REVIEW ARTICLE
Ribosomal RNA genes in eukaryotic microorganisms: witnesses of
phylogeny?
Ana Lilia Torres-Machorro, Roberto Hernández, Ana Marı́a Cevallos & Imelda López-Villaseñor
Departamento de Biologı́a Molecular y Biotecnologı́a, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México,
México D.F., Mexico
Correspondence: Imelda López-Villaseñor,
Departamento de Biologı́a Molecular y
Biotecnologı́a, Instituto de Investigaciones
Biomédicas, Universidad Nacional Autónoma
de México, Apartado postal 70-228, 04510
México D.F., Mexico. Tel.: 152 55 5622 8952;
fax: 152 55 5622 9212; e-mail:
imelda@biomedicas.unam.mx
Received 4 September 2009; revised 25
October 2009; accepted 27 October 2009.
Final version published online 23 November
2009.
DOI:10.1111/j.1574-6976.2009.00196.x
MICROBIOLOGY REVIEWS
Editor: Colin Berry
Keywords
rRNA; 5S rRNA; unicellular eukaryote;
ribosomal cistron organization;
extrachromosomal gene; repeated sequence.
Abstract
The study of genomic organization and regulatory elements of rRNA genes in
metazoan paradigmatic organisms has led to the most accepted model of rRNA
gene organization in eukaryotes. Nevertheless, the rRNA genes of microbial
eukaryotes have also been studied in considerable detail and their atypical
structures have been considered as exceptions. However, it is likely that these
organisms have preserved variations in the organization of a versatile gene that
may be seen as living records of evolution. Here, we review the organization of the
main rRNA transcription unit (rDNA) and the 5S rRNA genes (5S rDNA). These
genes are reiterated in the genome of microbial eukaryotes and may be coded
alone, in tandem repeats, linked to each other or linked to other genes. They may
be found in the chromosome or extrachromosomally in linear or circular units.
rDNA coding regions may contain introns, sequence insertions, protein-coding
genes or additional spacers. The 5S rDNA can be found in tandem repeats or
genetically linked to genes transcribed by RNA polymerases I, II or III. Available
information from about a hundred microbial eukaryotes was used to review the
unexpected diversity in the genomic organization of rRNA genes.
Introduction
The most recent phylogenetic model for relationships among
eukaryotes clusters them into six supergroups, probably
monophyletic (Simpson & Roger, 2004; Adl et al., 2005).
Microbial eukaryotes are found in all six groups and have
considerable morphological, ultrastructural and genetic diversity. Several unique features have been described in these
organisms, such as trans-splicing and RNA editing in trypanosomatids (Madison-Antenucci et al., 2002; Haile & Papadopoulou, 2007) as well as DNA splicing and rearrangements
in the ciliate Tetrahymena (Prescott, 2000). Microsporidia
(Encephalitozoon cuniculi) possess genomes in the size range
of bacteria (Keeling & Slamovits, 2004), while the genomes of
dinoflagellates lack histones and nucleosomes (Moreno Dı́az
de la Espina et al., 2005). Cryptomonad and chlorarachniophyte unicellular algae conserve a relict miniaturized nucleus
of a formerly independent alga (nucleomorph) (CavalierSmith, 2002) and specialized infection organelles (rhoptries
and micronemes) are present in apicomplexans such as
FEMS Microbiol Rev 34 (2010) 59–86
Plasmodium (Kats et al., 2006) and Toxoplasma parasites
(Boothroyd & Dubremetz, 2008). Unusual characteristics
extend to the organization of rRNA genes, which evidence
the peculiarities, diversity and divergence of the genome
structure in microbial eukaryotes. An overview of the biology
of key microbial eukaryotes is given in Box 1.
The typical eukaryotic translation machinery, the ribosome,
is composed of two subunits with four rRNA species and 4 70
proteins. The large subunit (LSU) contains the 28S, the 5.8S and
the 5S rRNAs. The small subunit (SSU) contains the 18S rRNA
(SSU rRNA). The four rRNA mature molecules are coded in
two rRNA genes transcribed by two different RNA polymerases.
The 18S, 5.8S and 28S rRNAs are coded in a single transcription
unit called a ribosomal cistron or the main transcription unit,
transcribed by RNA polymerase I (pol I). The 5S rRNA gene is
not usually linked to the ribosomal cistron and is transcribed by
pol III (Mandal, 1984; Paule & White, 2000).
rRNA genes were among the first genes to be studied in
detail due to their highly repetitive nature, ease of manipulation and biological importance (Miller & Beatty, 1969;
2009 Federation of European Microbiological Societies
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c
60
A.L. Torres-Machorro et al.
Box 1. Characteristics of some microbial eukaryotes
We used the most recent and accepted classification of eukaryotes, based on multiple gene molecular phylogenies and structural analyses. This system divides
eukaryotes into six supergroups: Amoebozoa, Opisthokonta, Rhizaria, Plantae, Chromalveolata and Excavata (Simpson & Roger, 2004; Adl et al., 2005; Dacks
et al., 2008). Here, we describe key microorganisms of each supergroup (Margulis & Schwartz, 2000).
1. AMOEBOZOA: Organisms that show amoeboid locomotion with pseudopodia.
Pelomyxa palustris: Giant anaerobic amoeba that contains three types of bacterial endosymbionts that replace the functions of some lacking organelles such as
the mitochondria.
Acanthamoeba castellanii (Acanthamoebidae): Freeliving soil amoeba.
Entamoeba histolytica (Entamoebida): Uninucleate amitochondriate amoeba that infects the intestine of animals, causing amoebiasis.
Physarum polycephalum (Eumycetozoa, Myxogastria): Amoeboid cells that can differentiate into fungus-like reproductive structures. During its life cycle, a
diploid zygote divides repeatedly to form a multinucleated cytoplasmic mass called the plasmodium. Under dry conditions, the plasmodium may mature into
spore-producing organs.
Didymium iridis (Eumycetozoa, Myxogastria): Plasmodial slime mold.
Dictyostelium discoideum (Eumycetozoa, Dictyostelia): Land-dwelling cellular slime mold. Independent amoebas may aggregate into a slimy mass (slug) that
eventually transforms into a reproductive body that produces spores.
2. OPISTHOKONTA: Organisms with a single posterior flagellum in at least one stage of the life cycle.
Fungi: Dominant osmotrophs that play crucial roles as decomposers and as symbionts or parasites.
Ascomycota: Hold a microscopic reproductive structure called ascus.
Pneumocystis carinii (Ascomycota, Taphrinomycotina): Causes fatal pneumonia in immunocompromised humans.
Schizosaccharomyces pombe (Ascomycota, Taphrinomycotina, Schizosaccharomycetes): Fission yeast.
Saccharomyces cerevisiae (Ascomycota, Saccharomycetes): Budding yeast that ferments sugars to ethyl alcohol.
Candida albicans (Ascomycota, Saccharomycetes): Causes infections in humans.
Microsporidia: Intracellular asexual parasites that lack mitochondria. The microsporan resting stages are the chitinous spores, which contain a polar
filament and an infective body.
Encephalitozoon cuniculi: Parasites of warm-blooded vertebrates, holds one of the smallest known eukaryotic genomes (2.9 Mbp) (Biderre et al., 1997).
Nosema bombycis: causes disease in insects.
3. RHIZARIA: Organisms with pseudopodia of various types.
Foraminifera: Planktonic or benthic free-swimming organisms that have pore-studded shells. They show nuclear dimorphism and complex life cycles.
4. PLANTAE (ARCHAEPLASTIDA): Organisms that hold a photosynthetic plastid derived from a primary endosymbiosis with a cyanobacterium.
Rhodophyceae (Red algae): Mostly marine organisms that hold rhodoplasts (red plastids).
Cyanidoschyzon merolae: Unicellular organisms that inhabit sulfate-rich hot springs.
Chloroplastida: Organisms that hold green chloroplasts.
Acetabularia mediterranea (Chlorophyta, Ulvophyceae): Syncytial green algae.
Chlorella (Chlorophyta, Trebouxiphyceae).
Chlamydomonas (Chlorophyta, Chlorophyceae).
5. CHROMALVEOLATA: Organisms that contain a plastid that comes from a secondary endosymbiosis with an ancestral archaeplastid.
Bacillariophyta (Stramenopiles): Single cells or colonies covered by an elaborate, symmetrical two-part shell.
Alveolata:
Dinoflagellata (Dinozoa): Mostly unicellular marine plankton, holding two undulipodia and complex rigid walls (tests). Some species produce toxins.
Pfiesteria piscicida (Dinophyceae, Peridinophyceae).
Ciliophora: Unicellular organisms covered with cilia (short undulipodia). They have two types of nuclei: small genetic micronuclei (MIC, containing
standard chromosomes) and large transcriptionally active macronuclei (MAC, it develops from the micronuclei).
Euplotes (Intramacronucleata, Spirotrichea, Hypotrichia).
Paramecium (Intramacronucleata, Oligohymenophorea, Peniculia).
Tetrahymena (Intramacronucleata, Oligohymnophorea, Hymenostomatia).
Apicomplexa: Specialized obligate intracellular parasites named for the ‘apical complex’ that hold structures such as rhoptries and micronemes, the
specialized machinery used for invasion (Kats et al., 2006; Boothroyd & Dubremetz, 2008).
Plasmodium (Aconoidasida, Haemosporida): The causative agent of malaria exists in association with an invertebrate host (sexual stage in the mosquito)
and a vertebrate host (asexual stage). Plasmodium falciparum and Plasmodium vivax infect human red blood cells, while Plasmodium berghei infects rodents.
Babesia bovis (Aconoidasida, Piroplasmorida).
Theileria parva (Aconoidasida, Piroplasmorida).
Cryptosporidium (Conoidasida, Coccidiasina).
Eimeria (Conoidasida, Coccidiasina).
6. EXCAVATA: Organisms that typically have a suspension-feeding groove and flagella.
Giardia intestinalis (Fornicata, Eopharyngia, Diplomonadida, Giardiinae): A parasite of the small intestine of vertebrates through infective cysts. It has two
transcriptionally active karyomastigonts (nuclei attached to undulipodia by thin fibers), and lacks mitochondria and the Golgi apparatus.
Trichomonas vaginalis (Parabasalia, Trichomonadida): Amitochondriate parasite causative of trichomoniasis in humans. The organelles known as parabasal
bodies are involved in the synthesis, storage and transport of proteins.
Trichomonas tenax (Parabasalia, Trichomonadida): Infects the human mouth.
Tritrichomonas foetus (Parabasalia, Trichomonadida): Infects the urogenital tract of cattle.
Naegleria gruberi: (Heterolobosea, Vahlkampfiidae) Soil and freshwater freeliving amoeba that transforms into unduliopodiated cells.
Euglena gracilis (Euglenozoa, Euglenida, Euglenea): Unicellular organism living in stagnant water. It can be found with or without chloroplasts.
Kinetoplastea (Euglenozoa): Contain a large mitochondrion called a kinetoplast.
Trypanosoma (Metakinetoplastina, Trypanosomatida): The change of host and some differentiation steps are associated with characteristic movements of
the kinetoplast along the cell. Trypanosoma brucei infection (transmitted to humans through the bite of infected tsetse flies) causes the sleeping sickness,
while Trypanosoma cruzi infection (transmitted through the bite of infected reduviid bugs) leads to Chagas disease in humans.
Leishmania (Metakinetoplastina, Trypanosomatida): Parasite responsible for the leishmaniasis disease. It multiplies within the lysosomes of vertebrate
macrophages and within the digestive system of sand-flies.
Bodo saltans (Metakinetoplastina, Eubodonida): Freeliving bi-undulipodiated cell.
Crithidia (Metakinetoplastina, Trypanosomatida).
Trypanoplasma (Metakinetoplastina, Parabodonia).
c 2009 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
FEMS Microbiol Rev 34 (2010) 59–86
61
Ribosomal RNA genes in eukaryotic microorganisms
Fig. 1. General organization of the ribosomal main transcription unit (rDNA) and 5S rDNA. (a) Schematic representation of Xenopus laevis rDNA. About
600 U of the ribosomal cistron are encoded in the chromosome in head-to-tail tandem repeats. Each unit contains a coding region (red) and an IGR. (b)
A single unit of the X. laevis rDNA. The 18S, 5.8S and 28S rRNA molecules are transcribed as a single RNA precursor that is post-transcriptionally
processed to produce the mature rRNA molecules. Transcription regulatory elements for RNA polymerase I are found in the NTS: tandem-repeated
sequences (R), spacer promoters (SP), transcription terminators (T) and the promoter (P). The IGR comprises both the NTS and the ETS. (c) Organization
of somatic 5S rDNA in X. laevis. The 5S rDNA is organized in tandem head-to-tail repeats that include a coding region (green box) and an intergenic
sequence (black line). The 5S rDNA promoter is internal to the coding region (light green box). Arrows represent the transcription start point. ETS,
external transcribed spacer.
Long & Dawid, 1980; Sollner-Webb & Mougey, 1991). The
thorough study and description of genomic organization
and regulatory elements in the rRNA genes of Xenopus,
Drosophila and mouse led to the most accepted model of
rRNA gene organization in eukaryotes (Long & Dawid,
1980; Mandal, 1984; Sollner-Webb & Mougey, 1991) (Fig.
1). The rRNA genes of microbial eukaryotes have also been
intensively studied, although they were considered to be the
exception to the rule, as their organization differs from the
general models (Long & Dawid, 1980; Mandal, 1984). Here,
we focus on the rRNA gene organization of microbial
eukaryotes where many examples of gene diversity can be
found. This work also summarizes the variability of motifs
present in the rDNA intergenic region (IGR), which may
include general and species-specific elements. For simplicity,
in this review, the ribosomal cistron is referred to as rDNA
and the term 5S rDNA is used for the 5S rRNA gene.
Overview of the eukaryotic rRNA genes
The ribosomal cistron (rDNA)
In most species, the rDNA is present in multiple copies
organized as tandem head-to-tail repeats. The rDNA unit is
composed of a transcribed region and an IGR (also called
the intergenic spacer) consisting of a nontranscribed spacer
(NTS) 2–30 kbp long and an external transcribed spacer.
The NTS contains most of the regulatory elements for
FEMS Microbiol Rev 34 (2010) 59–86
transcription, while the external transcribed spacer is part
of the primary transcript (pre-rRNA, 7–14 kb long) (Sollner-Webb & Mougey, 1991) (Fig. 1).
Several regulatory elements may be found in the IGR such
as enhancers, spacer promoters, a proximal terminator and
the gene promoter. This region may also contain several
repetitive sequences that may improve the transcription
efficiency, with additive effects (Paule & White, 2000). A
schematic representation of the Xenopus laevis rDNA is
shown in Fig. 1a and b as an example of the ‘typical’
eukaryotic rDNA organization (Sollner-Webb & Mougey,
1991). The rDNA pol I core promoter and other nonrepeated rDNA regulatory elements have been described and
studied in detail in some unicellular eukaryotes such as
Trypanosoma cruzi, Acanthamoeba castellanii and yeast
(Kownin et al., 1985; Neigeborn & Warner, 1990; Wai et al.,
2000; Figueroa-Angulo et al., 2006).
Transcription of the rDNA proceeds from the promoter
through the 5 0 external transcribed spacer – 18S rRNA –
internal transcribed spacer-1 (ITS-1) – 5.8S rRNA – ITS-2
and 28S rRNA, until pol I comes across a transcription
termination signal (Long & Dawid, 1980). In most cases, the
rDNA primary transcript is post-transcriptionally processed
in three rRNA mature molecules: 18S, 5.8S and 28S, resulting from the elimination of the external transcribed spacers
ITS-1 and ITS-2 (Fig. 1) (Long & Dawid, 1980). Additional
processing of the rRNAs into several smaller molecules has
also been described. As most eukaryotic LSU rRNAs (eLSU
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c
62
rRNAs) are fragmented by removal of ITS-2, the eLSU rRNA
should be defined as 5.8S128S rRNA. Because the term LSU
rRNA has been used as equivalent to bacterial 23S rRNA,
here we refer to the 5.8S128S rRNA as eLSU rRNA.
A.L. Torres-Machorro et al.
to maintain the 18S, 5.8S, 28S and 5S rRNA homeostasis for
the efficient synthesis of ribosomes.
The ribosomal cistron: localization, gene
linkage and IGR
The 5S rRNA gene (5S rDNA)
The typical rDNA organization
The 5S rDNA is reiterated in the eukaryotic genome in
tandem head-to-tail arrays (Paule & White, 2000) (Fig. 1c).
The 5S rDNA promoter (Internal Control Region) is found
downstream of the transcription start point and within the
transcribed region. Upstream regulatory elements can also
be found in some 5S rDNAs that may be necessary for
transcription. The Internal Control Region is sufficient for
transcription of 5S rRNA in Xenopus (Bogenhagen et al.,
1980; Sakonju et al., 1980) whereas in Saccharomyces cerevisiae two upstream regulatory elements (start site element
and upstream promoter element) are necessary for its
efficient transcription in vivo (Lee et al., 1997).
Redundancy and relative ratio of the rRNA genes
rRNA genes are reiterated in almost every eukaryotic
genome studied, and the gene copy number is maintained
at a characteristic constant level for each organism. In most
organisms, not all of the rDNA copies are transcribed
(Conconi et al., 1989), suggesting that the total rDNA copy
number is not directly related to the synthesis of rRNA
(Kobayashi et al., 1998; Grummt, 2003; Raska et al., 2004). It
has been proposed that the rDNA may participate in roles
other than transcription, such as maintenance of the nucleolar structure and rDNA stability (Nogi et al., 1991;
Oakes et al., 1993).
A considerable variation in the rDNA and 5S rDNA gene
copy number exists among eukaryotes (Table 1): the rDNA
copy number can range from one and two copies in the
Ascomycota Pneumocystis carinii and the Apicomplexa Theileria parva to 4800 copies in the green alga Acetabularia
mediterranea and 9000 copies in the ciliate Tetrahymena
thermophila. The 5S rDNA copies can range from three in
the red alga Cyanidioschyzon merolae to about one million in
the ciliate Euplotes eurystomus. In the slime mold Dictyostelium discoideum and in the yeast S. cerevisiae, the rDNA and
5S rDNA genes are present in equal numbers, although a
strict relationship between both types of genes is not always
observed. For example, Euglena gracilis has 800–4000 copies
of rDNA and only 300 copies of the 5S rDNA; in contrast,
110 copies of the rDNA are present in the genome of the
kinetoplastid T. cruzi, while the 5S rDNA is repeated 1600
times. The copy number of rRNA genes in some microbial
eukaryotes and the rDNA/5S rDNA ratio are given in Table
1. The considerable variability in this ratio suggests that
different species may have particular regulatory mechanisms
c 2009 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
Chromosomally localized tandem head-to-tail repeats of
rDNA units containing a coding region and an IGR represent the typical rDNA organization in eukaryotes. Some
microbial organisms of various phylogenetic branches share
this general organization, as shown in Fig. 2 and Table 2.
Tandem rDNA units may be located in a single chromosome
and locus (e.g. Kluyveromyces lactis and Trichomonas vaginalis), or in various chromosomes and loci (e.g. T. cruzi).
Atypical tail-to-tail and head-to-head rDNA repeats (interspersed with typical tandem head-to-tail repeats) are observed in Acetabularia exigua (Berger et al., 1978; Spring
et al., 1978). In various yeast species such as K. lactis and
S. cerevisiae, tandem rDNA units are genetically linked to the
5S rDNA. In these cases, the 5S rDNA can be coded either in
a sense or an antisense orientation relative to the rDNA
coding strand (Table 2, Fig. 2b and c).
Depending on the species (and isolate) studied, several
tandemly repeated sequences may be found in the IGR with
variations in size, sequence and number (Table 3). For
example, the IGR of Leishmania species contain a 60–64 bp
repeated element reiterated between 16 and 275 times,
causing length variations in the IGR that range from 4 to
12 kbp. The size, number and sequence of these motifs are
species- and isolate-specific elements in Leishmania spp.
(Gay et al., 1996; Uliana et al., 1996; Yan et al., 1999;
Martı́nez-Calvillo et al., 2001; Orlando et al., 2002) (Table 3
and Fig. 2g).
Unlinked and heterogeneous rDNA
rDNAs with heterogeneous coding and intergenic sequences
are characteristic of the Apicomplexa group and may be
found unlinked (located in nonadjacent loci) and in low
copy number (Table 4). For example, Plasmodium spp. may
have four to eight rDNA copies per haploid genome.
Plasmodium falciparum and Plasmodium berghei have two
types of rDNA units (Waters et al., 1989; Waters, 1994): Atype and S-type (also known as C-type in P. berghei) code for
different SSU and eLSU rRNAs that correlate with the
production of ribosomes with different GTPase activity
(Rogers et al., 1996; Velichutina et al., 1998). The expression
of rDNA genes is tightly linked to the progression of
Plasmodium life cycle: the A-type rRNA is expressed predominantly in the vertebrate host (asexual development),
whereas the S-type rRNA is expressed in the mosquito stage
(sexual development) (Mercereau-Puijalon et al., 2002) (Fig.
FEMS Microbiol Rev 34 (2010) 59–86
63
Ribosomal RNA genes in eukaryotic microorganisms
Table 1. rRNA genes: copy number, unit size and organization
Organism
rDNA unit
size (kbp) and
organization
5S rDNA
copies
5S rDNA unit
size (kbp) and
organization
11–12 (T)
250–300
0.23 (T)
800–4000 (C),
4(chr)
11.5 (C)
330
0.68 and 1.7 (T)
0.6 (T)
166
12.5 (T)
rDNA
copies
Excavata
Crithidia fasciculata
Diplonema papillatum
Euglena gracilis
Herpetomonas
Leishmania donovani
rDNA/5S
rDNA copy
ratio
0.6 (T)
Leishmania major
63
14 (T)
Trypanosoma brucei
56
(T)
1500
0.75
30 (T)
1600
0.48 (T)
330
0.9
0.72
14 (C)
5.6 (T)
(T)
6 (T)
0.01%
0.1%
0.307 and 0.316 (T)
0.334 and 0.335 (T)
6 (T)
0.04%
0.86 and 1.3 (T)
Trypanosoma cruzi
Trypanosoma rangeli
Trypanosoma vivax
Naegleria gruberi
Giardia intestinalis
Trichomonas tenax
Trichomonas vaginalis
Tritrichomonas foetus
110
3000–5000 (C)
60 (H), 300
12
Trypanoplasma borreli
Chromalveolates
Babesia bigemina
0.59
3
Reddy et al. (1991)
3
4
5 (H)
6.5 (T)
6
Eimeria tenella
140
(T)
4
Plasmodium falciparum
500
0.55 and 0.79, 3 (T),
3(U)
0.73 (T)
0.28
(U)
3
(T)
1.33
5–8
(U)
3
1.67–2.67
6
7
2
(U)
(U)
(U)
3
0.67
110
1
Plasmodium lophurae
Plasmodium vivax
Theileria parva
Toxoplasma gondii
Perkinsus andrewsi
Euplotes crassus
Euplotes eurystomus
Glaucoma chattoni
Oxytricha fallax
Köck & Cornelissen (1990),
Schnare et al. (2000)
Sturm et al. (2001)
2.42–12.12 Ravel-Chapuis (1988), Keller
et al. (1992)
Aksoy (1992)
Yan et al. (1999), León et al.
(1978)
Martı́nez-Calvillo et al. (2001),
Ivens et al. (2005)
0.04
Hasan et al. (1984), Berriman
et al. (2005)
0.07
Castro et al. (1981),
Hernández-Rivas et al. (1992),
Hernández et al. (1993)
Aksoy et al. (1992)
Roditi (1992)
Clark & Cross (1988)
Le Blancq et al. (1991)
Torres-Machorro et al. (2009)
López-Villaseñor et al. (2004),
Torres-Machorro et al. (2006)
Chakrabarti et al. (1992),
Torres-Machorro et al. (2009)
Maslov et al. (1993)
10.65, 10.8 and
13.35 (U)
7
Babesia bovis
Babesia canis
Cryptosporidium parvum
Plasmodium berghei
110
Paramecium tetraurelia
Tetrahymena pyriformis
200 MAC
(H), 1 MIC
Tetrahymena thermophila 9000 MAC,
1MIC
FEMS Microbiol Rev 34 (2010) 59–86
7.5 (T)
7.7–7.8
7 (L)
1 000 000
9.3 (L)
7.49 (L)
0.83
0.93 (L)
0.69 (L)
9 (T,L,C)
21 (L,P)
References
350 MAC,
350 MIC (H)
150 MAC,
150 MIC (H)
0.28
0.29
0.25–0.29
30
Dalrymple (1990)
Dalrymple et al. (1992)
Taghi-Kilani et al. (1994), Le
Blancq et al. (1997)
Stucki et al. (1993), Shirley
(2000)
Dame & McCutchan (1983),
Waters (1994)
Shippen-Lentz & Vezza
(1988), Gardner et al. (2002)
Unnasch & Wirth (1983)
Li et al. (1997)
Kibe et al. (1994), Gardner
et al. (2005)
Guay et al. (1992)
Pecher et al. (2004)
Erbeznik et al. (1999)
Roberson et al. (1989)
Challoner et al. (1985)
Rae & Spear (1978), Swanton
et al. (1982)
Preer et al. (1999)
Kimmel & Gorovsky (1976),
Kimmel & Gorovsky (1978)
Yao & Gall (977), Allen et al.
(1984), Eisen et al. (2006)
2009 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
64
A.L. Torres-Machorro et al.
Table 1. Continued.
Organism
rDNA unit
size (kbp) and
organization
5S rDNA
copies
5S rDNA unit
size (kbp) and
organization
12 (T)
480
(U)
1.25
180 (L), 1 (chr)
88 (L,P)
180
88 (L,P)
1
200 (C), 0 (chr)
20 (L)
24.5 (C)
1 1011
4 60 (L,P)
3500–4800
3
(T)
(U)
rDNA
copies
Amoebozoa
Acanthamoeba castellanii 24(H), 600
Dictyostelium discoideum
Didymium iridis
Entamoeba histolytica
(HM-1:IMSS)
Physarum polycephalum
Plantae
Acetabularia mediterranea
Cyanidioschyzon merolae
Opisthokonta
Candida albicans
Candida glabrata
100 (C), 200 (chr) 11.6–12.5 (T)
4 115
(T)
rDNA/5S
rDNA copy
ratio
0.68
3
(U)
1
4 230
0.5
Hansenula polymorpha
Kluyveromyces lactis
Pneumocystis carinii
50–60
60
1
8 (T)
8.6 (T)
50–60
60
1
1
Saccharomyces cerevisiae
100–200
9.1 (T)
100–200
1
Schizosaccharomyces
pombe
Yarrowia lipolytica
100–120
10.4 (T)
30
(U)
3.33–4
100
7.7 and 8.7 (T)
108
(U)
0.93
8.9 (tel)
3
(U)
7.33
Encephalitozoon cuniculi
22
Nosema apis
Nosema bombycis
18 (T)
4.3 (T)
References
Zwick et al. (1991), Yang et al.
(1994)
Cockburn et al. (1978),
Hofmann et al. (1993)
Johansen et al. (1992)
Huber et al. (1989), Bagchi
et al. (1999)
Campbell et al. (1979)
Spring et al. (1978)
Maruyama et al. (2004),
Matsuzaki et al. (2004)
Huber & Rustchenko (2001)
Maleszka & Clark-Walker
(1993), Bergeron & Drouin
(2008)
Ramezani-Rad et al. (2003)
Verbeet et al. (1984)
Tang et al. (1998), Fischer
et al. (2006)
Rubin & Sulston (1973),
Rustchenko & Sherman (1994)
Wood et al. (2002)
van Heerikhuizen et al. (1985),
Acker et al. (2008)
Peyretaillade et al. (1998),
Katinka et al. (2001)
Gatehouse & Malone (1998)
Huang et al. (2004)
All copy numbers are approximate, mainly based on quantitative hybridization analyses. If the rDNA is chromosomal, the unit size corresponds to the
complete unit containing the rDNA coding region and the intergenic spacer.
If the rDNA unit is extrachromosomal, the unit size corresponds to the whole molecule size.
In trichomonads the percentage of the genome sequence that corresponds to the 5S rRNA coding region is indicated.
C, extrachromosomal circle; chr, chromosomal; H, haploid; L, extrachromosomal linear molecule; MAC, macronucleus; MIC, micronucleus; P,
palindrome; T, tandem; tel, telomeric; U, unlinked and nontandem.
3). During the transfer of Plasmodium from the vertebrate
host to the mosquito, drastic changes in glucose concentration and temperature are involved in regulating the expression of A- and S-type rDNA genes from different promoter
elements (Mack et al., 1979; Fang & McCutchan, 2002; Fang
et al., 2004). A third type, the O-type rDNA (oocyst), has
been described in the human malaria parasite Plasmodium
vivax, whose synthesis takes place in ookinetes inside the
mosquito’s gut (Li et al., 1997). Comprehensive reviews of
rRNA genes from Plasmodium describe in detail the characteristic organization and function of these genes (Waters,
1994; McCutchan et al., 1995). Other Apicomplexa species
c 2009 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
with a similar rDNA organization are described in Table 4, as
well as the non-Apicomplexa red alga C. merolae. This
organism has only three unlinked rDNA units in two
different chromosomes, with similar rRNA coding sequences (Matsuzaki et al., 2004).
Telomeric rDNA
The microsporidian obligate intracellular parasite E. cuniculi
has 22 rDNA units located as single copies in all telomeres of
its 11 chromosomes (Brugère et al., 2000). Candida albicans
rDNA is found in two subtelomeric loci (Dujon et al., 2004),
FEMS Microbiol Rev 34 (2010) 59–86
65
Ribosomal RNA genes in eukaryotic microorganisms
Fig. 2. Different organization of tandem head-to-tail rDNA repeats in microbial eukaryotes. (a) Eimeria tenella (Apicomplexa) exemplifies microbial
eukaryotes with the typical rDNA organization. (b, c) rDNA copies in Saccharomyces cerevisiae (Ascomycota) and Toxoplasma gondii (Apicomplexa) are
linked to the 5S rDNA (green), but in opposite polarities. Intergenic short direct repeats present in S. cerevisiae are shown as colored bars (see also Table
3). (d) In Giardia intestinalis (Diplomonadida), a 32-kDa antigenic protein (dark blue arrow) is coded in the complementary rDNA strand. (e) In
Acanthamoeba castellanii (Acanthamoebidae), the mature eLSU rRNA is fragmented into three molecules: 5.8S, 26Sa (2.4 kb) and 26Sb (2 kb); the IGR
contains six repeats of a 140-bp element (R, aqua boxes). (f, g) In Trypanosoma cruzi and Leishmania major (kinetoplastids), the eLSU rRNA is
fragmented into seven molecules. The T. cruzi IGR contains a 172-bp repeated sequence (orange boxes). In Leishmania spp., the IGR is characterized by
the presence of multiple repeated units (yellow). Leishmania major Friedlins e region is duplicated once. Drawings are not to scale. The size of rDNA units
is shown in Table 1. Arrows show the polarity of transcription.
while Yarrowia lipolytica and Giardia intestinalis rDNA units
are positioned in seven and six subtelomeric loci, respectively (Le Blancq et al., 1991; Dujon et al., 2004). The
G. intestinalis chromosome I varies 5–20% in size due to
subtelomeric rearrangements including variations in rDNA
copy number and size (Hou et al., 1995), while some
subtelomeric rDNA copies are linked to transcriptional gene
units, including protein-coding genes such as ankyrin. Some
of these regions may also hold incomplete rDNA sequences
(Upcroft et al., 2005). Interestingly, fragments of the rDNA
unit are found in all chromosomal ends in D. discoideum.
These regions encode complex repeated sequences (transposable elements–rDNA junctions) that generate novel telomeric structures (Eichinger et al., 2005).
The subtelomeric localization of rDNA sequences as those
found in E. cuniculi, D. discoideum and G. intestinalis
suggests a physiological role for these elements. Telomeres
have an ordered structure in the nucleus and can be
clustered or associated with the nuclear matrix, at least in
FEMS Microbiol Rev 34 (2010) 59–86
some stage during the life cycle (Pryde et al., 1997).
Telomeres are regions of great plasticity within a heterochromatic context, with dynamics that allow for the amplification
and/or variation in the number of telomeric genes and
repeated sequences. It is not known whether subtelomeric
rDNA is involved in the maintenance of the characteristic
telomeric structure or whether the rDNA exploits this
particular structure to regulate its expression and to maintain the sequence and copy number (Pryde et al., 1997).
rDNA may be located extrachromosomally
Extrachromosomal rDNA has been found in ciliates,
cellular and plasmodial slime molds and in yeasts (Table 5).
The polyploid somatic macronucleus of the ciliate T. thermophila contains about 9 000 copies of a palindromic selfreplicating linear minichromosome, which codes for two
rDNA units (Fig. 4a and Table 5). The IGR of this palindrome contains six types of repeated sequences (Fig. 4a and
2009 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
66
A.L. Torres-Machorro et al.
Table 2. Organisms with typical rDNA organization
Organism
Localization
IGS repeated
elements
5S
linkage
Excavata
Crithidia fasciculata
Leishmania spp.
1Ch, 1L
Yes
Yes
X
X
X
Yes
intestinalis and
muris
X
X
X
X
X
X
X
X
X
Schnare et al. (2000)
Uliana et al. (1996), Yan et al. (1999), Martı́nez-Calvillo et al.
(2001), Orlando et al. (2002), de Andrade Stempliuk & FloeterWinter (2002)
Hasan et al. (1984), Melville et al. (1998)
Hernández et al. (1993)
Edlind & Chakraborty (1987), Boothroyd et al. (1987), van Keulen
et al. (1992), Upcroft et al. (1994)
Torres-Machorro et al. (2009)
López-Villaseñor et al. (2004), Torres-Machorro et al. (2009)
Torres-Machorro et al. (2009)
X
X
X
Sense
Shirley (2000)
Guay et al. (1992)
Yes
X
D’Alessio et al. (1981), Yang et al. (1994)
1Ch, 1L
1Ch, 1L
X
X
Yes
Linked
Antisense
Antisense
Schizosaccharomyces pombe 1Ch, 2L
Torulopsis utilis
Yarrowia lipolytica
7L
Nosema apis
X
X
Yes
X
X
Antisense
X
Sense
Klabunde et al. (2002)
Verbeet et al. (1984)
Rubin & Sulston (1973), Skryabin et al. (1984), Srivastava &
Schlessinger (1991), Dujon et al. (2004), Kim et al. (2006)
Schaak et al. (1982), Wood et al. (2002)
Tabata (1980)
van Heerikhuizen et al. (1985)
Gatehouse & Malone (1998), Iiyama et al. (2004)
Trypanosoma brucei
Trypanosoma cruzi
Giardia spp.
4Ch
Z2Ch
6L (intestinalis)
Trichomonas tenax
Trichomonas vaginalis
Tritrichomonas foetus
1Ch, 1L
1Ch, 1L
Chromalveolates
Eimeria tenella
Toxoplasma gondii
1Ch, 1L
Amoebozoa
Acanthamoeba castellanii
Opisthokonta
Hansenula polymorpha
Kluyveromyces lactis
Saccharomyces cerevisiae
References
IGS, intergenic spacer; X, not identified or not present; Ch, chromosome; L, locus or loci.
Table 3). The rDNA organization in Tetrahymena pyriformis
is similar to the T. thermophila rDNA palindrome, with
variations in the intergenic repeated motifs (Tables 3 and 5).
Dictyostelium discoideum and Physarum polycephalum
rDNA is also encoded in palindromic extrachromosomal
molecules (Fig. 4 and Table 5). Both rDNA minichromosomes contain several repeated sequence elements (Table 3).
In the D. discoideum rDNA palindrome, two 5S rDNA copies
are present near the telomeric ends, in the same polarity as
the rDNA unit (Fig. 4b) (Cockburn et al., 1978). Additionally, a single rDNA palindrome is located in chromosome IV
(Sucgang et al., 2003). Even though D. discoideum has six
chromosomes, a seventh ‘chromosome’ can be observed in
some chromosomal spreads. This additional ‘chromosome’
corresponds to a chromosome-sized cluster of palindromic
rDNA minichromosomes (Sucgang et al., 2003), which
suggests a physical interaction of the extrachromosomal
rDNA. This particular organization may play a role in the
expression and segregation of mitotic rDNA.
Extrachromosomal linear molecules containing one
rDNA unit are found in Didymium iridis (Fig. 4 and Table
5). Ciliates such as Euplotes crassus and Glaucoma chattoni
have extrachromosomal rDNA copies in single genec 2009 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
sized linear molecules within the macronucleus with
characteristic intergenic repeated elements (Tables 3 and 5).
Finally, tandem rDNA genes in Paramecium tetraurelia
can be found both in circular and in linear extrachromosomal molecules (Table 5), which can contain 4 13 rDNA
copies.
rDNA units coded in extrachromosomal circular plasmids may be found in Amoebozoa (Entamoeba histolytica)
and Excavata (E. gracilis and Naegleria gruberi) (Fig. 5 and
Table 5). The most-studied E. histolytica isolate HM-1:IMSS
lacks an rDNA chromosomal copy (Bagchi et al., 1999) but
possesses about 200 copies of an extrachromosomal circular
molecule, with two inverted rDNA units and repeated
sequences in the IGR (Table 3 and Fig. 5a). This molecule
starts replication at multiple sites; the primary replication
origins are located near the pol I promoters, but other
replication origins found all the way through the circle are
activated under stress conditions (Ghosh et al., 2003).
Interestingly, a 0.7-kb RNA of unknown function is encoded
in the upstream region of one rDNA unit (Bhattacharya
et al., 1998) (Fig. 5a). Depending on the isolate, variations
are found in the size and organization of the rDNA circular
molecules: the 200:NIH E. histolytica isolate has a
FEMS Microbiol Rev 34 (2010) 59–86
67
Ribosomal RNA genes in eukaryotic microorganisms
Table 3. Repeated sequences in the ribosomal cistron intergenic spacer
Organism
Excavata
Crithidia fasciculata
Euglena gracilis
Leishmania major
Size of repeated
sequences
Number of
repeated sequences
19 bp
55 bp
14 bp, imperfect repeat
30 bp imperfect
palindromes
63 bp
28
4
6
2
35–70
9
40
Leishmania donovani
39
12
40
2–8
2 in GS strain, 3 in GK
strain
Varies, 6 min
Leishmania hoogstraali
Trypanosoma cruzi
Giardia intestinalis
Giardia muris
Chromalveolates
Euplotes crassus
73 bp
I. 30 bp
IV. 17 bp
Glaucoma chattoni
I. 32 bp (5 0 )
II. 14–24 bp (5 0 )
III. 18 bp (5 0 )
IV. 17 bp (3 0 )
V. 130 bp (3 0 )
Tetrahymena thermophila I. 32 bp (5 0 )w
II.20–21 bp (5 0 )
III. 20 bp (5 0 )
430 bp (5 0 )
Tetrahymena pyriformis
IV. 17 bp (3 0 )
V. 130 bp (3 0 )z
I. 33 bp (5 0 )
II. 10–24 bp (5 0 )
III. 14–21 bp (5 0 )
IV. 17 bp (3 0 )
V. 130 bp (3 0 )
15 bp approximately
9 bp
Perkinsus andrewsi
Pfiesteria piscicida
Amoebozoa
Acanthamoeba castellanii 106–174 bp
Dictyostelium discoideum 29 bp (3 0 )
50
Entamoeba histolytica
DraI 170 bp (3 0 )
ScaI 144 bp (3 0 )
ScaI 144 bp (5 0 )
PvuI 145 bp (5 0 )
HinfI 653 bp (5 0 )z
74 bp (5 0 )
AvaII 153 bp (5 0 )
1408 bp (5 0 )
FEMS Microbiol Rev 34 (2010) 59–86
Function
References
Schnare et al. (2000)
3.5
Greenwood et al. (2001)
16–275
Leishmania amazoniensis 60 bp
Leishmania chagasi
64 bp
Leishmania infantum
63 bp
LiR3 348 bp
64 bp
63 bp
63 bp
172 bp
78 bp
Intergenic
region
size (kb)
4–12
Enhancer-like
4–12
Martı́nez-Calvillo et al.
(2001)
Uliana et al. (1996)
Gay et al. (1996)
Requena et al. (1997)
Transcription
termination?
5.8
4
5.5
Recombination?
Yan et al. (1999)
Orlando et al. (2002)
Pulido et al. (1996)
Upcroft et al. (1994)
van Keulen et al. (1992)
Erbeznik et al. (1999)
3
7
1
9
3
4
13
7
4 (2 tandem in each
chromosome half)
5
4
3
11
5
ND
ND
8
6
4
Spacer promoter?
Enhancer?
Termination?
Gene packing?
Spacer promoter?
Enhancer?
Challoner et al. (1985)
1.9
Replication, include
type I and III repeats
Termination?
Gene packing?
Spacer promoter?
Enhancer?
Challoner et al. (1985)
Challoner et al. (1985)
Coss et al. (2001)
Saito et al. (2002)
UBF binding
2.33
Yang et al. (1994)
Sucgang et al. (2003)
9.2 (5 0 )
3.5 (3 0 )
Huber et al. (1989), Mittal
et al. (1992), Bhattacharya
et al. (1998)
‰
10
7
6
11
2 and 4
2
5
2
ARS-like sequences
Pathogenic-specific
sequence
Recombination
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c
68
A.L. Torres-Machorro et al.
Table 3. Continued.
Organism
Physarum polycephalum
Size of repeated
sequences
I. 130 bp (5 0 )
I 0 . 130 bp (3 0 )
II. 50 bp (5 0 )
II 0 . 52 bp (3 0 )
Opisthokonta
Saccharomyces cerevisiae I. 6 bp (5 0 )
II. 16 bp (5 0 )
III. 8 bp (5 0 )
IV. 11 bp (3 0 )
V. 9 bp (3 0 )
VI. 9 bp (3 0 )
Yarrowia lipolytica
140–150 bp
11 bp
Encephalitozoon cuniculi
29 bp (5 0 )
19 bp (5 0 )
51 bp (3 0 )
43 bp (3 0 )
Number of
repeated sequences
Function
Intergenic
region
size (kb)
References
Z22
Hattori et al. (1984)
16
6
1.1 (5 0 ) Skryabin et al. (1984)
1.25 (3 0 )
3
2
3
2
2
2
Varies
14
van Heerikhuizen et al.
(1985), Fournier et al.
(1986)
Peyretaillade et al. (1998)
2
8
Repeated sequences may not be identical.
The 55-bp repeat has an internal repeated inverted sequence.
w
Type I and III repetitions are within a 430-bp repeated segment involved in the replication of the minichromosome.
z
Inverted repeat.
‰
Two blocks of highly repetitive DNA bracket the transcribed region.
z
Polymorphic locus with a 65-bp internal inverted repeated sequence.
ND, not determined; UBF, upstream binding factor; IGS, intergenic spacer.
Table 4. Organisms with unlinked rDNA units
Organism
Copy number
Chromalveolates
Babesia bigemina
Babesia bovis
Babesia canis
Cryptosporidum parvum
Theileria parva
Plasmodium berghei
Plasmodium falciparum
3
3
4
5
2
4
5–8
Plasmodium lophurae
Plasmodium vivax
Plantae
Cyanidioschyzon merolae
6 (7–9)
7
3
Chromosomal localization and rDNA types
Two in chr III and one in chr IV
Two in one chr and two in the other chr
3 tandem, 2 alone in different chr
A and B units in different chr
Type A in chr XII and VII; Type C in chr VI and V
Subtelomeric: Type A in chr V and VII; Type S in
chr XI and XIII
A, S and O rDNA types
3 different loci, two in chr XVII and one in chr
XVIII
References
Reddy et al. (1991)
Dalrymple (1990), Brayton et al. (2007)
Dalrymple et al. (1992)
Le Blancq et al. (1997)
Kibe et al. (1994), Bishop et al. (2000)
Waters (1994)
Langsley et al. (1983), Mercereau-Puijalon
et al. (2002), Gardner et al. (2002)
Unnasch & Wirth (1983)
van Spaendonk et al. (2000)
Maruyama et al. (2004)
Chr, chromosome.
palindromic circular organization (25.9 kb), while the HK-9
(15.3 kb) and the Rahman (18.3 kb) isolates possess single
rDNA units in their circular extrachromosomal molecules
(Sehgal et al., 1994; Bhattacharya et al., 1998).
Most E. gracilis rDNA is found in extrachromosomal
circular molecules that code for a single rDNA unit (Fig. 5b
and Table 5). The whole E. gracilis rDNA circle is transcribed, suggesting a read-around transcription without the
c 2009 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
need for transcriptional terminators (Greenwood et al.,
2001). Naegleria gruberi rDNA plasmid contains two ORFs:
a large one downstream of the 28S rRNA (similar to a
homing endonuclease gene, HE gene) and a short one that
codes for a hypothetical protein (Maruyama & Nozaki,
2007) (Fig. 5c and Table 5).
The yeast C. albicans possesses both chromosomal and
extrachromosomal rDNA. About 200 copies in tandem,
FEMS Microbiol Rev 34 (2010) 59–86
69
Ribosomal RNA genes in eukaryotic microorganisms
Fig. 3. rDNA organization in Plasmodium berghei. Four unlinked rDNA copies are coded in the telomeres of P. berghei. Type-A rDNAs contain a mature
eLSU rRNA fragmented into three molecules (5.8S, 28Sa and 28Sb) and are expressed during the asexual stage in vertebrate hosts. Type-C rDNAs (with a
nonfragmented 28S rRNA) are expressed during the sexual development in mosquitoes. The differential expression of rDNAs is regulated by specific
promoter sequences (purple and pink boxes). Drawings are not to scale. The size of rDNA units is shown in Table 1.
Table 5. Extrachromosomal rDNA units
Organism
Lineal/
circular
Size
Organization
Copy
number
Excavata
Euglena gracilis
C
11.5 kbp
Single
800–4000
4
C
14 kbp
Single
300–5000
None
L
L
7 kbp
9.3 kbp
Single gene
Single gene
MIC
MIC
L
L
7.49 kbp
Single gene
Single gene
MIC
MIC
Single gene
Tandem
Single gene
Palindrome
200/haploid MAC
MIC
MIC
MIC
1
Naegleria gruberi
Chromalveolates
Euplotes crassus
Glaucoma chattoni
Nyctotherus ovalis
Oxytricha fallax
Oxytricha nova
L
Paramecium tetraurelia C and L
Stylonychia mytilus
L
Tetrahymena pyriformisL
7.49 kbp
Additional copies
in chromosome
References
Ravel-Chapuis (1988), Schnare
et al. (1990)
Clark & Cross (1987)
Erbeznik et al. (1999)
Katzen et al. (1981), Challoner
et al. (1985)
Ricard et al. (2008)
Rae & Spear (1978), Swanton
et al. (1982)
Swanton et al. (1982)
Findly & Gall (1978)
Lipps & Steinbrück (1978)
Engberg et al. (1976), Yao & Gall
(1977), Niles et al. (1981)
Engberg (1985), Eisen et al. (2006)
Tetrahymena
thermophila
Amoebozoa
Dictyostelium
discoideum
L
21 kbp
Palindrome
9000 MAC
L
88 kbp
Palindrome
90
1 Palindrome
Didymium iridis
Entamoeba histolytica
HM-1:IMSS
Physarum
polycephalum
Opisthokonta
Candida albicans
L
C
20 kbp
24.5 kbp
Single
Palindrome
200/haploid
None
L
4 60 kbp
Palindrome
1 1011
C and L
1.2 Mbp
Tandem
100
200
Cockburn et al. (1978), Hofmann
et al. (1993), Eichinger et al.
(2005)
Johansen et al. (1992)
Huber et al. (1989), Bhattacharya
et al. (1998), Bagchi et al. (1999)
Vogt & Braun (1976), Campbell
et al. (1979)
Huber & Rustchenko (2001)
The size depends on the number of rDNA repeats.
MAC, macronucleus; MIC micronucleus.
varying in size, are present in chromosome R while roughly
100 copies are found in an 1.2 Mbp autonomously replicating circle. Some rDNA sequences are also found in
50–150-kbp linear molecules (Huber & Rustchenko, 2001).
rDNA plasmids have only been observed in old
S. cerevisiae cell cultures. During the aging process, the
tandem rDNA copies are excised from the chromosome
and replicate autonomously. The accumulation of rDNA
circles leads to yeast sterility and shortening of the life span.
An association between rDNA locus instability and loss of
FEMS Microbiol Rev 34 (2010) 59–86
epigenetic silencing has also been observed (Sinclair &
Guarente, 1997).
The ribosomal cistron: the coding region
The typical eukaryotic rDNA coding region is composed of
the 18S, 5.8S and 28S rRNA coding sequences separated by
ITS-1 and ITS-2. The rDNA coding sequence consists of a
common core of domains that may be interspersed with a
distinct set of variable regions (also called expansion
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c
70
A.L. Torres-Machorro et al.
Fig. 4. Linear extrachromosomal rDNA units in microbial eukaryotes. (a) rDNA is coded in extrachromosomal linear palindromes in Tetrahymena
thermophila. Two head-to-head rDNA units are coded in a macronuclear minichromosome that contains typical telomeric sequences (black dots).
Upstream and downstream IGRs contain various types of repeated sequences (colored bars; see also Table 3). A group I intron (blue square) is found
within the eLSU rRNA. (b) The rDNA and 5S rDNA in Dictyostelium discoideum are linked and coded in extrachromosomal linear palindromes containing
telomeres (black dots). (c) rDNA in Physarum polycephalum is coded in palindromic head-to-head units in a minichromosome with various repeated
sequences both in the 5 0 and 3 0 IGRs (see also Table 3). The eLSU rRNA is interrupted by two group I introns (blue squares), and a third group I intron that
includes an HE gene (purple square). (d) In Didymium iridis the rDNA is coded in linear minichromosomes containing one rDNA unit. The SSU rRNA
contains a twintron (purple box) and two group I introns are found within the 28S rRNA (blue boxes). Drawings are not to scale. The size of rDNA units is
shown in Table 1. Arrows show the polarity of transcription.
segments; Dover, 1988). Ten and 18 variable regions have
been identified in the SSU and LSU rRNAs of all organisms
(Raué et al., 1988). Three types of sequence insertions have
been found within these variable regions: (1) expansion
segments, encoding RNA sequences conserved in the mature
molecule; (2) group I introns, located within highly conserved regions and removed after transcription; and (3)
transcribed spacers, sequences removed from the mature
rRNA, thus producing fragmented eLSU rRNA molecules
(Clark et al., 1984). Babesia bovis (Dalrymple et al., 1992),
Cryptosporidium parvum (Le Blancq et al., 1997), D. discoideum (Frankel et al., 1977), E. histolytica (Huber et al.,
1989), G. intestinalis (Healey et al., 1990), K. lactis (Verbeet
et al., 1984), S. cerevisiae (Bell et al., 1977), Toxoplasma
gondii (Gagnon et al., 1996) and T. vaginalis (LópezVillaseñor et al., 2004) are microbial eukaryotes with the
typical rDNA organization of the coding region (Fig. 2a–e).
Insertions of expansion segments in the SSU
The average length of eukaryotic SSU rRNA is 2 kb. Unusually long SSU rRNAs have been found in Pelobionta
(Pelomyxa palustris), Foraminifera (Hemisphaerammina bradyi) and Euglenozoa (Distigma sennii) (Table 6). The longest
SSU rRNA known is found in the Euglenid D. sennii,
comprising 4 4.5 kb. In most cases, the insertions are
found in the SSU rRNA variable regions V2, V4 and V7.
The only exceptions are an extended V5 region in
c 2009 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
A. castellanii and an expansion in a nonvariable region in
P. palustris (Gunderson & Sogin, 1986; Milyutina et al.,
2001). The rRNA variable regions are located in the mature
ribosome surface and their evolutionary implications are
unknown (Katz & Bhattacharya, 2006).
Group I introns and twintrons
Group I introns can be found as insertions in the SSU and
eLSU rRNA coding regions that are removed from the
mature molecule by means of a self-splicing reaction (Einvik
et al., 1998), generating a completely functional rRNA
molecule (Mandal, 1984). The ribozymes encoded in group
I introns have conserved secondary structures of 10 basepaired segments, as well as some additional paired segments
depending on the intron subclass (Michel & Westhof, 1990).
The splicing reaction initiates with a nucleophilic attack of a
guanosine cofactor at the 5 0 splice site and, after two
sequential transesterification reactions, the exons are ligated
and the RNA intron is removed (Einvik et al., 1998).
Group I introns are widely distributed in nature and can
be found in bacteria, mitochondrial and chloroplast genomes, and in the eukaryotic nucleus (Johansen et al., 2007).
Group I introns may interrupt the SSU rRNA coding
sequence in 40 distinct conserved sites of several microbial
eukaryotes (Jackson et al., 2002) such as Acanthamoeba
griffini and the green alga Characium saccatum. These
introns may also be present in the eLSU rRNA, as is the case
FEMS Microbiol Rev 34 (2010) 59–86
71
Ribosomal RNA genes in eukaryotic microorganisms
Table 6. Variations in the size of the SSU rRNA due to insertions
SSU
size (kb) References
Organism
Excavata
Astasia curvata
Astasia torta
Distigma curvatum
Distigma elegans
Distigma sennii
Ploeotia costata
Euglena gracilis
Amoebozoa
Acanthamoeba castellanii
Acanthamoeba griffini
Acanthamoeba lenticulata
Pelomyxa palustris
Phreatamoeba balamuthi
Entamoeba histolytica
Plantae
Ankistrodesmus stipitatus
Rhizaria
Foraminifera
Fig. 5. Circular extrachromosomal rDNA units in microbial eukaryotes.
(a) In Entamoeba histolytica extrachromosomal self-replicating molecules
encode two palindromic rDNA units (red). The upstream and downstream IGRs contain several repeated sequences (coloured boxes, detailed in Table 3). The 5 0 IGR also encodes a 0.7-kb mRNA (grey arrow). In
the HM1 strain, four hemolysine virulence proteins (HLYs) are coded
within the rRNA coding region, in antisense orientation relative to the
rRNA coding strand (navy blue arrows). (b) In Euglena gracilis the rDNA is
coded in circular plasmids and the eLSU is fragmented in 14 segments. (c)
The Naegleria gruberi rDNA plasmid encodes one rDNA unit containing
one twintron in 18S rRNA (purple box) and three type-I introns in the
eLSU rRNA (blue boxes). The IGR contains two ORFs (dark blue arrows).
Black arrows show the polarity of transcription.
for P. falciparum A-type eLSU rRNA and the 26S rRNA of
some Tetrahymena isolates (Fig. 4a, Table 7). It is interesting
that some organisms may have both the SSU and the eLSU
rRNAs interrupted by group I introns (e.g. P. carinii,
Chlorella ellipsoidea and D. iridis, Fig. 4d). Table 7 describes
some of the introns found in the rDNA of several microbial
eukaryotes.
Twintrons are more complex insertions in the rDNA that
consist of two group I introns (ribozymes) and an ORF
encoding an HE (Einvik et al., 1998; Johansen et al., 2007).
The D. iridis and N. gruberi SSU twintrons contain a small
ribozyme (GIR1), followed by the HE ORF inserted into a
second ribozyme (GIR2). Two different isolates of D. iridis
FEMS Microbiol Rev 34 (2010) 59–86
2.56
2.9
3.4–3.7
3.9
4.5
2.4
2.3
Busse & Preisfeld (2002)
Busse & Preisfeld (2002)
Busse & Preisfeld (2002)
Busse & Preisfeld (2002)
Busse & Preisfeld (2002)
Busse & Preisfeld (2003)
Gunderson & Sogin (1986)
2.3
2.9
3
3.5
2.74
2.3
Gunderson & Sogin (1986)
Gast et al. (1994)
Schroeder-Diedrich et al. (1998)
Milyutina et al. (2001)
Hinkle et al. (1994)
Loftus et al. (2005)
2.2
Dávila-Aponte et al. (1991)
2.3–4
Katz & Bhattacharya (2006)
have two types of introns, containing an HE gene in both
polarities relative to the SSU rRNA gene (Fig. 6) (Johansen
et al., 2007). The twintron contains the HE ORF (I-DirI) in
the same polarity as the 18S rRNA coding region. GIR2 is a
self-splicing ribozyme that releases the HE transcript. A
second intron encoding a ribozyme (GIR1) is also found
within the twintron. GIR1 modifies the 5 0 end of the HE
transcript to form a 2 0 5 0 cap that increases its translational
efficiency (Fig. 6a) (Einvik et al., 1998; Johansen et al.,
2007). In contrast, the intron II contains an HE gene (IDirII) in opposite polarity relative to the SSU rRNA and
ribozyme-coding sequences (Johansen et al., 2006). Transcription of I-DirII is established from a pol II-like promoter
located immediately upstream of the HE gene (Fig. 6b)
(Johansen et al., 2006). Both D. iridis HE transcripts are
processed through the nuclear spliceosomal complex to
remove a 50-nt noncoding spliceosomal intron, found within the HE coding sequences, and are polyadenylated (Vader
et al., 1999; Johansen et al., 2007).
Physarum polycephalum eLSU rDNA contains an optional
group I intron holding an HE gene (Ruoff et al., 1992). The
full-length RNA intron can be excised or alternatively
processed (immediately downstream of the HE gene) to
produce a smaller transcript. Only the full-length RNA
intron (lacking a 5 0 cap and a poly-A tail) is translated into
the HE I-PpoI protein (Ruoff et al., 1992). The cleavage of
this transcript in the internal processing site seems to
downregulate HE I-PpoI expression by decreasing the
stability of the transcript in yeast transintegrated introns
(Johansen et al., 2007). Table 7 summarizes rDNA group I
introns and HE gene insertions.
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A.L. Torres-Machorro et al.
Table 7. Group I rDNA introns
Organism
Excavata
Ploeotia costata
Naegleria gruberi
Location
Number
of introns
SSU rRNA
SSU rRNA
LSU rRNA
1
1
3
Size
HE gene
References
I-NgrI
Busse & Preisfeld (2003)
Wikmark et al. (2006)
Einvik et al. (1998)
494 bp
Chromalveolates
Plasmodium falciparum
Tetrahymena pigmentosa
Tetrahymena thermophila
Amoebozoa
Acanthamoeba griffini
Acanthamoeba lenticulata
Didymium iridis
LSU rRNA
LSU rRNA
LSU rRNA
1
1
1
400 bp
370–410 bp
SSU rRNA
SSU rRNA
SSU rRNA
1
1
1
519 bp
656 bp
1.43 kbp
I-DirI and I-DirIIw
Physarum polycephalum
LSU rRNA
LSU rRNA
2
3
688 and 573 bp
0.7, 0.6 and 0.94 kbp
I-PpoIz
Dunaliella parva
SSU rRNA
SSU rRNA
SSU rRNA
LSU rRNA
SSU rRNA
1
1
1
1
2
334 bp
477 bp
442 bp
445 bp
381 and 419 bp
Dunaliella salina
SSU rRNA
1‰
LSU rRNA
SSU rRNA
LSU rRNA
SSU rRNA
SSU rRNA
1
1
1
1
1
Plantae
Ankistrodesmus stipitatus
Characium saccatum
Chlorella ellipsoidea
Opisthokonta
Candida albicans
Pnemocystis carinii
Histoplasma capsulatum
Nosema bombycis
397/8 bp
379 bp
390 bp
403–425 bp
Langsley et al. (1983)
Wild & Gall (1979)
Sogin et al. (1986)
Gast et al. (1994)
Gast et al. (1994)
Johansen & Vogt (1994), Johansen et al.
(2006), Johansen et al. (2007)
Johansen et al. (1992)
Ruoff et al. (1992), Johansen et al.
(2007)
Dávila-Aponte et al. (1991)
Wilcox et al. (1992)
Aimi et al. (1994)
Aimi et al. (1993)
Van Oppen et al. (1993), Wilcox et al.
(1992)
Van Oppen et al. (1993), Wilcox et al.
(1992)
Miletti-González & Leibowitz (2008)
Sogin & Edman (1989)
Lin et al. (1992), Liu et al. (1992)
Okeke et al. (1998), Lasker et al. (1998)
Iiyama et al. (2004)
Only in one of the eight LSU rRNA copies.
w
Depends on the isolate.
Coded in the 0.94-kbp intron.
‰
Can have two types of intron differing in sequence.
z
The ITS-1 and -2
The rDNA transcript is generally post-transcriptionally
processed in three rRNA mature molecules: 18S, 5.8S and
28S rRNAs that result from elimination of ETS, ITS-1 and
ITS-2 from the precursor transcript (Fig. 1). In microbial
eukaryotes, ITS-1 ranges from 100 to 400 bp, while ITS-2 is
200–500 bp. Unusually long ITSs are found in the red alga
C. merolae (Maruyama et al., 2004), where ITS-1 and ITS-2
average sizes are 862 and 1738 bp, respectively. Euglena
gracilis has the largest known ITS-1, 1188 bp in length
(Schnare et al., 1990). The dinoflagellate Cochlodinium
polykrikoides ITS-1 contains a 101-bp sequence in six
tandem repeats, resulting in an ITS-1 length of 813 bp (Ki
& Han, 2007). Yarrowia and Giardia have the shortest
known ITSs in microbial eukaryotes: the sum of ITS-1 and
ITS-2 lengths in Y. lipolytica is only 150 bp (van Heerikhuic 2009 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
zen et al., 1985), while the G. intestinalis ITS-1 and ITS-2 are
37 and 52 bp in length, respectively (Boothroyd et al., 1987).
Some Microsporidia species completely lack the ITS-2 (Fig.
7) (Vossbrinck & Woese, 1986), as discussed below. The
biological relevance of the ITSs’ length and the presence of
internal repeats are currently unknown, although their
sequence has been useful in molecular phylogenetic studies
of closely related species.
Additional ITSs generate fragmented eLSU rRNA
Some microbial eukaryotes process the pre-rRNA into more
than three mature molecules due to the presence of additional ITSs. Well-known examples of fragmented rRNA are
found among kinetoplastids, with the eLSU rRNA fragmented in seven molecules. The nomenclature of these rRNAs
varies according to the organism, the size of the rRNA
FEMS Microbiol Rev 34 (2010) 59–86
73
Ribosomal RNA genes in eukaryotic microorganisms
molecule and the position in the coding region. The eLSU
rRNA of Leishmania spp. is fragmented into seven elements,
which are cotranscribed in the pre-rRNA and processed by
exo- and endonucleolytic activities to produce the functional eLSU fragments: 5.8S, LSUa, g, LSUb, d, z and e
(Martı́nez-Calvillo et al., 2001) (Fig. 2g). Trypanosoma cruzi
and Crithidia fasciculata also code for an eLSU rRNA
fragmented into seven elements: 5.8S, 24Sa, S1, 24Sb, S2,
S6 and S4 in T. cruzi (Fig. 2f) (Hernández et al., 1988), and
rRNAs 5.8S, c, d, e, f, g and j in C. fasciculata (Spencer et al.,
1987).
Processing of the eLSU rRNA into several fragments has
also been found in nonkinetoplastid eukaryotes (e.g.
P. berghei and Plasmodium chabaudi blood stages and
A. castellanii) (D’Alessio et al., 1981; da Silveira & Mercereau-Puijalon, 1983; Johansen et al., 1992) (Figs 2e and 3).
Euglena gracilis has the most fragmented eLSU rRNA
currently known with 14 mature molecules that result from
the processing of 14 ITSs (ITS-1 to ITS-14, Fig. 5b) (Schnare
et al., 1990).
Protein-coding regions within the rDNA coding
region
A correlation has been observed between the virulence of
E. histolytica isolates and the sequence composition of the
rDNA circular molecule described above (Clark & Diamond, 1991; Zindrou et al., 2001). Virulence associates with
the striking presence of genes encoding hemolysins (proposed as virulence factors) within and overlapping the rRNA
coding sequence, but in opposite polarity. Three hemolysins
overlap with the eLSU coding region, while the fourth
(HLY4) is coded in the ITS-1 between the SSU and 5.8S
rRNAs (Jansson et al., 1994) (Fig. 5a). In G. intestinalis, a
gene coding for a 32-kDa flagellum antigen has been
identified in the rDNA IGR that overlaps the 3 0 region of
the 28S rRNA (Fig. 2d). The motif that directs transcription
of this gene seems to be a hybrid pol II/pol III promoter
(Upcroft et al., 1990).
Unusual rDNA coding regions
Fig. 6. Group I introns that contain an HE gene. (a) Twintron present in
Didymium iridis: the DiGIR2 intron (purple) is encoded in the SSU rRNA
and transcribed by pol I as part of the pre-rRNA; it self-splices to generate
the HE pre-mRNA (splicing sites are represented as black bars). Subsequently, DiGIR1 intron (blue) self-splices and processes the I-Dir I HE premRNA in the 5 0 side, producing a 2 0 5 0 -cap. The I-DirI HE pre-mRNA is
additionally processed by the removal of spliceosomal intron SI (white
box) and polyadenylation of the 3 0 side to generate a functional I-Dir I HE
mRNA (yellow region). (b) Intron II present in D. iridis: the I-Dir II HE RNA
found within the DiGIR2 intron is coded in antisense orientation and is
transcribed from a pol II promoter. The HE pre-mRNA is processed by pol
II-associated factors to generate a typical 5 0 -cap and a 3 0 polyadenylated
tail. The spliceosomal intron SI is removed by the spliceosome machinery.
Microsporidia are obligate intracellular eukaryotes that possess many prokaryotic characteristics in their rRNA genes
(Weiss, 2001). The rDNA units are smaller than the standard
eukaryotic size and lack the ITS-2; consequently, the 5.8S
rRNA is fused to the 5 0 region of the 28S rRNA, as is found
in bacteria (Vossbrinck & Woese, 1986) (Fig. 7). Microsporidia are the only eukaryotes known to lack an individual
5.8S rRNA molecule (e.g. E. cuniculi and Vairimorpha
necatrix (Vossbrinck & Woese, 1986; Peyretaillade et al.,
1998). The relevance of this eukaryotic 5.8S–28S rRNA
fusion is unknown. In addition to these characteristics,
Nosema bombycis and Nosema spodopterae have an unusual
rDNA gene organization (Huang et al., 2004; Iiyama et al.,
2004; Tsai et al., 2005) because the LSU rRNA is coded and
transcribed upstream to the SSU rRNA (Fig. 7b) in contrast
to the almost universal order of the rRNA coding regions
(Fig. 1).
Fig. 7. Unusual rDNA organization in Microsporidia. Microsporidia lack a 5.8S rRNA mature molecule and the typical 5.8S rRNA sequence is fused to
the 23S rRNA. (a) Single telomeric rDNA units are surrounded by different repeated sequences in Encephalitozoon cuniculi (see also Table 3) and the
rDNA lacks ITS-2. (b) Some Nosema species have an atypical rDNA coding organization, with the LSU rRNA coded upstream of the 16S rRNA. The typical
5.8S rRNA sequence is fused to the 23S rRNA and the 5S rDNA is linked to the rDNA unit.
FEMS Microbiol Rev 34 (2010) 59–86
2009 Federation of European Microbiological Societies
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74
A.L. Torres-Machorro et al.
Table 8. Variability found in the rDNA
Organism
Chromalveolates
Babesia bigemina
Babesia bovis
Cryptosporidium parvum
Plasmodium berghei
Plasmodium falciparum
Plasmodium vivax
Theileria parva
Toxoplasma gondii
Oxytricha fallax
Paramecium tatraurelia
Perkinsus andrewsi
Plantae
Dunaliella salina
Opisthokonta
Saccharomyces cerevisiae
Candida albicans
Yarrowia lipolytica
Nosema apis
Nosema bombi
rDNA types
Localization of variability
References
2
A, B and C
At least 2
A and C
A and S
A, S and O
2
2
6 MAC/ 4 MIC
A and B
IGR and SSU coding region
ITS and SSU coding region
ITS and coding region
IGR, promoter and coding regions
IGR, promoter and coding regions
IGR, promoter and coding regions
ITS and LSU coding region
IGR and SSU coding region
LSU coding region
IGR
IGR and coding regions
Reddy et al. (1991), Dalrymple et al. (1992)
Laughery et al. (2009)
Le Blancq et al. (1997), Spano & Crisanti (2000)
Waters (1994)
Waters (1994)
Li et al. (1997)
Bishop et al. (2000)
Fazaeli et al. (2000)
Doak et al. (2003)
Preer et al. (1999)
Pecher et al. (2004)
2
Group I introns
Wilcox et al. (1992)
2
2–5
IGR
Intron-containing and intronless LSU
IGR
At least 3
At least 2
IGR
ITS
Skryabin et al. (1984), Jemtland et al. (1986)
Miletti-González & Leibowitz (2008)
van Heerikhuizen et al. (1985), Fournier et al.
(1986), Clare et al. (1986)
Gatehouse & Malone (1998)
O’Mahony et al. (2007)
The number or names of the rDNA types for each species are shown.
IGR, intergenic region; MAC, macronucleus; MIC, micronucleus.
Different rDNA genes may be found within an
organism
The 5S rDNAs are found as tandem head-to-tail
repeats
As has been mentioned, the rRNA genes within one organism are generally conserved in the coding region with an
occasional sequence variation in the IGRs and with little
variation in the coding sequences. Sequence variability in
the IGRs may result from sequence divergence or disparity
in the number of repeated sequences, involved in both upand downregulation of rDNA transcription. Therefore, the
heterogeneous composition of rDNA units may influence
rDNA expression. Sequence divergence in the coding region
and/or IGR within the same organism has led to a classification of rDNA units. For example, different types of rDNA
may be found in Paramecium, Y. lipolytica and the Apicomplexa group. A detailed description of this variability is
included in Table 8.
The 5S rDNA in T. cruzi, Trypanosoma brucei, T. vaginalis,
Trichomonas tenax, C. fasciculata, Eimeria tenella and
C. parvum is typically organized in tandem head-to-tail
repeats. Tritrichomonas foetus has two types of 5S rDNAs,
while P. falciparum has only three 5S rDNA copies in
tandem, differing in the length of the IGRs. The main
characteristics of the 5S rDNA tandem head-to-tail repeats
of several organisms are described in Table 9.
The 5S rDNA
The organization of the 5S rDNA is simpler than that of the
rDNA. Most 5S rDNAs are found in tandem head-to-tail
repeats consisting of a conserved 120-bp coding region
and an IGR of variable size and sequence. An internal pol III
promoter is present in all 5S rDNA studied to date
(Schramm & Hernandez, 2002) (Fig. 1c).
c 2009 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
The 5S rDNA may be interspersed with genes
transcribed by any of the three RNA polymerases
The 5S rDNA has been found linked to the rDNA (transcribed by pol I) in an alternate distribution in T. gondii,
Hansenula polymorpha, Perkinsus andrewsi and various
Nosema species (Guay et al., 1992; Coss et al., 2001;
Klabunde et al., 2002; Huang et al., 2004; Iiyama et al.,
2004; Tsai et al., 2005; Liu et al., 2008) (Figs 2c and 7b). Two
tandem 5S rDNA copies are linked to each repeated rDNA
unit in Candida glabrata (Dujon et al., 2004). In contrast,
the 5S rDNA is linked to the rDNA in opposite polarity in
various yeast species, such as Torulopsis utilis, K. lactis and
S. cerevisiae (Fig. 2b, Table 2). Two copies of the 5S rDNA are
coded in the extrachromosomal DNA molecule in
FEMS Microbiol Rev 34 (2010) 59–86
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Ribosomal RNA genes in eukaryotic microorganisms
Table 9. Typical 5S rDNA organization
Organism
5S rDNA types
Localization of variability
Other
References
Excavata
Trichomonas tenax
Trichomonas vaginalis
Tritrichomonas foetus
A and B
A and B
A and B
IGR and coding region
IGR
IGR, repeated sequences vs.
ubiquitin gene
Type B IGR palindrome
IGS 10-bp palindrome
Torres-Machorro et al. (2009)
Torres-Machorro et al. (2006)
Torres-Machorro et al. (2009)
Trypanosoma brucei
Trypanosoma cruzi
Chromalveolates
Cryptosporidium parvum
Eimeria tenella
Plasmodium falciparum
Tetrahymena pyriformis
Tetrahymena thermophila
6 Sp1 binding sites in IGR
IGR
3
IGR lengths
IGR
IGR lengths
Lenardo et al. (1985)
Hernández-Rivas et al. (1992)
Taghi-Kilani et al. (1994)
Stucki et al. (1993)
Shippen-Lentz & Vezza (1988)
Some linked to ubiquitin genes Guerreiro et al. (1993)
IGR 12 and 16-bp palindromes Allen et al. (1984)
The number or names of the 5S rDNA types for each species are shown.
IGR, intergenic region.
Table 10. 5S rRNA gene linkage to pol II transcribed genes
Organism
Excavata
Trypanosoma vivax
Trypanosoma rangeli
Bodo saltans
Bodo caudatus
Diplonema papillatum
Herpetomonas spp.
Trypanoplasma borreli
Trypanosoma avium
Euglena gracilis
Tritrichomonas foetus
Chromalveolates
Tetrahymena pyriformis
Pol II gene
Orientation
References
Spliced leader
Spliced leader
Spliced leader
Spliced leader
Spliced leader
Spliced leader
Spliced leader
Spliced leader
Spliced leader
Ubiquitin
Sense
Sense
Sense
Sense
Sense
Sense
Antisense
Antisense
Sense
Sense
Roditi (1992)
Aksoy et al. (1992)
Santana et al. (2001)
Campbell (1992)
Sturm et al. (2001)
Aksoy (1992)
Maslov et al. (1993)
Santana et al. (2001)
Keller et al. (1992)
Torres-Machorro et al. (2009)
Ubiquitin
Sense
Guerreiro et al. (1993)
D. discoideum, in the same polarity as the two rDNA copies
(Hofmann et al., 1993) (Fig. 4b). Finally, a S. cerevisiae 5S
rDNA variant is found in five repeats of 3.6 kbp, located next
to the rDNA tandem cluster locus, in the centromere-distal
side (McMahon et al., 1984).
Some Trypanosoma species such as Trypanosoma vivax
and Typanosoma rangeli have the 5S rDNA copies linked to
the spliced-leader (SL) tandem repeated genes, transcribed
by pol II. SL transcripts are necessary to process the mRNAs
in kinetoplastids by a trans-splicing reaction (Simpson et al.,
2006). A similar linkage has been found in other Euglenozoa
such as Diplonema papillatum and Bodo caudatus. In Trypanoplasma borreli and Trypanosoma avium, the 5S rDNA is
coded in opposite polarity relative to the SL gene (Table 10).
Interestingly, the T. borreli SL can also be linked to 5S rRNA
pseudogenes (with a truncated 5 0 end) (Maslov et al., 1993).
Some 5S rDNA units in T. pyriformis and T. foetus are
associated with ubiquitin genes transcribed by pol II (Fig.
FEMS Microbiol Rev 34 (2010) 59–86
8b). Table 10 describes the relative polarity of the 5S rDNA
linked to the genes transcribed by pol II.
Some 5S rDNA copies in E. histolytica and one copy in
Leishmania tarentolae are linked to tRNA genes (Shi et al.,
1994; Clark et al., 2006), also transcribed by pol III.
Interestingly, 48 of the 108 5S rDNA copies of Y. lipolytica
produce pol III dicistronic transcripts: tRNA–5S rRNA
hybrid molecules. The synthesis of an 200-nt transcript is
driven by the tRNA pol III promoter, resulting in a
transcription independent of the 5S rDNA-specific transcription factor, TFIIIA. The dicistronic transcripts, as
well as a unique tricistronic transcript [Lys(CTT) tRNA–
Glu(CTC) tRNA–5S rDNA] are post-transcriptionally processed to generate the typical mature RNA molecules: tRNAs
and 5S rRNA (Acker et al., 2008) (Fig. 8c).
Nontandem 5S rDNA copies are found dispersed
throughout the genome of some microbial eukaryotes.
Some examples are A. castellanii (Zwick et al., 1991),
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76
A.L. Torres-Machorro et al.
Fig. 8. The 5S rDNA may be linked to pol II or pol III transcribed genes. (a) In Tritrichomonas foetus the 5S rDNA is linked to a multigenic ubiquitin family.
(b) In Euglena gracilis the 5S rDNA is linked to the SL gene. (c) In Yarrowia lipolytica dicistronic genes consisting of a tRNA gene (pink) and a 5S rDNA
(green) are dispersed in the genome. One tricistronic gene: Lys(CTT) tRNA–Glu(CTC) tRNA–5S rDNA is also found. These genes are transcribed from the
pol III promoter of the tRNA gene. Dispersed, single 5S rDNAs are also found (green).
Y. lipolytica and Schizosaccharomyces pombe (Tabata, 1981;
Dujon et al., 2004). The 5S rDNA may also be found in
extrachromosomal DNA. Noteworthy, ciliate organisms
such as Oxytricha fallax have single 5S rDNA copies coded
in macronucleus extrachromosomal molecules (Rae &
Spear, 1978; Roberson et al., 1989). Moreover, about one
million copies of the 5S rDNA are coded in linear minichromosomes flanked by telomeres in E. eurystomus (Roberson et al., 1989).
Concluding remarks
Ribosomes are complex organelles that require the intricate
collaboration of three types of RNAs (rRNA, mRNA and
tRNA) and 4 70 proteins for the synthesis of proteins.
rRNAs must maintain their convoluted structural motifs in
order to be functional. It is therefore not surprising that
their sequence is highly conserved among related organisms
and this similarity is gradually lost as organisms diverge. For
this reason, sequence comparison of the SSU rRNA has been
widely used in the field of molecular phylogeny (Van de Peer
et al., 2000).
The ‘typical’ eukaryotic rDNA genomic organization was
proposed 4 30 years ago, based on the analysis of the rDNA
in higher eukaryotes (Long & Dawid, 1980). The tandemly
repeated head-to-tail organization has been considered the
standard for eukaryotic rDNA. Surprisingly, analyses of the
genomic organization of ribosomal genes in microbial
eukaryotes demonstrate that although some organisms do
hold the typical rDNA configuration, the majority reveal
c 2009 Federation of European Microbiological Societies
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unusual characteristics. As shown in this review, the eukaryotic rDNA may be arranged in a wide variety of genomic
configurations, suggesting the existence of several regulatory
mechanisms (probably species-specific) within a conserved
rDNA regulatory context.
Reiteration is one of the most conserved rDNA characteristics. The rDNA copy number is extremely variable and
appears to be highly regulated within species. Nevertheless,
the total number of rDNA repeats does not always correlate
with the rate of rRNA synthesis (French et al., 2003),
implying that individual rDNA units may hold different
epigenetic marks that result in variable transcriptional rates
(Grummt, 2007). rDNA structure and transcription are also
important in the establishment of the nucleolar structure,
which also plays regulatory roles at the cellular level (Carmo-Fonseca et al., 2000). Dictyostelium discoideum, T. gondii
and some yeast species have equal numbers of rDNA and 5S
rDNA copies. This organization was considered coherent, as
the pool for rRNA molecules was supposed to hold equimolar amounts of 18S, 5.8S, 28S and 5S rRNA mature molecules for the efficient synthesis of ribosomes (Prokopowich
et al., 2003). However, rDNA and 5S rDNA are transcribed
by different RNA polymerases with dissimilar transcription
rates and number of transcriptionally open rDNA units.
Therefore, the rDNA/5S rDNA dosage is not directly related
to the stoichiometry of the total rRNA pool and expression
process. The rDNA/5S rDNA dosage varies widely throughout evolution. The processes that allow the maintenance of
the pool of rRNA mature molecules in appropriate stoichiometry must be a complex network including epigenetic,
FEMS Microbiol Rev 34 (2010) 59–86
77
Ribosomal RNA genes in eukaryotic microorganisms
transcriptional, post-transcriptional and structural mechanisms that may vary according to the rDNA/5S rDNA dosage.
Additionally, the location of the rDNA and 5S rDNA in the
genome may be related to its expression and physiology. The
chromosomal context, together with different chromatin
environments, may be involved in the maintenance of gene
copy number, recombination frequency, sequence conservation and transcription regulation of rDNA.
The organization of rDNA in extrachromosomal molecules may be associated with the cellular need for quick
changes in the rDNA copy number under stress conditions.
Some organisms have most of their rDNA in self-replicating
extrachromosomal molecules, but retain additional copies
in the chromosome probably as a backup. Interestingly,
some organisms hold the totality of their rDNA in extrachromosomal molecules. It has been shown that the accumulation of extrachromosomal rDNA copies in old S.
cerevisiae cultures affects cells’ health. Therefore, cells that
hold most or all rDNA copies extrachromosomally may have
special mechanisms to allow for the accumulation of large
rDNA minichromosomes without affecting the cell fitness.
However, it is possible that yeasts lack this mechanism,
resulting in cell damage when episomes accumulate.
Post-transcriptional processing of ITS-1 and ITS-2 is
observed in almost all eukaryotes. However, some organisms
possess additional ITS sequences in the 28S rRNA that
generate fragmented rRNA molecules that maintain the core
rRNA active elements in the mature ribosome. It has been
found that some organisms possess additional sequences in
variable regions internal to the SSU rRNA coding sequence
that remain in the mature molecule, thus generating unusually large SSU rRNAs. It is interesting to note that the SSU
rRNA has not been found as a fragmented molecule in
nuclear genomes; in contrast, the fragmented eLSU rRNA
could be regarded as 28S molecules that have processed their
variable regions. The structure and functionality of these
rRNAs in the ribosome may help to understand the importance of variable regions and the differences/restrictions
between subunits.
The rDNA coding region of several microbial eukaryotes
is interrupted by group I introns. Different transcriptional
and post-transcriptional mechanisms are involved in the
processing of introns and HE transcripts. Some C. albicans
strains have heterogeneous rDNA populations with both
intron-containing and intron-less rDNA units. Because no
function related to rDNA expression has been proposed for
group I introns, C. albicans may provide a good model to
study the role (if any) of these introns in rRNA expression,
processing and stability.
The linkage of 5S rDNA to a variety of tandem repeated
families may be the result of homogenizing mechanisms
responsible for concerted evolution (Drouin & de Sá, 1995).
The finding that the 5S rDNA can be linked to all polymerFEMS Microbiol Rev 34 (2010) 59–86
ase transcribed genes, coded alone in different chromosomal
loci or coded in extrachromosomal molecules underscores
the possibility of various mechanisms acting to regulate the
expression of different types of 5S rDNAs. The simultaneous
expression of pol I, pol II and pol III transcribed genes in a
particular locus may alter the chromatin context as well as
the availability of transcription factors in the proximity.
Nevertheless, the significance of the linkage between multigenic families and 5S rDNA has not been studied. The
presence of unlinked 5S rDNA copies is also interesting
because the chromosomal context for each gene may affect
its regulation.
Widespread rDNA characteristics present among eukaryotic supergroups as well as particular features predominating in some eukaryotic subgroups reflect the complexity of
evolution. The typical tandem head-to-tail organization of
the rDNA and 5S rDNA is found in all eukaryotic supergroups (Fig. 9), suggesting that the eukaryotic common
ancestor held this organization. Later on, the evolutionary
process probably led to a specialization and divergence of
the rDNA structure, resulting in the different variants
described here. Other features, such as the group I introns,
were acquired by horizontal transfer and are therefore widespread among microbial eukaryotes (Fig. 9) (Sogin et al.,
1986; Van Oppen et al., 1993).
Some particular rDNA characteristics are conserved
among related species, suggesting that the common ancestor
for each group held these traits before current speciation.
Examples of this can be found in the unlinked differentially
expressed rDNA units in Apicomplexa, the extralong SSU
rRNAs in Amoebozoa and Foraminifera, the extrachromosomal rDNA and 5S rDNA in ciliates and Amoebozoa, the
5.8S–23S rRNA fusion in Microsporidia and both the SL–5S
rDNA linkage and the eLSU fragmentation in Euglenozoa.
The 5S rDNA nontandem organization as well as the linkage
between 5S rDNA with the ribosomal cistron can also be
seen as predominant in the Fungi group (Fig. 9). Particular
traits such as the 28S rRNA fragmentation could have
appeared more than once and independently, leading to
non-Euglenozoa organisms containing fragmented rRNAs
such as A. castellanii and Plasmodium. Distinctive characteristics shared among non-closely related species may represent phylogenetic evidence of yet unknown linkages among
eukaryotic subgroups and species. Nevertheless, a thorough
and integrated comparative characterization of rRNA genes
in poorly or nonstudied eukaryotes may help to understand
the diversity and relationship among different forms of life.
Many unanswered questions regarding the regulation of
rRNA gene expression still remain, for example, the mechanism(s) that determine which rRNA gene copies will be
transcriptionally and/or epigenetically active (Lawrence
et al., 2004; Grummt, 2007) or the relevance of the genomic
context that surrounds the rRNA genes. Finally, it should be
2009 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
78
A.L. Torres-Machorro et al.
Fig. 9. Schematic phylogenetic tree describing the prevailing rDNA structures in subgroups of microbial eukaryotes. Phylogeny of eukaryotes is based
on the six supergroup classification (Box 1). Conserved rDNA structures that predominate in eukaryotic subgroups are boxed in color. Some of the
described characteristics may be shared by more than one subgroup.
pointed out that the rDNA organization is only one fundamental step in its regulation, because its expression is
interrelated with most, if not all, of the cell’s regulation
levels (Paule & White, 2000; Schramm & Hernandez, 2002;
Grummt, 2003).
Acknowledgements
This work was supported by grants IN214006 from Programa de Apoyo a Proyectos de Investigación e Innovación
Tecnológica (PAPIIT), Universidad Nacional Autónoma de
México (UNAM) and P45037-Q from Consejo Nacional de
Ciencia y Tecnologı́a (CONACYT), Mexico. A.L.T.-M. was
supported by a scholarship from CONACYT Mexico.
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