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Expanding RNA physiology: microRNAs in a unicellular organism
Haruhiko Siomi and Mikiko C. Siomi
Genes & Dev. 2007 21: 1153-1156
Access the most recent version at doi:10.1101/gad.1559707

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PERSPECTIVE

Expanding RNA physiology: microRNAs
in a unicellular organism
Haruhiko Siomi1 and Mikiko C. Siomi
Institute for Genome Research, University of Tokushima, 3-18-15 Kuramoto, Tokushima, 770-8503 Japan

Until recently, the world viewed through the eyes ofmolecular biologists seemed simple with a well-known
slogan hailing a central dogma: “DNA makes RNA
makes protein.” This has been interpreted to mean thatgenetic output is entirely or almost entirely transacted
by proteins (Mattick 2004). However, within a few yearsof the discovery of RNA interference (RNAi) (Fire et al.
1998), an explosion of data has lifted the veil off many
previous puzzling findings. Thus, our view of the field
was irrevocably changed with a new slogan—that went
alongside the classical central dogma—“RNA makes
small RNA makes no protein” (Fig. 1). Sequence information in long RNA transcripts is converted into
small, ∼20- to 30-nucleotide (nt)-long, non-protein-coding RNAs such as microRNAs (miRNAs) and small interfering RNAs (siRNAs). These small RNAs trigger
various forms of sequence-specific gene silencing, including RNA cleavage and degradation, cleavage-independent mRNA decay, translational repression, methylation of protein-coding DNA, and heterochromatin formation in a variety of eukaryotic organisms ranging
from fission yeast, plants, fungi, nematode, and fly tohumans. This process is now collectively referred toas RNA silencing (Tomari and Zamore 2005; Zaratiegui
et al. 2007). In addition to classical small non-proteincoding RNAs—including tRNAs, small nuclear RNAs
(snRNAs), and small nucleolar RNAs (snoRNAs)—recent studies have shown that our cells express a surprisingly large number of new regulatory small RNAs thatseem to be particularly abundant in roles that direct thebinding of protein complexes to specific nucleic acid sequences (Hüttenhofer and Schattner 2006). These include miRNAs, several groups of natural or endogenous
siRNAs, and Piwi-interacting RNAs (piRNAs) (Kim
2006). The ongoing efforts to uncover the full biologicalscope of RNA silencing have already modified our notions about how gene expression is controlled. It is becoming clear that RNA silencing comprises an important tier of gene expression in eukaryotes, enabling the
integration and networking of complex suites of geneactivity, thereby elaborating multicellular complexity(Bartel 2004; Kloosterman and Plasterk 2006).
The first small RNA trigger discovered in eukaryotes
was the lin-4 miRNA, which was found by mapping a
1

Corresponding author.
E-MAIL siomi@genome.tokushima-u.ac.jp; FAX 81-88-6339451.
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1559707.

developmental timing (heterochronic) mutant Caenorhabditis elegans locus (Lee et al. 1993). The discoveryof the phylogenetic conservation of let-7 miRNA thenreally opened up the field (Pasquinelli et al. 2000). Thousands of miRNAs have now been identified in various
multicellular organisms (miRBase; Griffiths-Jones et al.
2006), and an astonishing number of miRNA genes havebeen predicted to exist in our genome (Miranda et al.
2006). Transcripts of genes encoding self-complementary
RNA that forms imperfect hairpins, termed primary
microRNA (pri-miRNA), are cleaved by the Drosha–
Pasha/DGCR8 complex in the nucleus to generate premiRNA. The pre-miRNA is exported to the cytoplasm
and further processed by Dicer into an ∼21- to 23-nt
miRNA duplex. Then, one strand over the other is often
preferentially loaded into a RNA-induced silencing complex (RISC), which contains at its core a member of the
Argonaute family of proteins (Parker and Barford 2006).
Once incorporated into the silencing complex, mature
miRNAs interact with target mRNAs at specific sites to
induce cleavage of the message, cleavage-independent
mRNA decay, or inhibit translation (Tomari and Zamore
2005; Pillai et al. 2007). Cleavage of mRNA targets iscatalyzed by Argonaute proteins (Liu et al. 2004; Parker
and Barford 2006). In both animals and plants, miRNAs
play important roles in diverse developmental processes,
including developmental timing, cell death, cell proliferation, asymmetric cell fate decision, organogenesis,
and patterning of the nervous system. However, the biological functions of most miRNAs remain unknown (Alvarez-Garcia and Miska 2005; Jones-Rhoades et al. 2006).
Recent studies have shown that miRNAs target 30% ormore of all animal protein-coding genes, most of which
are regulated through base pairing between specific sites
in the 3⬘ untranslated region of the messenger RNAs(mRNAs) and a small region termed the “seed” (nucleotides 1–8, from the 5⬘ end) near the 5⬘ end of the miRNA(Brennecke et al. 2005; Lewis et al. 2005; Xie et al. 2005).
These rather lax criteria can result in numerous targets
being predicted for a given miRNA. Many genes haveseveral target sites for either one miRNA or a few different miRNAs. Additionally, many miRNAs are expressedin a cell type- or tissue-specific manner, and miRNAs
and their targets have, to a large extent, mutually exclusive expression patterns (“para-expression”) (Farh et al.
2005; Stark et al. 2005). Therefore, the general functionof miRNAs may be that miRNAs diversify cell types ororgan types by fine-tuning the proteome, and then retain

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Siomi and Siomi

Figure 1. RNA physiology. Recent studies show that the vast
majority of our genome is transcribed (e.g., Carninci et al. 2005;
Manak et al. 2006). This in turn produces a surprisingly large
number of functional non-protein-coding RNAs, including
small regulatory RNAs (Prasanth and Spector 2007). These observations have led to a new concept: that lying behind theorigin of complexity of higher organisms is the dramatic expansion of the non-protein-coding RNA inventory, including theabsolute number of siRNAs and miRNAs, rather than an increase in the protein-encoding inventory (Mattick 2004).

terminal differentiation programs in a stable end state bydumping expression of genes that should not be expressed in particular cell types (Kloosterman and Plasterk 2006). The rapidly expanding repertoire of miRNAs
and their targets suggests that miRNAs played a role in
the adaptive diversification of pre-existing transcriptional and cell–cell signaling pathways required for theevolution of new regulatory circuits and, ultimately,
new patterns within body plans. Thereby, miRNAs appear to drive animal and plant diversity (Carthew 2006;
Ambros and Chen 2007; Niwa and Slack 2007). Consistent with this notion that miRNAs are global regulatorsof cell differentiation and patterning, miRNAs have thus
far only been identified in multicellular organisms. They
are notably absent from unicellular organisms including
Schizosaccharomyces pombe and Tetrahymena thermophila, in which RNA silencing mechanisms are known
to operate. However, in this issue of Genes & Development, Qi and coworkers (Zhao et al. 2007) add an exciting new twist to this developing story, showing that theunicellular green alga Chlamydomonas reinhardtii encodes many miRNAs.
Long known for their roles in motility, flagella (orcilia) are increasingly being recognized as sites for transmitting signals to the cytoplasm and nuclei to control
gene expression, cell function, animal development, andbehavior (Snell et al. 2004; Scholey and Anderson 2006).
The unicellular biflagellate alga C. reinhardtii has been a
productive model system for molecular genetic studies

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GENES & DEVELOPMENTof flagellar assembly and function, as well as of basal
body function, for more than five decades, mainly because its flagellated vegetative cells are haploid and its
mutants can be genetically analyzed by sexual crosses
between different strains (Schroda 2006). Mating is initiated when the cells are starved for nitrogen and involves the shedding of the cell wall prior to cytoplasmic
fusion of opposite mating types. After nuclear and chloroplast fusion, flagella are lost and a zygotic cell wall
begins to form. Meiosis is initiated in zygotes by reintroduction of nutrients and is completed to produce four
meiotic zoospores. The development of reliable procedures for transformation has also facilitated the study ofvarious aspects of cell biology of C. reinhardtii. Additionally, RNAi has been shown to be an effective reverse
genetic approach in this organism. This indicates that C.
reinhardtii possesses functional RNAi machinery.
Zhao et al. (2007) now report the identification andcharacterization of small RNAs in C. reinhardtii using ahighly parallel pyrosequencing methodology (Margulies
et al. 2005). Computational studies show that among
4182 unique small RNA sequences identified, 200 species were derived from genomic sequences with the potential to form hairpin structures that meet the requirements for an miRNA precursor. These RNAs share several common features with miRNAs, including having a5⬘ phosphate and a preference for uracil at the 5⬘ end. In
addition, several of the miRNAs identified have a sequenced pairing miRNA* located in the opposite arm of
the precursor, which is capable of forming a duplex bearing the 2-nt 3⬘ overhang—the hallmark of Dicer cleavage(Tomari and Zamore 2005; Kim 2006). These findings
show that C. reinhardtii encodes miRNAs—the first evidence of miRNAs in a unicellular organism.
None of the C. reinhardtii miRNAs had an ortholog inother green algae, plants, or animals, suggesting that C.
reinhardtii miRNA genes may have arisen independently after splitting from a common lineage. A substantial number of the C. reinhardtii miRNAs are located inrelatively long hairpins, many of which have the potential to produce multiple small RNAs. These observations
are similar to recent results from plants that suggest thatsome miRNA genes evolve de novo through inverted duplication of their future target genes (Allen et al. 2004).
This hypothesis predicts that recently evolved, andtherefore presumably nonconserved, miRNAs might
still contain long segments with high similarity to thetarget gene sequence. Indeed, a number of nonconserved
Arabidopsis thaliana miRNA loci have been identified
to fulfill these criteria (Allen et al. 2004; Rajagopalan etal. 2006). Whether some of the C. reinhardtii miRNAs
fulfill the criteria remains to be determined. Given theirhighly evolving nature, newly evolved miRNAs are ideally suited to convey rapid adaptation to an environment
and, therefore, will probably be more abundant in theirstress responses (Voinnet 2004). Interestingly, computational studies have also predicted many genes encoding
flagellar-associated proteins as targets for the C. reinhardtii miRNAs. Chlamydomonas cells shed their flagella in response to many stressful conditions, presum-Downloaded from www.genesdev.org on May 15, 2007

RNA physiology

ably serving a protective function for cells in the particular environment. Then, after the shedding, Chlamydomonas cells begin to regenerate flagella (Lefebvre 1995).
Are the C. reinhardtii miRNAs functional? Zhao et al.
(2007) fractionated total extracts from C. reinhardtii cultures by gel filtration and found that the C. reinhardtii
miRNAs exist in complexes with a molecular size of∼150 kDa, a size that is similar to that of minimal RISC
in human and Drosophila cells (Martinez et al. 2002). If
miRNAs have sufficient complementarity to the target
RNA, they can direct cleavage of the target between 10
and 11 nt from the 5⬘ end of the miRNA, as is the casefor siRNAs in RNAi (Yekta et al. 2004). C. reinhardtii
miRNAs were cofractionated with the site-specificcleavage activity of their predicted mRNA targets, much
like “Slicer” activity of some Argonaute proteins (Parker
and Barford 2006). The Chlamydomonas genome
encodes three Dicer-like and at least two Argonaute
proteins (Schroda 2006). It remains to be determined
whether an Argonaute protein resides in C. reinhardtii
miRNP complexes and which cellular processes they are
involved in. Do the C. reinhardtii miRNAs act to regulate genes involved in, for example, specification of flagellar assembly, functioning in a manner analogous tocell fate decisions in multicellular organisms that are
triggered by signal transduction activity (Bartel 2004;
Carthew 2006; Kloosterman and Plasterk 2006)? It is
probably noteworthy that Chlamydomonas gametes use
their flagella as sensory transducers during fertilization(Snell et al. 2004; Scholey and Anderson 2006). The interaction between the flagella of cells of opposite mating
types initiates a series of morphological changes, including the release of their extracellular matrix (cell wall),modification and elongation of the flagellar tip, and erection of microvillus-like cell fusion organelles. Some of
the C. reinhardtii miRNAs appear to be differentiallyexpressed in a vegetative state and in gametes, suggesting that they might play roles in gametic differentiation.
It will be interesting to see how these processes are affected when some or all of the C. reinhardtii miRNAs
are depleted, and to analyze whether certain transcripts
or proteins are differently expressed in such miRNA-depleted and control gametes.
In addition to miRNAs, Zhao et al. (2007) identified alarge number of endogenous siRNAs. Their 5⬘ ends also
show the strong preference for uracil that is characteristic of small RNAs (Kim 2006). They are derived fromvarious locations including protein-coding genes and intergenic regions. Many of them are enriched in several
genomic loci and phased relative to each other, reminiscent of plant trans-acting siRNAs (tasiRNAs), whosebiogenesis requires miRNA-directed cleavage (Allen etal. 2005; Yoshikawa et al. 2005; Axtell et al. 2006). Although many endogenous siRNAs are products of RNAdependent RNA polymerases (RdRPs) in plants and C.
elegans (Cerutti and Casas-Mollano 2006; Vazquez2006), Chlamydomonas lacks an RdRP in its genome(Schroda 2006). Very few C. reinhardtii siRNAs arise
from repetitive sequences and transposons. In fact, repetitive sequences are underrepresented. This situation

appears similar to mammalian piRNAs (Aravin et al.
2006; Girard et al. 2006; Lau et al. 2006). It remains to be
determined whether these endogenous siRNAs in C. reinhardtii use a molecular mechanism similar to that of
mammalian piRNAs to silence portions of the genome(Kim 2006) or whether they use a molecular mechanism
similar to that of plant tasiRNAs to silence messages
from loci that are unrelated to those from which thesiRNAs derive (Vazquez 2006).
There are many more questions ensuing from thesestudies. Are miRNAs found in unicellular organisms
other than C. reinhardtii? The giant unicellular alga Acetabularia may be one such candidate. Control of developmental morphogenesis in this enormous cell (2–4 cm)
was studied by J. Hämmerling in the 1930s (Hämmerling
1934; Mandoli 1998), but the morphogenetic signals involved in his intriguing experiments are still to be explored using modern molecular techniques (Mandoli
1998). Are other classes of small RNAs found in C. reinhardtii? Zhao et al. (2007) isolated small RNAs from
C. reinhardtii cultures under vegetative growth conditions. By analogy with piRNAs in the mammalian germline, C. reinhardtii gametes may express specificclasses of small RNAs. Is there a class of siRNAs in C.
reinhardtii that can direct chromatin modifications in
the newly replicated “zygotic” nucleus and thus induce
heritable silencing? Perhaps the most crucial question is
how the surprisingly large number of siRNAs and miRNAs in C. reinhardtii connect between genotype andphenotype. Further investigation should reveal thephysiology of small RNAs in C. reinhardtii and help expand our understanding of the physiology of small RNAsin multicellular organisms.
Acknowledgments
We thank Yukihide Tomari and Takafumi Sunohara for comments. Work in our laboratories was supported by the Ministryof Education, Culture, Sports, Science, and Technology of Japan(MEXT) and the New Energy and Industrial Technology Development Organization (NEDO).

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