleic acid partners of bacterial Argonautes
in vivo
we expressed
and purified 6xHis-tagged Argonaute protein from host strain
R. sphaeroides
ATCC17025.
RsAgo is a 777 amino-acid protein and has the same domain structure as eukaryotic
Argonautes, namely PAZ, Mid and Piwi domains responsible for binding small nucleic acid
guides and endonucleolytic cleavage (slicing) of the target (Fig. 1A). However, like the
majority of prokaryotic Argonautes, RsAgo is likely not active as a slicer nuclease due to a
substitution of the critical DEDH residues responsible for the nuclease activity to GGHE
residues (Makarova et al., 2009). Similar to many other bacterial Argonautes, RsAgo resides
within one operon with a predicted DNA nuclease (Fig. 1A) (Makarova et al., 2009).
Tagged RsAgo complex purified from
R. sphaeroides
cells harvested at stationary phase
contains small nucleic acid species with major sizes around 15-24 nt and
45 nt (Fig 1B,
S1). The majority of the RsAgo-bound nucleic acids are eliminated by RNase A treatment
indicating that these are small RNAs. A minor fraction of 20-24 nt species was resistant to
RNase A treatment but susceptible to DNase I, indicating that RsAgo associates with short
DNA molecules. To gain further insight into the nature of Ago-associated nucleic acids, we
cloned and sequenced two size fractions of RsAgo-bound RNAs (15-24 nt and
45 nt)
using an established method that relies on the presence of a 5
phosphate and 3
hydroxyl in
the cloned molecules (Aravin et al., 2007; Lau et al., 2001). Sequence analysis of 45 nt long
RsAgo-associated RNA showed that this fraction consists almost exclusively of the 3
halves
of two types of tRNA
met
cut 1nt upstream of the anticodon (data not shown). It is difficult to
determine the biological significance of co-purification of RsAgo with tRNA halves; this
association could be caused by contamination by abundant RNA species, though it should be
noted that mammalian Argonaute was also found in complex with tRNA fragments
(Burroughs et al., 2011; Maute et al., 2013). Sequence analysis of the shorter (15-24 nt)
RNA fraction revealed RNA population between 15 and 19 nt in length (Fig. 2A). These
small RNAs show strong enrichment for uridine residue at the first position (Fig. 2B), a
distinctive feature of several classes of eukaryotic Argonaute-associated small RNAs
(Brennecke et al., 2007; Lau et al., 2001). A strong enrichment of a pyrimidine (U or C)
residue at position 2 was also apparent. The RsAgo-bound smRNA population was
moderately complex: the roughly 35 million sequenced reads correspond to about 1.7
million unique sequences (Table S1). The diversity of the 15-19 nt small RNA library is
corroborated by the absence of nucleotide biases at any position except of the first and
second nucleotides.
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Presence of small RNA population in wild-type cells depends on RsAgo expression
Purification of Ago-RNA complexes from bacterial cell lysate takes several hours during
which cellular RNA is exposed to nucleases that might generate small RNA fragments.
These could possibly be loaded into RsAgo (Riley et al., 2012). To verify that RsAgo-bound
small RNA species are present in native cells we purified total RNA from two
R.
sphaeroides
strains: ATCC17025 (referenced as ‘strain 25’ later in the text) that expresses
Argonaute endogenously and ATCC17029 (referenced as ‘strain 29’) that lacks the gene
encoding Ago. We also transformed both strains with a plasmid expressing RsAgo or an
empty control plasmid lacking Ago. Deep sequencing of the 13-30 nt range of small RNA
from non-transformed strain 25 revealed a prominent peak at 15-19 nt corresponding to the
size of RsAgo-associated small RNAs (Fig. 2C). Importantly, this peak was absent in strain
29 that lacks endogenous Argonaute protein. Analysis of sequence composition showed that
while small RNAs cloned from strain 29 do not show any bias for uridine at the first base
position, small RNAs from strain 25 are strongly enriched in 5
U (Fig. 2D). Expression of
RsAgo from the plasmid further increased the bias for 5
U in 15-19 nt RNA in strain 25.
These results indicate that an endogenous small RNA population with features identical to
purified RsAgo-bound small RNA is present in the
R. sphaeroides
strain that expresses non-
tagged RsAgo on a physiological level. Interestingly, expression of RsAgo from the plasmid
in strain 29 lacking the whole Ago operon leads to the appearance of 15-19 nt 5
U-rich RNA
in the total RNA population (Fig. 2C,D). Furthermore, expression of RsAgo significantly
changes the small RNA profile in strain 29: without RsAgo expression, 50% of small RNA
in the size range of 13 to 30 nt are mapped to rRNA genes and likely represent degradation
products of abundant rRNA species, compared to only 15% after RsAgo expression. This
result suggests that expression of RsAgo changes the small RNA profile by binding to a
select pool of small RNAs and that the putative nuclease that is encoded by the second gene
in the operon (Rsph17025_3695) is not required for generation of RsAgo-bound small RNA.
RsAgo-associated small RNAs match the majority of cellular transcripts
To further analyze RsAgo-associated small RNAs, we mapped them to the
R. sphaeroides
genome (strain 25) and the pSRKKm-RsAgo plasmid used for protein expression. Small
RNAs mapped to all six
R. sphaeroides
circular chromosomes as well as the expression
plasmid with 54.3% of genomic and 98.7% of plasmid nucleotides covered by at least one
read. 60.3% of the small RNA reads map to the largest 3.2 Mb chromosome, while 24.4% of
RNA reads map to the 8 kb expression plasmid (Fig. 3A). Normalized to chromosome
length RsAgo-bound small RNAs show a strong bias for the expression plasmid. This
enrichment of plasmid-derived sequences was also clear in strain 25 transformed with the
‘empty’ pSRKKm plasmid lacking the RsAgo gene, demonstrating that it is not caused by
the propensity of Argonaute to generate small RNAs in cis (Fig. S2A,B).
Annotation of small RNAs shows that the majority map to protein-coding and non-coding
genes in sense orientation (Fig. 3B). The distribution of small RNAs over genes is not
uniform: the top 10 genes, including three located on the expression plasmid (RsAgo itself,
Kan and lacI), contribute 37% of all RNA reads. Among the top genes located on the host
chromosomes were rRNAs and the IS4 family of DNA transposons that has more than 20
nearly identical copies scattered over the six chromosomes (Fig. 3C). To understand the
relationship between the RsAgo-bound small RNA population and normal cellular
transcripts, we analyzed the transcriptome of strain 25 using RNA-Seq of total and rRNA-
depleted RNA samples. Comparison of long RNA transcripts and short RsAgo-bound RNAs
shows a significant but relatively weak correlation between gene expression and the amount
of small RNAs mapping to each gene (Pearson correlation coefficient r
0.35, p <
2.2×10
-16
, Fig. 3D). Many genes are greatly depleted or enriched in the small RNA
population relative to the amount of their longtranscripts (Fig. 3E). Interestingly, the RsAgo
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gene expressed from the plasmid was among the top 30 genes with the ratio of small to long
RNA 30-fold higher than median. In contrast, non-coding RNAs, such as RNaseP and SRP
were among the 10 most depleted genes with the ratio of small to long RNA being 246- and
15,500-fold lower than median (Fig. 3E, Fig.S2C).
The most parsimonious interpretation of the sense bias of small RNAs and the general
correlation between their amount and that of long genic transcripts is that small RNAs are
processed from full-length mRNAs or their degradation products by nuclease activity.
However, the significant variation in the ratio of small to long RNA indicates that the
efficiency of such processing varies for different transcripts. Except for the depletion of non-
coding RNA genes and enrichment for plasmid-encoded genes (Fig. 3E), we were unable to
identify distinct gene features that could explain their enrichment or depletion in the small
RNA population. RsAgo-associated small RNAs are derived from many different transcripts
and many different positions inside each transcript. We did not detect any strong sequence
or structure motif upstream or downstream of mature small RNAs in potential precursor
transcripts, implying that there is no requirement for distinct primary or secondary structure
for processing of small RNA from long precursors (data not shown). Overall, our results
show that the population of RsAgo-bound small RNAs represents a sampling of the whole
bacterial transcriptome with significant enrichment for individual transcripts and selection
against others such as non-coding RNAs.
RsAgo-associated small DNAs are complementary to small RNAs and gene transcripts
To analyze the small DNA species associated with RsAgo we developed a method for
directional cloning of short single-stranded DNA molecules using bridged ligation that is
dependent on the presence of a 5
phosphate and 3
hydroxyl groups in the cloned molecule
(Fig. S3). Analysis of small DNA deep sequencing data revealed that they are 22-24 nt long
(Fig. 2A). The complexity of the small DNA population is comparable to that of small
RNAs:
16 million sequenced reads corresponds to
1.1 million unique sequences. Similar
to small RNA, RsAgo-bound small DNA map to all
R. sphaeroides
circular chromosomes
and the expression plasmid, but the bias for the expression plasmid is even more
pronounced: 87.6% of DNA reads map to the 8 kb expression plasmid, while 9.1% map to
the largest 3.2 Mb chromosome (Fig. 4A).
Annotation of small DNAs shows that, similar to small RNAs, the majority map to protein-
coding genes (Fig. 4B). A significant portion of small DNAs mapped to non-annotated loci;
the majority of these sequences match non-coding regions of the expression plasmid. The
orientation of small DNAs is opposite to that of small RNA species: for plasmid-mapped
sequences 95% of small DNAs are in antisense orientation to genes, while 97% of small
RNAs are in the sense orientation (Fig. 4C, D). A similar bias for antisense orientation is
apparent in small DNAs mapped to host chromosomes (Fig. S4B). The strong orientation
bias of small DNAs that map to the genomic strand corresponding to the antisense strand of
genes suggests that they are generated in an RNA- or, more generally, transcription-
dependent fashion.
To gain further insight into the relative positions of small RNA and DNA molecules we
performed genome-wide analysis of the distances between the ends of small RNAs and
small DNAs that mapped within 30 nt of each other. This analysis revealed a striking
enrichment for complementary RNA/DNA pairs with both ends of DNA protruding 3 nt
over small RNA sequence (Fig. 4E). Interestingly, while the 3
DNA overhang is almost
invariably 3 nt, the length of the 5
DNA overhang is less precise (Fig. 4E), suggesting that
the mechanisms of 5
and 3
end formation are different and specific. Small DNA species
did not show significant nucleotide biases in the first and second residues where small
RNAs had strong biases. However, when the 23-24 nt small DNA sequences were aligned
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by their 3
end, a strong enrichment for adenine and purine residues was seen 4 and 5 nt
away from the 3
end, respectively (Fig. 4F, Fig. S4C). This result supports the analysis of
relative positions of small RNA and DNA molecules that indicates that the majority of small
RNAs and small DNAs are complementary to each other with the small DNA having a 3 nt
overhang on each end (Fig. 4G). Indeed, mapping has shown that 79.9% of small RNA reads
are fully complementary to at least one sequence in small DNA library. To emphasize this
relationship, we propose to refer to single-stranded small RNA and DNA such as found in
the RsAgo complex as DNA-interacting (di)RNA and RNA-interacting (ri)DNA,
respectively. Considering that diRNA are
2-fold more abundant in purified RsAgo
complexes than riDNA (Fig. 1B) it is clear that some RsAgo complexes are loaded with
small RNA alone. Other complexes might be loaded with riDNA or a diRNA-riDNA
duplex.
RsAgo-associated small DNAs are enriched in foreign sequences
The amount of riDNA mapped to each gene correlates with the amount of diRNA (Pearson
correlation coefficient r
0.73, p < 2.2×10
-16
, Fig. S5A,B). However there are significant
variations in the ratio of riDNA to diRNA for individual genes: for example the five genes
encoded on the expression plasmid have on average 15-fold higher ratio compared to
median. Analysis of genes with high ratio of riDNA/diRNA shows that this set is enriched in
sequences that are annotated as transposons and phage genes (Fig. 5A). Many, but not all of
these genes are present in multiple copies in the
R. sphaeroides
genome. Similarly, genes
located on the expression plasmid that have 30 to 60 copies per cell and genes of unknown
origin that are present as multi-copy sequences in the genome are enriched in riDNA (Fig.
5A). To test if the disproportionally large amount of riDNA derived from some genes is
caused by greater availability of substrate DNA due to its high copy-number, we normalized
the amount of small DNA to the number of positions to which each sequence can be mapped
in the genome. Even after this normalization, genes with a high ratio of riDNA to diRNA are
enriched in phage genes and multi-copy genes of unknown origin (Fig. S5C). Importantly,
even single-copy phage genes have high ratio of riDNA to diRNA, while multi-copy host
genes do not have this bias. Average phage genes have a ratio of riDNA to diRNA that is
6.6- or 5-fold higher than that of an average single-copy host gene, before and after
normalization to copy number, respectively (Fig. 5B, Fig.S5D). Together these results
suggest that sequences that can be classified as ‘foreign DNA’ and not simply multi-copy
sequences generate disproportionally more riDNA. Considering that
90% of the riDNA
map to the expression plasmid, it is plausible that discrimination of foreign DNA against
host genes is achieved by recognition of extra-chromosomal DNA molecules as a substrate
for small DNA processing. In agreement with this hypothesis, transposons and DNA phages
that show enrichment in riDNA have extra-chromosomal DNA stages in their life cycles.
Expression of RsAgo in
E. coli
causes degradation of plasmid DNA
In order to further understand the ability of RsAgo to recognize foreign nucleic acids we
expressed it in the heterologous
E. coli
system. As a control we also expressed the point
mutant, denoted as RsAgo-YK, with two amino acid substitutions in the 5
end binding
pocket that should render it unable to bind 5
phosphorylated nucleic acids (Ma et al., 2005;
Parker et al., 2005). Purification of wild-type RsAgo from
E. coli
cells showed that it binds
both small RNA and DNA species of the same sizes as in
R. sphaeroides
(Fig. 6A)
suggesting that either RsAgo is autonomous in producing and loading small nucleic acids or
it requires factors that are conserved between the two species. As expected, the RsAgo-YK
mutant was devoid of both small RNA and DNA species (data not shown). Cloning of small
RNA and DNA isolated from RsAgo expressed in
E. coli
showed that they are almost
exclusively derived from the expression plasmid (90.2% and 99.2% of small RNA and DNA
reads, respectively) (Fig. 6B). Induction of expression of the wild-type protein, but not the
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RsAgo-YK mutant, leads to significant decrease in plasmid yield (Fig. 6C, S6).
Furthermore, plasmid DNA isolated from cells expressing wild-type RsAgo was strongly
degraded (Fig. 6D) suggesting that the riDNAs found in the RsAgo complex are generated
by excision from plasmid DNA.
RsAgo represses plasmid-encoded genes in
R. sphaeroides
To gain further insight into the function of
R. sphaeroides
Argonaute, we used directional
insertion of the omega transcription termination cassette upstream of the Piwi domain of
RsAgo to generate a mutant of strain 25 deficient in RsAgo expression (Fig. S11). RT-PCR
of the RsAgo transcript confirmed the lack of RsAgo expression in mutant cells (Fig. S7).
Furthermore, cloning of small RNAs from RsAgo-deficient cells showed the absence of the
15-19 nt peak, which is present in wild-type 25 cells indicating that RsAgo protein is indeed
functionally impaired (Fig. 7A). Measuring growth of the wild type and RsAgo-deficient
cells under standard conditions did not reveal significant differences between the two strains
indicating that RsAgo is not required for general cellular fitness (Fig. S7D). To study the
effect of RsAgo deficiency on gene expression, we used RNA-Seq to profile the
transcriptome of wild-type and mutant cells grown under identical conditions. Duplicate
experiments analyzed by the DESeq (Anders and Huber, 2010) showed no significant
reproducible difference in gene expression between the two strains (Fig. S7E). Taken
together, these data indicate that despite being loaded with small RNAs derived from a
variety of chromosomal transcripts, RsAgo does not strongly affect gene expression. RsAgo-
associated diRNA and riDNA are particularly enriched in sequences derived from the
exogenously introduced plasmid (Fig. 3A, 4A) suggesting that RsAgo might specifically
affect expression of genes encoded on foreign extrachromosomal DNA. To test this
hypothesis we introduced the pSRKTc-Fluc plasmid encoding the firefly luciferase and lacI
genes into wild-type strain 25 and RsAgo mutant cells. Measurement of luciferase activity in
three independent experiments showed that its expression was
2-fold higher in the RsAgo
mutant cells (Fig. 7B). Furthermore, abundance of both luciferase and lacI plasmid-derived
transcripts were also
2-fold higher in mutant cells as measured by RT-qPCR, while the
amount of plasmid DNA measured by PCR was identical (Fig. 7B). These data indicate that
RsAgo is able to repress gene expression from an exogenous plasmid.
Discussion
The finding of nucleic acid partners of bacterial Ago protein sheds new light on the
evolution and function of RNAi pathways. RsAgo-bound small RNAs (diRNAs) are shorter
(15-19 nt) compared to all known classes of Argonaute-associated RNA in eukaryotes
(19-31 nt). However, similar to many eukaryotic small RNAs, RsAgo-bound RNAs have a
strong bias for uridine at the first position, a feature that is likely determined by the structure
of the 5
binding pocket of the Argonaute (Frank et al., 2012). Unlike miRNAs and siRNAs
in eukaryotes, RsAgo-associated diRNAs are derived from RNA precursors that lack distinct
secondary structure. This fact, together with a remarkable sequence diversity of RsAgo-
bound RNAs, make them similar to another class of eukaryotic RNA, piRNA that is
generated from single-stranded non-structured RNAs of any sequence (Brennecke et al.,
2007; Muerdter et al., 2012). Indeed, RsAgo-associated diRNAs can be mapped to the
majority of cellular transcripts in sense orientation with a bias against structural non-coding
RNAs. Therefore diRNAs are either directly processed from mRNAs or generated from the
products of their degradation (Fig. 7C). The discrimination against structural RNA favors
the latter explanation, sampling of degradation products (so called degradome), as structural
RNAs are significantly more stable compared to mRNAs. The strong bias for uridine at the
first position might be introduced by the processing machinery, or, alternatively, the result
of RsAgo selecting 5
U-RNAs from a pool of available sequences. diRNAs with correct size
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and 5
U-bias are formed upon RsAgo expression in the heterologous
E. coli
system arguing
against involvement of species-specific proteins in small RNA processing. Overall,
generation of diRNAs can be explained without postulating the existence of a complex
processing machinery. In the simplest scenario, RsAgo would bind products of mRNA
degradation that possess 5
phosphate. Subsequently, a nuclease would trim the bound RNA
from the 3
end until it reaches the region protected by the footprint of the RsAgo protein,
the mechanism proposed for formation of the 3
end of Metazoan piRNAs (Kawaoka et al.,
2011).
The finding of small single-stranded DNA (riDNA) species associated with RsAgo is very
intriguing as small DNAs have not been found in association with Argonaute proteins in
eukaryotes. Natural short single-stranded DNAs, to our knowledge, have not been found in
any cellular pathway, though short DNA sequences, Okazaki fragments, paired with long
DNA are synthesized during replication of the lagging DNA strand. riDNAs found in
association with RsAgo complex have two distinct and surprising features. First, they are
largely complementary to diRNAs and gene transcripts. Second, the riDNAs have a peculiar
arrangement with the complementary diRNAs: they form duplexes that have 3 nt DNA
overhang on each side of the duplex (Fig. 4G). Both features strongly argue that riDNAs are
generated in a RNA-dependent fashion as only transcribed RNA can provide such strand
information. It is unlikely that riDNAs are generated by direct reverse transcription of
riRNAs as DNAs are longer and harbor nucleotide residues that match genomic sequences
but are lacking in complementary RNA molecules (Fig. 4G).
The observed arrangement of diRNAs and riDNAs resembles the ping-pong cycle in the
piRNA pathway where formation of secondary piRNAs is guided by primary piRNA
(Brennecke et al., 2007). However, the ping-pong cycle uses the endonucleolytic (slicer)
activity of Argonaute to generate secondary piRNA. As a result of slicer cleavage, the 5
end
of the secondary piRNA is shifted by 10 nt compared to the 5
end of the primary piRNA.
The residues necessary for slicer activity are mutated in RsAgo and we did not detect
cleavage activity in an
in vitro
assay (data not shown). Equally important, observed
arrangement of diRNA/riDNA pairs is not compatible with Argonaute-dependent cleavage.
Instead we propose that recognition of DNA by small RNA-loaded RsAgo leads to DNA
cleavage by an unknown nuclease(s) on both sides of the targeted region leading to
formation of riDNA with 3 nt overhands (Fig. 7C). Indeed, we observe strong degradation of
the plasmid upon expression of RsAgo in
E. coli
cells that correlates with production of
small DNAs mapping almost exclusively to the plasmid. Argonautes and associated small
RNA can cause transcriptional silencing and even DNA elimination of cognate sequences in
eukaryotes (Kataoka and Mochizuki, 2011), however, it is believed that Argonaute
complexes target nascent transcripts followed by recruitment of chromatin-modifying
machinery to the locus. In contrast, our model proposes direct targeting of DNA by RsAgo/
small RNA complexes.
Generation of riDNAs by cutting them out of double-stranded DNA will lead to damage of
genomic DNA unless it is tightly controlled and restricted to specific sequences. Indeed, we
observed that riDNA are highly enriched in sequences that can be characterized as foreign to
the cell: artificially introduced plasmid as well as phages and transposons. Therefore, we
postulate the existence of an additional mechanism that allows small RNA-loaded RsAgo
complexes to discriminate and target foreign DNA while avoiding normal genomic
substrates. The specific molecular determinant that allows such discrimination should be a
subject for future studies. The extra-chromosomal nature of DNA can be used to
discriminate proper targets: indeed plasmids are extrachromosomal by definition and both
phages and transposons go through an extrachromosomal step during their life-cycle.
However, the extra-chromosomal nature by itself is not sufficient to explain the observed
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specificity as endogenous plasmids similar in size to the artificially introduced plasmid do
not serve as preferred substrate for riDNA generation (Fig. 4A). High rate of DNA
replication might be a feature for recognition of proper targets as replicating DNA provides
readily available single-stranded DNA substrate for recognition by small RNA-loaded
RsAgo. According to this model, effective RsAgo targeting requires both a high level of
gene expression (to generate small RNA) and a high level of DNA replication (to provide
single-stranded DNA target). Such a combination is not typical for host genes and would
allow RsAgo to discriminate against invasive DNA.
The enrichment of riDNAs in foreign sequences implicates RsAgo in repression of foreign
genomic elements. Indeed, expression of genes encoded on a foreign (but not endogenous)
plasmid is suppressed by RsAgo (Fig. 7B). What is the molecular mechanism of gene
silencing by RsAgo? Though we did not observe strong degradation of plasmid DNA at the
physiological expression level of RsAgo in
R. sphaeroides
cells (data not shown), it is
possible that a low level of plasmid DNA damage associated with excision of riDNAs is
sufficient to cause moderate repression (Fig. 7C). Alternatively, the repression might be
achieved by transcriptional interference as elongating RNA polymerase collides with
RsAgo/diRNA complexes that are bound to the template strand of the plasmid DNA.
According to both models, that are not mutually exclusive, riDNAs are by-products of a
rather rare event of DNA degradation and do not play an independent role in the silencing
process. Alternatively, after their generation antisense riDNA might be loaded into RsAgo
and repress complementary plasmid transcripts post-transcriptionally (Fig. 7C). Unlike
Argonautes of
Aquifex aeolicus
and
Thermus thermophilus
that are able to use DNA guides
to cleave RNA in vitro (Wang et al., 2008a; Yuan et al., 2005), RsAgo lacks slicer activity
and would need to employ different mechanism. Independently of the specific molecular
mechanism, which future studies will address, our findings suggest that bacterial Argonautes
and their nucleic acid partners play a function similar to that of CRISPR, metazoan piRNA
and plant siRNA, namely, protection of the genome against invading genetic material.
Experimental Procedures
Bacterial strains
Rhodobacter sphaeroides
strains ATCC17025 and ATCC17029 were kindly provided by
Timothy Donohue (University of Wisconsin–Madison). Cells were grown on Sistrom's
minimal medium A at 30C under aerobic conditions. Kanamycin was used at concentration
25
g/ml for
R. sphaeroides
and 25
g/ml for E. coli BL21(DE3), Tetracyclin at 1
g/ml for
R. sphaeroides
.
RsAgo expression and purification in R. sphaeroides
N-6xHis-tagged ORF of RsAgo (Rsph17025_3694) was amplified from genomic DNA of
strain ATCC17025 and cloned into broad-host-range expression vector pSRKKm (Khan et
al., 2008). Plasmid was mobilized into
R. sphaeroides
by biparental mating with
E. coli
BW29427. Protein synthesis was induced by 1mM IPTG for 5-10 hours. RsAgo was isolated
using Talon beads (Clontech). A detailed protocol is provided in the Supplemental
Experimental Procedures.
RsAgo expression in E. coli
For expression of RsAgo in E. coli BL21(DE3) vector pSRKKm-RsAgo was used. For
experiments shown in Fig. 6 C, D and Fig. S10, 6xHis-Flag-tagged RsAgo was cloned into
vector pET30a(+), which contains T7 RNA polymerase-driven promoter. RsAgo-YK
isoform impaired in small RNA binding contained mutations Y463G and K467G in the
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RsAgo MID domain. As a control Flag-tagged GFP was cloned into pET30a(+). Protein
synthesis was induced with 1mM IPTG for 5 hours at 37C.
Small RNA and DNA isolation and sequencing
For total small RNA cloning (13-30 nt range) RNA was isolated using the Amresco Phenol-
Free Total RNA Purification Kit after fixation of the cell culture with Ambion RNAlater
reagent. Small RNA and DNA species were extracted from purified RsAgo complex using
proteinase K treatment followed by neutral phenol:chloroform extraction. Small RNAs were
cloned according to published protocol (Brennecke et al., 2007; Lau et al., 2001) that
requires the presence of 5
phosphate and 3
hydroxyl termini in RNA using linkers and
primers from the Illumina TrueSeq Small RNA Sample Prep kit. Half of the sample of
RsAgo-bound small RNA was radioactively labeled using sequential treatment with CIP and
PNK prior to ligation of the adapters allowing cloning of RNA molecules with other termini.
No such treatment was used during cloning of small RNAs from total RNA samples.
To clone small DNA we used bridged (splinted) ligation approach (Fig. S4). A simultaneous
5
and 3
linker ligation reaction was performed in 15
l volume and contained varying
amounts of small DNA (1:3-1:5 mixture of 5
‵
-
32
P labeled: non-labeled DNA), 100 pmoles
of 5
‵
and 3
‵
linker and 5
‵
and 3
‵
bridge oligonucleotides, 5% PEG8000, 1x T4 DNA ligase
buffer (50 mM Tris-HCl, 10 mM MgCl2,1 mM ATP, 10 mM DTT, pH 7.5) and 1
l of T4
DNA ligase (NEB, 400,000 units/ml). The reaction was incubated at room temperature for 1
to 10 hours, followed by electrophoresis in 12% urea-PAGE. Standard Illumina primers
were used to create indexed libraries.
Details on cloning procedures are given in the Supplemental Experimental Procedures.
Generation of RsAgo mutant strain ATCC17025
To create the mutagenesis vector a 1 kb homology arm corresponding to 5
‵
half of RsAgo
CDS was joined with an omega transcription termination cassette (Prentki and Krisch, 1984)
and cloned into the suicide vector pK18
mobsacB
, containing kanamycin resistance gene
(Schafer et al., 1994) (Fig. S11). After conjugation cells were selected on kanamycin and
correct insertion was verified with PCR.
Long RNA sequencing and analysis
For regular RNA sequencing samples were processed according to the Illumina TrueSeq
RNA prep kit. rRNA depletion was performed using the RiboZERO gram-negative bacterial
rRNA depletion kit (EpiBio). To profile the transcriptomes of wild-type and RsAgo mutant
strain 25, duplicate RNA-Seq libraries were prepared from rRNA-depleted RNA isolated
from two independent experiments.
Sequence analysis
For sequence analysis of small RNA and DNA we used the small RNA dashboard server
(Olson et al., 2008) and Galaxy tools (Blankenberg et al., 2010).
Accession numbers
RNA-seq, small RNA and small DNA data were deposited in the Gene Expression Omnibus
database under accession number ###.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
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Acknowledgments
We thank Katalin Fejes Tóth and members of the Aravin lab for helpful discussion and comments on the
manuscript. We thank Chia-Hung Wu, Shannon Park and Gargi Kulkarni from Dianne Newman's lab for guidance
with bacterial experiments and Konstantin Piatkov for helpful suggestions. We thank Timothy Donohue (University
of Wisconsin–Madison), Shulin Chen (WSU), Rebecca Parales (UC, Davis) and Carlos Ríos-Velázquez (UPR-
Mayagüez) for provided reagents and bacterial strains. We are grateful to Todd Lowe (UCSC) for providing access
to the genome browser. We thank Sailakshmi Subramanian (Mount Sinai), Georgi Marinov and Sergei Manakov
(Caltech) for help with bioinformatic and statistical analysis and Igor Antoshechkin (Caltech) for help with RNA
sequencing. We thank Gregory Donaldson and Vishnu Manoranjan for assistance with experiments. IO is a CEMI
(Center for Environmental Microbiology Interactions) fellow at Caltech. This work was supported by grants from
the National Institutes of Health (R01 GM097363, R00 HD057233 and DP2 OD007371A) and the Searle Scholar
Award to AAA. DKN is an Investigator of the Howard Hughes Medical Institute.
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Highlights
RsAgo associates with 15-19 nt RNA and 22-24 nt DNA molecules in vivo.
RsAgo-associated small RNAs correspond to the majority of cellular transcripts.
Small DNAs are complementary to the small RNAs and enriched in foreign sequences.
RsAgo degrades plasmid DNA and represses expression of plasmid-encoded genes.
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Figure 1.
R. sphaeroides
Argonaute protein associates with small RNA and small DNA molecules
(A)
R. sphaeroides
Argonaute protein and its operon structure. Gene Rsph17025_3695
encodes a predicted nuclease and overlaps with the last 4 nt of the RsAgo coding sequence.
PAZ, MID and PIWI are conserved domains shared by all Argonautes, while N (N-
terminal), L1 (linker 1) and L2 (linker 2) are not conserved.
(B)
Purified RsAgo is
associated with small RNA (15-19 nt) and small DNA (22-24 nt) species. RsAgo-associated
nucleic acids were 5
-radioactively labeled, treated with RNase A or DNase I and resolved
on a 15% urea PAGE. The
45 nt band is composed primarily of tRNAmet 3
fragments.
Densitometric measurement of three independent RsAgo samples indicates that the ratio of
small RNA to small DNA in RsAgo complexes is 2.12±0.66.
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Figure 2. Analysis of RsAgo-associated small RNA
(A)
Read length distribution of sequenced small RNA and small DNA associated with
RsAgo. Only sequences that could be mapped to the genome or the expression plasmid were
considered.
(B)
Nucleotide bias in small RNA extracted from purified RsAgo. 16-19 nt
reads were trimmed from the 3
end to 16 nt and analyzed with WebLogo.
(C)
Length profile
of total small RNAs extracted from two strains of
R. sphaeroides
- one encoding Ago
(17025) and one not (17029) - carrying either an empty plasmid or a plasmid expressing
RsAgo.
(D)
Bias for the uridine residue in position 1 in libraries shown in panel C. 61% of
reads in total small RNA from wild type strain 17025 lacking expression plasmid had
uridine in position 1.
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Figure 3. RsAgo-associated small RNAs represent a broad sample of the transcriptome
(A)
Distribution of unique small RNA reads from purified RsAgo over the six chromosomes
of
R. sphaeroides
strain 25 plotted as a fraction of raw read numbers and read numbers
normalized to chromosome length.
(B)
Annotation of RsAgo-associated small RNAs. Genes
on the expression plasmid were included in the analysis.
(C)
Density of cloned RsAgo-
associated small RNAs along chromosome 2 of
R. sphaeroides
strain 25. The graph includes
reads that can be mapped without mismatch to multiple genomic positions. Note that the
large number of reads mapping to the RsAgo gene is likely originating from the expression
plasmid. In the top graph each genomic position corresponding to a cloned small RNA is
given equal weight; on the bottom graph each read is normalized for the number of times it
maps to the genome, resulting in proportionally less signal for reads that map many times
such small RNA matching IS4 transposon.
(D)
The correlation between long and small RNA
abundance for
R. sphaeroides
genes. rRNA-depleted long RNA library was prepared from
strain 25 containing the pSRKKm-RsAgo plasmid.
(E)
R. sphaeroides
genes are sorted by
their enrichment for small RNAs relative to long transcripts as measured by RNA-Seq.
Shown are fold enrichment (positive values) or depletion (negative values) of small to long
RNA normalized to the mean small RNA/long RNA ratio for all genes. The 100 genes most
strongly depleted in small RNA (right side of the distribution) are enriched in non-coding
RNA relative to the rest (12% compared to 1.2%).
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Figure 4. Analysis of RsAgo-associated small DNA
(A)
Distribution of unique small DNA reads from purified RsAgo over the six chromosomes
of
R. sphaeroides
strain 25 plotted as a fraction of raw read numbers and read numbers
normalized to chromosome length.
(B)
Annotation of RsAgo-associated small DNA
sequences.
(C)
The profile of RsAgo-associated small RNA and DNA over the expression
plasmid pSRKKm-RsAgo. Reads that mapped to the plus or minus strand are shown above
and below the axis, respectively.
(D)
Strand bias of RsAgo small RNAs and DNAs mapped
to genes encoded on the expression plasmid pSRKKm-RsAgo.
(E)
Relative distance
between ends of small RNAs and DNAs mapped to opposite genomic strands of plasmid
pSRKKm-RsAgo. Graphs indicate that RNA and DNA have a high tendency to map as pairs
with the DNA molecule protruding 3 nt on each side. Note that this distance is more rigid on
the left side (5
end of RNA and 3
end of DNA) compared to the right side (3
end of RNA
and 5
end of DNA)
(F)
Nucleotide bias in small DNA aligned by the 3
end and analyzed
with WebLogo.
(G)
Model of small diRNA-riDNA pairs.
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