On the origin and functions of RNA-mediated silencing: from protists to man.

TLDR: The authors' analyses suggest that a relatively complex RNAi machinery was already present in the last common ancestor of eukaryotes and consisted, at a minimum, of one Argonaute-like polypeptide, one Piwi-like protein, one Dicer, and one RNA-dependent RNA polymerase.

Abstract: Double-stranded RNA has been shown to induce gene silencing in diverse eukaryotes and by a variety of pathways. We have examined the taxonomic distribution and the phylogenetic relationship of key components of the RNA interference (RNAi) machinery in members of five eukaryotic supergroups. On the basis of the parsimony principle, our analyses suggest that a relatively complex RNAi machinery was already present in the last common ancestor of eukaryotes and consisted, at a minimum, of one Argonaute-like polypeptide, one Piwi-like protein, one Dicer, and one RNA-dependent RNA polymerase. As proposed before, the ancestral (but non-essential) role of these components may have been in defense responses against genomic parasites such as transposable elements and viruses. From a mechanistic perspective, the RNAi machinery in the eukaryotic ancestor may have been capable of both small-RNA-guided transcript degradation as well as transcriptional repression, most likely through histone modifications. Both roles appear to be widespread among living eukaryotes and this diversification of function could account for the evolutionary conservation of duplicated Argonaute-Piwi proteins. In contrast, additional RNAi-mediated pathways such as RNA-directed DNA methylation, programmed genome rearrangements, meiotic silencing by unpaired DNA, and miRNA-mediated gene regulation may have evolved independently in specific lineages.

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May 2006
On the Origin and Functions of RNA-Mediated Silencing: From On the Origin and Functions of RNA-Mediated Silencing: From
Protists to Man Protists to Man
Heriberto D. Cerutti
University of Nebraska - Lincoln
, hcerutti1@unl.edu
Juan Casas-Mollano
University of Nebraska - Lincoln
, jcasasmollano2@unl.edu
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Cerutti, Heriberto D. and Casas-Mollano, Juan, "On the Origin and Functions of RNA-Mediated Silencing:
From Protists to Man" (2006).
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Abstract
Double-stranded RNA has been shown to induce gene silencing in
diverse eukaryotes and by a variety of pathways. We have exam-
ined the taxonomic distribution and the phylogenetic relationship
of key components of the RNA interference (RNAi) machinery in
members of ve eukaryotic supergroups. On the basis of the par-
simony principle, our analyses suggest that a relatively complex
RNAi machinery was already present in the last common ances-
tor of eukaryotes and consisted, at a minimum, of one Argonaute-
like polypeptide, one Piwi-like protein, one Dicer, and one RNA-
dependent RNA polymerase. As proposed before, the ancestral
(but non-essential) role of these components may have been in
defense responses against genomic parasites such as transposable
elements and viruses. From a mechanistic perspective, the RNAi
machinery in the eukaryotic ancestor may have been capable of
both small-RNA-guided transcript degradation as well as tran-
scriptional repression, most likely through histone modications.
Both roles appear to be widespread among living eukaryotes and
this diversication of function could account for the evolution-
ary conservation of duplicated Argonaute-Piwi proteins. In con-
trast, additional RNAi-mediated pathways such as RNA-directed
DNA methylation, programmed genome rearrangements, meiotic
silencing by unpaired DNA, and miRNA-mediated gene regula-
tion may have evolved independently in specic lineages.
Keywords: RNA interference, transposon silencing, heterochro-
matin, RNAi phylogenetics
Introduction
RNA-mediated silencing is an evolutionarily con-
served mechanism(s) through which double-stranded RNA
(dsRNA) induces the inactivation of cognate sequences.
The role of dsRNA in triggering repression was initially
characterized in Caenorhabditis elegans and termed RNA
interference (Fire et al. 1998). However, silencing phenom-
ena had already been described in a number of eukaryotes
and the connection to dsRNA helped to unify several, ap-
parently disparate, processes involving post-transcriptional
RNA degradation, transcriptional gene silencing via hetero-
chromatin formation and/or DNA methylation, DNA elimi-
nation, or meiotic silencing by unpaired DNA (Baulcombe
2004; Matzke and Birchler 2005; Meister and Tuschl 2004;
Ullu et al. 2004; Zamore and Haley 2005). In plants and an-
imals, the RNAi machinery is also involved in the produc-
tion of microRNAs (miRNAs), by the processing of ge-
nome encoded imperfect RNA hairpins, which play a role in
developmental regulation (Bartel 2004; Chen 2005; Wien-
holds and Plasterk 2005; Zamore and Haley 2005).
Currently, the most extensively characterized dsRNA-
mediated mechanism is targeted mRNA degradation
guided by small interfering RNAs (siRNAs). Genetic and
biochemical studies from diverse species have revealed
that long dsRNAs are processed into siRNAs by an RNas-
eIII-like endonuclease, named Dicer (Bernstein et al. 2001;
Published in Current Genetics 50 (2006), pp. 81–99; doi 10.1007/s00294-006-0078-x
Copyright © 2006 Springer-Verlag. Used by permission. http://www.springerlink.com/content/0172-8083
Submitted January 25, 2006; revised April 15, 2006; accepted April 19, 2006; published online May 12, 2006.
Communicated for Current Genetics by R. Bock
Review Article
On the Origin and Functions of RNA-Mediated Silencing:
From Protists to Man
Heriberto Cerutti* and J. Armando Casas-Mollano
School of Biological Sciences and Plant Science Initiative, University of Nebraska–Lincoln, Lincoln, NE 68588-0666, USA
* Correspondence—email: hcerutti1@unl.edu ; tel 402 472-0247; fax 402 472-8722
81

82 H. Cerutti and J. armando Casas-mollano in Current GenetiCs 50 (2006)
Meister and Tuschl 2004; Sontheimer 2005; Tomari and
Zamore 2005). siRNAs are then incorporated into a mul-
tiprotein complex, the RNA-induced silencing complex
(RISC) (Pham et al. 2004; Tomari et al. 2004). Members of
the Argonaute-Piwi (Ago-Piwi) family of proteins are core
components of the RISC and some of these polypeptides
function as siRNA-guided endonucleases (Baumberger and
Baulcombe 2005; Hammond et al. 2001; Liu et al. 2004a;
Meister and Tuschl 2004; Qi et al. 2005; Tomari and Za-
more 2005). Recent evidence suggests that a siRNA duplex
may be loaded into RISC and then Ago cleaves one of the
siRNA strands (the passenger strand) triggering its dissoci-
ation from the complex (Matranga et al. 2005; Miyoshi et
al. 2005; Rand et al. 2005). Activated RISC then functions
as a multiple-turnover enzyme that recognizes and cleaves
RNA molecules complementary to the incorporated sin-
gle-stranded guide siRNA (Meister and Tuschl 2004; Son-
theimer 2005; Tomari and Zamore 2005).
Members of the Argonaute-Piwi family fall into two
main classes, one named after Arabidopsis thaliana Ar-
gonaute and the other after Drosophila melanogaster Piwi
(Carmell et al. 2002). These proteins are highly basic, ap-
proximately 100-kD in size, and contain two conserved
motifs, the PAZ (after Piwi/Argonaute/Zwille) and the
PIWI domains (Cerutti et al. 2000; Lingel et al. 2004; Ma
et al. 2004; Song et al. 2004; Yuan et al. 2005). A number
of experiments have implicated certain argonautes, such as
human Ago2, as the catalytic unit (“slicer”) of the RISC
(Liu et al. 2004a; Okamura et al. 2004; Rivas et al. 2005).
However, several other Ago paralogs are not endonucleo-
lytically active (Liu et al. 2004a; Meister et al. 2004; Rivas
et al. 2005). In fact, in several species, there is evidence for
functional specialization of Ago-Piwi proteins (Grishok et
al. 2001; Lee et al. 2003; Matzke and Birchler 2005; Oka-
mura et al. 2004).
RNA-dependent RNA polymerases (RdRPs) also play
an important role in RNAi in some eukaryotes (Wasseneg-
ger and Krczal 2006). For instance, putative homologs of a
tomato RdRP are required for post-transcriptional gene si-
lencing (PTGS) triggered by sense transgenes in A. thali-
ana, for quelling (a phenomenon similar to PTGS) and for
meiotic silencing by unpaired DNA in Neurospora crassa,
as well as for RNAi in C. elegans and Dictyostelium discoi-
deum (Baulcombe 2004; Cogoni and Macino 2000; Mar-
tens et al. 2002; Shiu et al. 2001; Sijen et al. 2001; Wassen-
egger and Krczal 2006). It has been proposed that RdRPs
generate dsRNA from single-stranded transcripts either by
de novo, primer independent second-strand synthesis (uti-
lizing as template “aberrant” RNAs, presumably lacking
normal processing signals such as a 5′ cap or a polyA tail)
or by using siRNAs as primers to synthesize RNA comple-
mentary to the target mRNA (Baulcombe 2004; Sijen et al.
2001; Wassenegger and Krczal 2006). Thus, RdRP activ-
ity may initiate RNAi (by producing the trigger dsRNA)
or dramatically enhance the RNAi response (by amplify-
ing the amount of dsRNA) (Baulcombe 2004). However,
dsRNA-induced RNAi can occur in the absence of RdRP
activity (Schwarz et al. 2002; Stein et al. 2003).
The biochemical and genetic studies briey summa-
rized above have led to the identication of three key com-
ponents of the RNAi machinery, namely Dicer, Argonaute-
Piwi, and RdRP. However, the taxonomic distribution of
these proteins and their ancestry have not been explored in
detail. In this review we have examined the phylogenetic
relationship of Ago-Piwi, Dicer-like, and RdRP proteins
present in members of ve eukaryotic supergroups. On the
basis of the parsimony principle we have attempted to infer
the composition and function(s) of the RNAi machinery in
the last common ancestor of eukaryotes. We have also as-
sessed putatively derived RNAi functions that might have
evolved in specic lineages. Our ndings provide a frame-
work for predicting the existence of RNAi-related mecha-
nisms in uncharacterized eukaryotes.
Distribution of Argonaute-Piwi, Dicer-like, and RdRP
proteins in eukaryotes
Based on morphological, biochemical, and molecular
phylogenetic approaches, eukaryotes have recently been
classied into six supergroups: the Opisthokonta, including
animals and fungi; the Amoebozoa, including most tradi-
tional amoebae and slime moulds; the Excavata, grouping
diplomonads, several genera of heterotrophic agellates,
and possibly the Euglenozoa; the Rhizaria, including the
Foraminifera and the Cercozoa; the Archaeplastida, group-
ing red algae, green algae, and plants; and the Chromalveo-
lata, including dinoagellates, apicomplexan parasites, and
the Stramenopiles (brown algae, diatoms, and many zoo-
sporic fungi) (Adl et al. 2005; Medina 2005). In order to
evaluate the phyletic distribution of the RNAi machinery
components, we have surveyed 25 complete or near-com-
plete genomes that belong to ve eukaryotic supergroups
(with only Rhizaria remaining unsampled). Proteins con-
taining conserved Argonaute-Piwi, Dicer, or RdRP domains
were identied by either BLAST or PSI-BLAST searches
of protein and/or translated genomic DNA databases. Since
several of the examined genomes are in draft stage, an im-
portant caveat in our analyses is that some proteins may be

on tHe origin and funCtions of rna-mediated silenCing: from protists to man 83
missing from the databases whereas others may have errors
in the predicted gene structure. However, we only consid-
ered as potential homologs proteins that exhibited enough
sequence similarity to be aligned and used for phylogenetic
tree construction.
Argonaute-Piwi, Dicer-like, and RdRP proteins occur in
members of all the eukaryotic supergroups examined (Ta-
ble 1). This widespread taxonomic distribution, as well as
the direct demonstration of RNAi related phenomena in
most of these organisms (Table 2), suggests that the main
components of the RNAi machinery were already pres-
ent in the last common ancestor of eukaryotes. However,
Ago-Piwi, Dicer-like, and RdRP polypeptides (or a subset
of these proteins) also appear to have been lost from spe-
cic lineages. The RNAi machinery seems to be entirely
absent in Saccharomyces cerevisiae (Opisthokonta), Try-
panosoma cruzi and Leishmania major (Excavata), Cy-
anidioschyzon merolae (Archaeplastida), and Plasmodium
falciparum (Chromalveolata) (Table 1). For some of these
organisms there is also convincing evidence that they are
unable to utilize dsRNA to trigger degradation of target
RNA (DaRocha et al. 2004; Robinson and Beverley 2003;
Ullu et al. 2004). Thus, the RNAi mechanism appears to
have been lost independently several times during eukary-
otic evolution.
The greatest conservation among the examined poly-
peptides corresponded to Ago-Piwi proteins, which are
clearly identiable in all species where RNAi-related phe-
nomena have been experimentally demonstrated (Tables 1,
2). Moreover, the dual domain structure of the Ago-Piwi
polypeptides, namely a PAZ domain followed by a PIWI
domain, has also been well conserved. The only exception
Table 1 Distribution of RNAi machinery components in eukaryotes
Species Genome
a
Argonaute- Dicer-like RdRP Role in virus,
Piwi transposon, or
repetitive DNA
silencing
b
Excavata
Giardia intestinalis Assembly + + + ?
Trypanosoma brucei Assembly + − − +
Trypanosoma cruzi Complete − − − NA
Leishmania major Assembly − − − NA
Chromalveolata
Paramecium tetraurelia In progress + + + ?
Tetrahymena thermophila In progress + + + +
Plasmodium falciparum Assembly − − − NA
Phytophthora sojae In progress + + + ?
Thalassiosira pseudonana Assembly + − − ?
Rhizaria
Data not available
Archaeplastida
Cyanidioschyzon merolae Complete − − − NA
Chlamydomonas reinhardtii In progress + + − +
Arabidopsis thaliana Complete + + + +
Oryza sativa (japonica) Complete + + + +
Amoebozoa
Dictyostelium discoideum Assembly + + + +
Entamoeba histolytica Complete + − + ?
Opisthokonta
Saccharomyces cerevisiae Complete − − − NA
Schizosaccharomyces pombe Complete + + + +
Neurospora crassa Complete + + + +
Aspergillus nidulans Assembly + + + ?
Caenorhabditis elegans Complete + + + +
Drosophila melanogaster Complete + + − +
Anopheles gambiae Assembly + + − +
Strongylocentrotus purpuratus In progress + + − ?
Ciona intestinalis Assembly + + − ?
Homo sapiens Complete + + − +
a
Status of genome sequencing projects taken from http://www.ncbi.nlm.nih.gov/genomes/leuks.cgi
b
Inferred from the phenotype of mutant or RNAi knock-down strains defective in at least one of the RNAi effectors (?, lack of evidence; NA, not applicable)

84 H. Cerutti and J. armando Casas-mollano in Current GenetiCs 50 (2006)
Table 2 Taxonomic distribution of RNAi-mediated silencing pathways in eukaryotes
Species
Cloned endogenous
small RNAs (20–
30 nt)
dsRNA-induced
RNAi
RNAi-mediated
(hetero)chromatin
formation
Programmed genome
rearrangement (DNA
elimination)
RNA-directed DNA
methylation
Meiotic
silencing by
unpaired DNA
miRNA-mediated
gene regulation
Excavata
Giardia intestinalis Ullu et al. (2005)
Trypanosoma brucei
Djikeng et al. (2001)
Durand-Dubief and
Bastin (2003) and
Shi et al. (2004)
Indirect
a
Durand-Dubief and Bastin
(2003) and Shi et al.
(2004)
Chromalveolata
Paramecium tetraurelia
Galvani and Sperling
(2002)
Garnier et al. (2004) and
Nowacki et al. (2005)
Tetrahymena
thermophila
Lee and Collins
(2006)
Direct
b
Liu et al. (2004b) and
Mochizuki and Gorovsky
(2005)
Mochizuki and
Gorovsky (2005) and
Yao and Chao (2005)
Phytophthora infestans
Whisson et al.
(2005)
Archaeplastida
Chlamydomonas
reinhardtii
Rohr et al. (2004)
and Schroda (2006)
Arabidopsis thaliana
Llave et al. (2002)
and Reinhart et al.
(2002)
An et al. (2003) and
Watson et al. (2005)
Direct
b
Matzke and Birchler
(2005) and Zilberman et
al. (2003)
Chan et al. (2004)
and Matzke and
Birchler (2005)
Chen (2005) and Xie
et al. (2005)
Oryza sativa
(japonica) Sunkar et al. (2005)
Miki and Shimamoto
(2004) and Tang et
al. (2004)
Liu et al. (2005a)
Amoebozoa
Dictyostelium
discoideum
Kuhlmann et al.
(2005)
Martens et al. (2002)
Entamoeba histolytica
Kaur and Lohia
(2004) and Vayssie
et al. (2004)
Opisthokonta
Schizosaccharomyces
pombe
Reinhart and Bartel
(2002)
Sigova et al. (2004)
Direct
b
Volpe et al. (2002) and
Verdel et al. (2004)
Neurospora crassa
Chicas et al. (2004)
Chicas et al. (2005)
and Nakayashiki
(2005)
Shiu et al.
(2001) and Lee
et al. (2003)
Aspergillus nidulans
Hammond and
Keller (2005)

Frequently asked questions

Q1. What are the contributions in "On the origin and functions of rna-mediated silencing: from protists to man" ?

The authors have examined the taxonomic distribution and the phylogenetic relationship of key components of the RNA interference ( RNAi ) machinery in members of five eukaryotic supergroups. On the basis of the parsimony principle, their analyses suggest that a relatively complex RNAi machinery was already present in the last common ancestor of eukaryotes and consisted, at a minimum, of one Argonautelike polypeptide, one Piwi-like protein, one Dicer, and one RNAdependent RNA polymerase. 

Q2. What is the likely explanation for the bulged structure of miRNAs?

Progressive sequence degradation, under selective pressure, may eventually result in the bulged structure typical of miRNA precursors. 

Q3. In what organisms do RNAi-mediated (hetero)chromatin formation occur?

in S. pombe and chicken DT40 cells RNAi-mediated (hetero)chromatin formation may now play a critical role in determining chromosome structure and function during mitosis and/or meiosis (Fukagawa et al. 

Q4. In what organisms do RNAi-independent pathways exist?

RNAi-independent pathways for (hetero)chromatin formation and DNA methylation appear to exist in several RNAi-positive eukaryotes (Chicas et al. 

Q5. What is the role of RNAi in the elimination of excess DNA in protozoa?

Many of the eliminated sequences appear to be derived from transposons (Lee and Collins 2006; Yao and Chao 2005) and RNA-mediated DNA elimination may have evolved as an extension of the role of the RNAi machinery in the transcriptional silencing of transposon/repetitive sequences. 

Q6. What is the parsimonious interpretation of the data?

A parsimonious interpretation of these data suggests that the last common ancestor of eukaryotes contained both Argonaute-like and Piwi-like proteins and that specific lineages independently lost either one or the other. 

Q7. What could explain the more variable Dicer-like proteins found in other living organisms?

Domain deletion/truncation, domain fusion, as well as sequence divergence could explain the more variable Dicer-like proteins found in other living organisms (which contain various combinations of some of the putative ancestral motifs) (Figure 1). 

Q8. What is the significance of the expansion of Dicer-like sequences in plants?

plant Dicer-like sequences underwent significant expansion largely prior to the divergence of monocots and dicots. 

Q9. What are the main caveats associated with the analysis?

Besides the already discussed caveats associated with their analyses, the topology of the RdRP tree might also be affected by more prevalent lineage-specific losses of some of these proteins.