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. 2008 May;179(1):21–30. doi: 10.1534/genetics.107.086025

Genomewide Analysis of Box C/D and Box H/ACA snoRNAs in Chlamydomonas reinhardtii Reveals an Extensive Organization Into Intronic Gene Clusters

Chun-Long Chen *,†,‡,1, Chong-Jian Chen *,†,§,1, Olivier Vallon **, Zhan-Peng Huang *, Hui Zhou *, Liang-Hu Qu *,2
PMCID: PMC2390600  PMID: 18493037

Abstract

Chlamydomonas reinhardtii is a unicellular green alga, the lineage of which diverged from that of land plants >1 billion years ago. Using the powerful small nucleolar RNA (snoRNA) mining platform to screen the C. reinhardtii genome, we identified 322 snoRNA genes grouped into 118 families. The 74 box C/D families can potentially guide methylation at 96 sites of ribosomal RNAs (rRNAs) and snRNAs, and the 44 box H/ACA families can potentially guide pseudouridylation at 62 sites. Remarkably, 242 of the snoRNA genes are arranged into 76 clusters, of which 77% consist of homologous genes produced by small local tandem duplications. At least 70 snoRNA gene clusters are found within introns of protein-coding genes. Although not exhaustive, this analysis reveals that C. reinhardtii has the highest number of intronic snoRNA gene clusters among eukaryotes. The prevalence of intronic snoRNA gene clusters in C. reinhardtii is similar to that of rice but in contrast with the one-snoRNA-per-intron organization of vertebrates and fungi and with that of Arabidopsis thaliana in which only a few intronic snoRNA gene clusters were identified. This analysis of C. reinhardtii snoRNA gene organization shows the functional importance of introns in a single-celled organism and provides evolutionary insight into the origin of intron-encoded RNAs in the plant lineage.

SMALL nucleolar RNAs (snoRNA) are one of the largest classes of noncoding RNAs in eukaryotes. They play an essential role in ribosomal RNAs (rRNA) biosynthesis (Maxwell and Fournier 1995). A small fraction of snoRNAs such as U3, U8, U14, U22, U17, and RNase MRP RNA are involved in the cleavage of pre-rRNAs (Venema and Tollervey 1999). However, most of them guide the 2′-O-ribose methylation and pseudouridylation of rRNAs (Smith and Steitz 1997). On the basis of common sequence motifs and structural features, all snoRNAs except RNase MRP fall into two families: box C/D snoRNAs and box H/ACA snoRNAs, which guide site-specific 2′-O-ribose methylations and pseudouridylations of rRNAs, respectively, via base complementarity (Balakin et al. 1996; Bachellerie et al. 2000; Kiss 2001). The box C/D snoRNAs display two conserved motifs, the 5′-end C box (5′-RUGAUGA-3′) and the 3′-end D box (5′-CUGA-3′), usually flanked by short inverted repeats. In addition to the H box (ANANNA) in the hinge region and an ACA motif 3 nucleotides upstream of the 3′-end of the molecule, box H/ACA snoRNAs are characterized by a common hairpin–hinge–hairpin–tail secondary structure (Ganot et al. 1997; Ni et al. 1997). The spectrum of snoRNA targets is continuously growing. They are now known to guide post-transcriptional modifications of snRNAs (Tycowski et al. 1998; Zhou et al. 2002) and tRNAs (Clouet d'Orval et al. 2001; Zemann et al. 2006), as well as the alternative splicing of a pre-mRNA (Cavaille et al. 2000; Kishore and Stamm 2006). Furthermore, some “orphan” snoRNAs with no obvious target of rRNAs and snRNAs have also been reported (Huttenhofer et al. 2001; Chen et al. 2003).

The genomic organization of snoRNA genes displays a great diversity in various organisms. In human, almost all of the snoRNAs are encoded by single genes nested within introns and maturated by a splicing-dependent processing (Tycowski et al. 1993; Kiss and Filipowicz 1995; Bachellerie et al. 2000). A few of them are transcribed independently with their own promoter. In the intron-poor genome of Saccharomyces cerevisiae, only a few of the snoRNA genes are intronic. The majority of the snoRNAs are encoded by single genes, but 17 (20%) snoRNAs are encoded in five gene clusters each driven by an upstream promoter (Lowe and Eddy 1999; Qu et al. 1999). In contrast, snoRNA gene clusters dominate in the plant lineage. More than 80% of the snoRNA genes of Arabidopsis thaliana and Oryza sativa are organized into gene clusters (Leader et al. 1997; Brown et al. 2003a; Chen et al. 2003). Four intronic snoRNA gene clusters were found in A. thaliana, and one-half of the O. sativa clusters have been found within introns of protein-coding genes.

Chlamydomonas reinhardtii is a unicellular green alga belonging to Chlorophyta, a phylum that diverged from land plants over one billion years ago, following the eukaryotic radiation that gave rise to the animal, fungal, and plant kingdoms (Merchant et al. 2007). Comparison of the C. reinhardtii proteome to Homo sapiens and A. thaliana showed that C. reinhardtii proteins are overall slightly more similar to A. thaliana than to human proteins. However, C. reinhardtii shares many genes with animals, in particular those associated with flagellar functions. These proteins have been inherited from the common ancestor of plants and animals, but were lost in land plants (Li et al. 2004). The availability of the C. reinhardtii genome provides an intriguing opportunity to investigate the ancestor snoRNA gene constitution and organization in the plant lineage. Here we present a genomewide analysis of two major families of snoRNAs, i.e., box C/D and box H/ACA snoRNAs in the C. reinhardtii genome and compare them with those of Volvox carteri, a multicellular green alga diverged from C. reinhardtii ∼50 million years ago (Coleman 1999) and of other eukaryotes including plants, animals, and fungi.

MATERIALS AND METHODS

The draft sequence of the C. reinhardtii genome assembly v3.1, the Volvox carteri f. nagariensis genome assembly v1.0, and gene annotations provided in the GeneCatalog track were downloaded from genome browsers in the DOE Joint Genome Institute (JGI) (http://genome.jgi-psf.org). SnoRNA genes of C. reinhardtii were identified using the snoRNA mining platform (snoRMP) (Chen et al. 2003; Huang et al. 2007), which is based on the SnoScan (Lowe and Eddy 1999) and SnoGPS (Schattner et al. 2006) algorithms. Further characterization was based on secondary structure prediction, gene organization, and comparative genomic analysis.

The box C/D snoRNA search program identified segments <150 bp, with box C (RUGAUGA), box D (CUGA), at least 10 nucleotides complementary (Watson–Crick and G:U base pairs) to a rRNA (5.8S, 18S, or 26S rRNA) or snRNA (U1, U2, U4, U5, or U6 snRNA) sequence and terminal short inverted repeats. Among these parameters, we considered primarily the quality of the potential snoRNA-target duplexes (the duplex length, the number and position of the duplex mismatches, and those of the GU pairings). The box H/ACA snoRNA search program identified segments <200 bp exhibiting a typical hairpin–hinge–hairpin–tail secondary structure, with box H (ANANNA) in the hinge region and box ACA in the tail, as well as two sequences of the pocket of the hairpin with complementarity to a rRNA or snRNA sequence (each >3 nucleotides and the sum >9 nucleotides). Sequences flanking the snoRNA candidates were examined for other possible snoRNAs, and BLAST searches (Altschul et al. 1997) were performed within the C. reinhardtii genome to identify paralogs or within the V. carteri genome to identify orthologs. The Vista track (Mayor et al. 2000) constructed with the C. reinhardtii and V. carteri genomes with a window of 100 bp and a minimum percentage of conservation identity of 60% was used to recover orphan snoRNA genes in the regions flanking the newly discovered snoRNAs. SnoRNA secondary structures were predicted with the Mfold program (Mathews et al. 1999). Sequence alignments and phylogenetic trees were performed with ClustalW (Thompson et al. 1994) and T-coffee (Notredame et al. 2000). The interval sequences of snoRNAs were scanned to check for AGNN stem-loop structures by using the program designed to identify Rnt1p cleavage signals with a cutoff of final score = 0.8 (Ghazal et al. 2005).

RESULTS

Identification of 322 snoRNA genes in the C. reinhardtii genome:

Using the snoRMP to screen the C. reinhardtii genome (see materials and methods), 316 putative snoRNA genes have been identified, with 183 of the box C/D type and 133 of the box H/ACA type (Tables 1 and 2). On the basis of sequence similarity (Blast E value <0.01) and antisense element conservation, the genes defined 72 box C/D and 42 box H/ACA snoRNA families (see Figure 1 for an alignment of CD11 snoRNAs). The orthologs of 93 snoRNA families could be detected in the V. carteri genome by sequence homology search. Furthermore, comparative genomic analysis indicated that one-third of the C. reinhardtii snoRNA genes reside in regions that are highly conserved between C. reinhardtii and V. carteri: 71 genes from 48 box C/D snoRNA families and 36 genes from 21 box H/ACA snoRNA families could be viewed as peaks in the Vista track. Manual inspection of the C. reinhardtii–V. carteri conserved regions flanking the newly discovered snoRNA genes allowed further identification of 6 additional putative snoRNAs, representing 4 families that appeared as orphan snoRNAs with no obvious rRNA or snRNA target: 2 single-copy box C/D snoRNAs, 1 single-copy box H/ACA snoRNA, and 1 box H/ACA snoRNA family with 3 members. Overall, 55 of the 118 C. reinhardtii snoRNA families have only 1 member, and 63 contain multiple members ranging from 2 to 14.

TABLE 1.

Box C/D snoRNA genes in C. reinhardtii

Homology
snoRNA Iso Target Antisense (nt) Plants Fungi Animals Volvox iso
CrCD01 5 Cr26S-G2754 18 (5′) snoR1 SnR48 — 1
CrCD02 2 Cr26S-A1819 12 (5′) — — BII420 —
CrCD03 6 Cr26S-A2603 12 (5′) snoR68Y SnR68 — —
CrCD04 2 Cr26S-U1892 13 (5′) — — U50 1
CrCD05 2 CrU6-C55 11 (5′) — — U94 1
CrCD06 5 Cr18S-G1408 12 (5′) snoR69 — — 3
CrCD07 2 Cr26S-C2799 13 (3′) snoR24 — — 1
CrCD09 4 Cr26S-A795 13 (5′) U51 SnR39 U51 3
Cr18S-U1539 15 (3′) — — —
CrCD10 7 Cr26S-U1043 16 (5′) snoR41Y — — —
CrCD11 10 Cr18S-A419 13 (5′) snoR120 SnR52 U83 3
Cr26S-C1533 11 (3′) — — —
Cr26S-C2645 14 (3′) snoR162 — —
CrCD13 5 Cr18S-A539 14 (3′) snoR41Y SnR41 U62 2
CrCD14 1 Cr18S-G1567 10 (5′) — SnR57 — 1
CrCD15 6 Cr26S-C2443 13 (5′) — — — 3
Cr26S-A2432 11 (3′) — — —
CrCD16 6 Cr18S-U609 13 (5′) snoR13 — — 6
Cr26S-G1425 11 (3′) — — —
CrCD17 3 Cr26S-U1868 15 (5′) U34 SnR62 U34 1
CrCD18 3 Cr26S-C1454 13 (3′) Z270 — — 2
CrCD19 1 Cr18S-G593 11 (5′) U54 — U54 2
CrCD20 5 Cr26S-G1831 12 (3′) U59 — — —
CrCD21 2 Cr26S-C1492 15 (5′) U49 — — —
CrCD22 4 Cr26S-G2088 11 (5′) snoR60 — — 2
CrCD23 5 Cr18S-A1199 15 (5′) — — — 2
Cr18S-C1211 13 (3′) snoR130 — BII142
CrCD24 3 Cr18S-A1087 15 (3′) — — — 1
CrCD25 2 Cr18S-G632 12 (5′) J27 — BII108 2
Cr18S-A617 14 (3′) U36 SnR47 U36
CrCD26 2 Cr18S-C413 21 (3′) U14 U14 U14 5
CrCD27 1 Cr26S-C2922 15 (5′) U35 SnR73 U35 2
CrCD28 1 Cr26S-G2582 11 (5′) U35 SnR73 U35 2
CrCD29 1 Cr26S-A2909 13 (5′) U29 SnR71 U29 2
CrCD30 1 Cr26S-A639 15 (5′) U18 U18 U18 2
CrCD31 1 Cr26S-U2385 13 (5′) snoR37 SnR78 U52 —
Cr26S-C2329 13 (3′) snoR37 — U53
CrCD32 4 Cr18S-U122 12 (5′) — — Z17 3
Cr18S-G1122 12 (3′) snoR41YII SnR41 —
CrCD33 1 Cr18S-C38 13 (5′) snoR66 — — 1
CrCD34 1 Cr26S-A2874 14 (5′) snoR31 — — 1
Cr5.8S-A43 13 (3′) snoR9 — —
CrCD35 1 Cr26S-U2613 14 (5′) snoR10 — U58 1
CrCD36 1 Cr18S-A970 12 (5′) U59 SnR54 U59 2
CrCD37 1 Cr26S-A2285 13 (3′) U30 — U30 1
CrCD38 1 Cr18S-U598 13 (5′) Z267 — — 1
CrCD39 1 Cr26S-U2077 13 (5′) — — — 1
CrCD40 1 Cr26S-C2301 13 (5′) snoR44 SnoR64 Z18 1
Cr26S-A2290 13 (3′) snoR44 — Z22
CrCD41 6 Cr26S-G1907 13 (5′) — — U50 3
CrCD43 1 Cr18S-U1227 11 (5′) snoR14 SnR82 BII55 1
Cr18S-U1374 14 (3′) U61 — U61
CrCD44 1 Cr18S-U576 12 (5′) snoR77Y SnR77 — 1
CrCD45 1 Cr18S-U1438 15 (3′) snoR19 — — 1
CrCD46 2 Cr26S-C1836 12 (5′) snoR15 — U39 —
CrCD47 3 Cr18S-G560 14 (5′) — snR80 — —
Cr18S-C585 11 (3′) — — —
CrCD48 7 Cr26S-G2200 14 (5′) U36 — — 4
Cr26S-A2184 17 (3′) U36 snR47 U36
CrCD49 1 Cr26S-U2698 13 (3′) snoR68 — — 1
CrCD50 6 Cr26S-G2252 11 (5′) U15 snR75 U15 4
Cr26S-A2245 11 (3′) U15 — U15
CrCD51 1 Cr26S-A864 13 (5′) snoR72Y snR72 — 1
Cr18S-C147 13 (3′) — — — —
CrCD52 1 Cr26S-C1983 12 (5′) — — — —
CrCD53 1 Cr26S-G896 15 (3′) U80 snR60 Z15 2
CrCD54 1 Cr26S-A805 12 (5′) U80 snR60 Z15 —
CrCD55 1 Cr18S-A28 11 (5′) U27 snR74 U27 1
CrCD56 1 Cr18S-A795 13 (5′) snoR53Y snR53 — 1
Cr26S-G1419 13 (3′) — — —
CrCD57 1 Cr26S-A1118 13 (5′) U38 snR61 U38 1
CrCD58 1 Cr26S-C2268 15 (5′) — — — 1
CrU6-C70 14 (5′) — gU6-77 —
CrU6-A40 12 (3′) — gU6-47 —
CrCD59 1 Cr26S-A1434 11 (5′) U24 U24 U76 1
Cr26S-C1422 10 (3′) U24 U24 U24
CrCD60 2 Cr26S-U2560 12 (5′) — — — 2
CrCD61 1 Cr18S-G1424 12 (5′) snoR19 snR56 U25 1
CrCD62 4 Cr26S-U784 10 (5′) — — — 4
Cr26S-G793 11 (3′) snoR39BY snR39b snR39B
CrCD63 2 Cr18S-C1634 11 (5′) U43 snR70 U43 2
CrCD64 1 Cr26S-A2220 11 (3′) U40 snR63 U40 1
CrCD65 3 Cr26S-A905 12 (5′) snoR133 snR84 — 3
Cr26S-C849 13 (3′) — — —
CrCD66 7 Cr26S-A2641 15 (5′) — — — 2
CrCD67 2 Cr26S-A1352 12 (3′) snoR7 — — 2
CrCD68 1 Cr26S-G2778 12 (5′) snoR38Y snR38 snR38 2
Cr18S-A161 10 (3′) snoR18 — U44
CrCD69 1 Cr26S-G2373 12 (5′) — — — 1
Cr26S-G2355 11 (3′) snoR29 — —
CrCD70 1 Cr26S-A2090 12 (5′) snoR12 — — 1
CrCD71 1 Cr26S-G2756 14 (3′) — snR48 U60 —
CrCD72 1 Orphan — — — 4
CrCD73 6 Cr18S-A1322 10 (5′) snoR32 — — 3
CrCD74 1 Orphan — — — 1
CrCD75 1 Cr26S-A1875 13 (3′) — — — 1
CrCD76 3 Cr26S-U1253 11 (5′) snoR22 — — 2
CrCD77 2 Cr18S-G559 12 (3′) — — — —
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SnoRNA family names are listed with the numbers of isoforms in the C. reinhardtii (Iso) and V. carteri (Volvox iso) genomes and the rRNA and snRNA targets. Antisense sequence lengths are indicated and the modification sites are compared to those of plants (A. thaliana and O. sativa) (Brown et al. 2003b; Chen et al. 2003), fungi (S. cerevisiae and S. pombe) (Li et al. 2005; Piekna-Przybylska et al. 2007), and animals (H. sapiens and M. musculus) (Huttenhofer et al. 2001; Lestrade and Weber 2006). SnoRNAs that lack a target site are indicated as orphan. —, no corresponding snoRNA identified.

TABLE 2.

Box H/ACA snoRNA genes in C. reinhardtii

Homology
snoRNA Iso Target Antisense (nt) Plants Fungi Vertebrates Volvox iso
CrACA01 8 Cr26S-U2278 7 + 4 (5′) snoR83 snR86 ACA48 2
Cr26S-U2315 5 + 8 (3′) snR82 — —
CrACA02 1 Cr26S-U887 5 + 4 (5′) — — — 1
CrACA03 1 Cr26S-U46 5 + 6 (5′) — — — 1
CrACA04 7 Cr18S-U558 7 + 4 (3′) — — ACA24 3
CrACA05 3 Cr26S-U2175 7 + 5 (3′) snoR99 snR90 ACA27 2
CrACA06 2 Cr26S-U873 5 + 6 (5′) Osaca052a — — —
CrU6-U88 5 + 6 (3′) — — —
CrACA07 1 Cr18S-U831 6 + 5 (5′) — — — —
Cr26S-U1994 5 + 4 (3′) — — —
CrACA08 2 Cr26S-U857 4 + 5 (5′) — — — 1
Cr26S-U923 4 + 5 (5′) — — —
CrACA09 2 Cr26S-U2311 6 + 5 (3′) Osaca003a — — 1
CrACA10 10 Cr18S-U1301 6 + 5 (5′) — — — 3
Cr18S-U1707 3 + 6 (5′) — — —
CrACA13 2 Cr26S-U2789 6 + 3 (3′) snoR2 snR34 U65 —
CrACA15 1 Cr26S-U3281 6 + 3 (3′) — — ACA22 —
CrACA16 3 Cr18S-U110 6 + 5 (5′) snoR100 — ACA42 3
Cr18S-U206 7 + 4 (3′) — snR49 —
CrACA18 1 Cr18S-U1187 7 + 5 (5′) — snR35 ACA13 1
Cr18S-U708 5 + 4 (3′) — — —
Cr18S-U1246 7 + 3 (3′) — — —
CrACA19 1 Cr26S-U857 6 + 5 (5′) — — — 1
Cr26S-U1028 7 + 4 (3′) — — —
CrACA21 1 Cr26S-U2230 6 + 8 (5′) Osaca019a snR84 — 1
CrACA22 4 Cr26S-U2828 6 + 6 (5′) — snR46 ACA16 1
CrACA23 1 Cr26S-U1765 5 + 5 (3′) — — — 1
CrACA24 2 Cr18S-U942 5 + 8 (5′) — — — 1
Cr26S-U2311 6 + 5 (3′) Osaca003a — —
CrACA26 1 Cr18S-U757 5 + 5 (3′) snoR91 — ACA25 —
CrACA28 5 Cr18S-U1131 4 + 5 (5′) — — — —
Cr26S-U1820 5 + 4 (3′) — — —
Cr18S-U1598 5 + 5 (3′) — — —
CrACA29 1 Cr26S-U3074 7 + 4 (5′) Osaca019a — ACA17 1
Cr26S-U2938 6 + 9 (3′) Osaca003a snR42 ACA27
CrACA30 1 Cr18S-U1428 7 + 3 (5′) — — — 1
Cr18S-U1183 6 + 5 (3′) — — ACA36
CrACA31 3 Cr26S-U2100 6 + 5 (5′) snoR87 — ACA19 3
CrACA32 6 Cr26S-U1224 7 + 3 (5′) snoR96 — — 3
CrACA33 4 Cr18S-U600 6 + 7 (5′) snoR91 — ACA20 1
CrACA35 7 Cr26S-U2380 7 + 3 (5′) — snR11 ACA3 5
CrACA36 2 Cr18S-U1428 4 + 5 (5′) — — — —
Cr26S-U224 4 + 5 (5′) — — —
Cr18S-U200 5 + 5 (5′) — — —
Cr26S-U2592 6 + 8 (3′) snoR78 — —
CrACA37 1 Cr18S-U357 7 + 3 (5′) snoR86 — U71 —
CrACA38 5 Cr18S-U1556 5 + 7 (3′) Osaca053a — ACA5 4
CrACA39 14 Cr18S-U102 4 + 7 (3′) Osaca025a — — 5
CrACA40 4 Cr26S-U764 6 + 4 (5′) snoR82 snR80 — 7
Cr26S-U807 5 + 4 (3′) snoR77 — —
CrACA41 6 Cr18S-U1623 6 + 4 (5′) — — U70 2
CrACA42 6 Cr26S-U2685 4 + 5 (5′) — — ACA34 6
Cr18S-U301 6 + 6 (5′) — snR49 —
Cr18S-U378 7 + 5 (3′) — — —
CrACA43 1 Cr18S-U1210 7 + 5 (3′) Osaca069a — — —
CrACA44 1 Cr26S-U1667 5 + 8 (5′) — snR91 — 1
CrACA45 2 Cr18S-U985 7 + 3 (5′) — — — 2
CrACA46 3 Cr26S-U2278 5 + 8 (5′) snoR83 snR86 ACA48 —
CrACA48 1 CrU6-U24 5 + 8 (5′) — — ACA65 —
CrACA50 2 Cr26S-U2224 7 + 9 (5′) U19 snR191 — 3
CrACA51 1 Cr26S-U2817 6 + 4 (5′) — — ACA21 —
CrACA52 1 Cr26S-U2886 6 + 4 (3′) snoR74 snR10 ACA21 —
CrACA54 3 Orphan — — — 3
CrACA55 1 Orphan — — — 1
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SnoRNA family names are listed with the number of isoforms in the C. reinhardtii (Iso) and V. carteri (Volvox iso) genomes and the rRNA and snRNA targets. Antisense sequence lengths are indicated and the modification sites are compared to those of plants (A. thaliana and O. sativa), (Brown et al. 2003b; Chen et al. 2003), fungi (S. cerevisiae and S. pombe) (Li et al. 2005; Piekna-Przybylska et al. 2007), and animals (H. sapiens and M. musculus) (Huttenhofer et al. 2001; Lestrade and Weber 2006). SnoRNAs that lack a target site are indicated as orphan. —, no corresponding snoRNA identified.

a

Plant snoRNAs that are from our unpublished data.

Figure 1.—

Figure 1.—

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Evolution of C. reinhardtii snoRNA genes. (A) Organization of snoRNA genes. Host genes from C. reinhardtii (Cr) and V. carteri (Vc) are indicated by their protein identification from the JGI genome browser. SnoRNA families are represented by different colored boxes, and isoforms are denoted by their suffixal characters (a, b, c, …). Exons are indicated by thick gray boxes, UTRs by intermediate-sized gray bars, and introns by black lines. The ordinals of exons flanking the snoRNAs are given. For each gene, the Vista track is attached below, with pink for conserved noncoding regions and blue for conserved coding regions. All are drawn to scale. (B) Functional evolution of CD11 snoRNA genes in C. reinhardtii and V. carteri. A rectangular cladogram tree is placed to the left of the sequence alignment. Boxes C, D′, and D are shown in open boxes, antisense sequences are shaded, and related methylated sites are indicated.

Properties of C. reinhardtii box C/D and box H/ACA snoRNA functions:

The 74 box C/D snoRNA families of the C. reinhardtii genome were predicted to guide methylation at 96 sites of rRNAs, including 1, 33, and 62 sites of the 5.8S, 18S, and 26S rRNAs, respectively, and 3 sites of the U6 snRNAs (Table 1). More than 80% of the C. reinhardtii rRNA putative methylated sites have analogs in plants (A. thaliana and O. sativa), fungi (S. cerevisiae and Schizosaccharomyces pombe), or animals (Homo sapiens and Mus musculus) (Table 3A). These include 27 sites well conserved among all three groups, 7 shared with plants and fungi, 11 with plants and animals, and 1 with fungi and animals; 24 have analogs solely in plants, 2 in fungi, and 5 in animals. Remarkably, 19 rRNA putative methylated sites that did not have any analogs are probably C. reinhardtii specific. In addition, among the 3 U6 snRNA putative methylated sites, 2 are conserved with fungi, and 1 is C. reinhardtii specific.

TABLE 3.

Homology information on modification sites in the C. reinhardtii rRNAs and snRNAs

Homologous with PFA PA PF FA P F A Non Total
A. Methylated sites guided by the C. reinhardtii box C/D snoRNAs
rRNA sites 27 11 7 1 24 2 5 19 96
snRNA sites 2 1 3
B. Pseudouridylation sites guided by the C. reinhardtii box H/ACA snoRNAs
rRNA sites 6 7 3 3 9 3 6 23 60
snRNA sites 1 1 2
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The modification sites of the C. reinhardtii rRNAs and snRNAs were compared to those of plants (P), fungi (F), and animals (A).

The 44 Box H/ACA snoRNA families of the C. reinhardtii genome were predicted to guide pseudouridylation at 60 sites of rRNAs, including 26 and 34 sites of the 18S and 26S rRNAs, respectively, and 2 sites of the U6 snRNAs (Table 2). Among the 60 C. reinhardtii rRNA putative pseudouridylation sites, 37 have analogs in one of the six species of higher plants, fungi, and animals (Table 3B), including 6 sites common to the three kingdoms; 3 have analogs within both plants and fungi, 7 within both plants and animals, and 3 within both fungi and animals; 9 have analogs solely in plants, 3 in fungi, and 6 in animals. Twenty-three sites display as C. reinhardtii specific. Among the 2 snRNA putative pseudouridylation sites, 1 is conserved with animals, the other is C. reinhardtii specific.

The predicted modification pattern of C. reinhardtii rRNAs was closely related to that of land plants. More than 72% of the C. reinhardtii rRNA putative methylation sites are conserved with land plants compared to only 46 and 39% with animal and fungi, respectively. Pseudouridylation sites are also more conserved with land plants (42%) than with animal (37%) and fungi (25%). The fact that more pseudouridylation sites appear specific to C. reinhardtii is probably a consequence of the smaller number of box H/ACA snoRNAs that have been identified in other organisms, especially in higher plants than box C/D snoRNAs. It is worth noting that although we used all the U1, U2, U4, U5, and U6 snRNAs as target to look for the putative modification sites guiding by snoRNAs, only the snoRNAs guiding U6 snRNA modification were found. This correlates with the fact that synthesis and maturation processes are different between the RNA polymerase III-transcribed U6 snRNA and the polymerase II-transcribed snRNAs (U1, U2, U4, and U5). In mammals, the 2′-O-methylation and pseudouridylation of the U6 snRNA takes place in the nucleolus, and it is directed by snoRNAs (Tycowski et al. 1998; Ganot et al. 1999). While the RNA polymerase II-transcribed snRNAs undergo site-specific post-transcriptional modification in Cajal bodies directed by another type of ncRNAs called small Cajal body-specific RNA (scaRNAs) (Darzacq et al. 2002; Kiss 2004).

Prevalence of intronic snoRNA gene clusters in the C. reinhardtii genome:

Gene cluster is the main genomic organization of snoRNA genes in C. reinhardtii. Besides 80 singletons, 242 snoRNA genes are arranged into 76 gene clusters, with a <500-bp interval between 2 adjacent snoRNAs (supplemental Table S1). Remarkably, 67 singletons and 61 clusters were found to reside within introns of protein-coding genes presented on the GeneCatalog track. Examination of the 64 snoRNA genes that were initially predicted to lie between protein-coding genes revealed that only 30 of them were truly intergenic. The 34 others were found to lie within 16 genes that had been mispredicted. In each case, more appropriate gene models were placed in the catalog. Nine of them are supported by EST and/or homology data. Altogether 76 singleton and 70 snoRNA clusters, totaling 292 (>90%) snoRNA genes are encoded in introns of 98 host genes that function mainly in translation-related processes, such as ribosomal protein, tRNA synthetase, tRNA processing protein, and elongation factor. Athough some snoRNA genes may have escaped our analysis, the C. reinhardtii genome contains the highest number of known intronic snoRNA clusters among eukaryotes.

The C. reinhardtii snoRNA gene clusters are composed of 2–7 snoRNA genes. In contrast with the land plants in which about two-thirds of the snoRNA gene clusters consist of heterologous snoRNA genes (heterocluster) (Brown et al. 2003a; Chen et al. 2003), >77% of the C. reinhardtii snoRNA gene clusters are made up of homologous snoRNA genes (homocluster). This is probably the result of extensive local tandem duplications. All 25 box H/ACA snoRNA gene clusters and 34 of the 42 box C/D snoRNA gene clusters are homoclusters. Only 8 box C/D snoRNA gene clusters are heteroclusters. In addition, 9 snoRNA gene clusters containing both box C/D and H/ACA snoRNAs were also identified.

Duplication and evolution of the C. reinhardtii snoRNA genes:

The evolution of snoRNAs is thought to have occurred through a repeated series of duplications, accompanied by mutations and selection for their ability to associate into stable snoRNPs and to influence ribosome assembly and function (Brown et al. 2003a). We took advantage of the clustered organization of the C. reinhardtii snoRNA genes to investigate their evolution following gene duplication. In most cases, despite extensive sequence divergence among snoRNA paralogs, their functions presumably remain unchanged because nucleotide differences mainly map outside of the guiding sequences. However, the function of duplicates may differ from the original one by subfunctionalization (where the snoRNA duplicates a fraction of the ancestral function) or by neofunctionalization (where the duplicate evolves to gain a novel function). One such example is the bifunctional CrCD11 family. A cluster with five CrCD11 snoRNAs (CrCluster 13) and a cluster with two VcCD11 snoRNAs reside in the corresponding introns of C. reinhardtii–V. carteri orthologs, Cr142296 and Vc102784 (Figure 1A). The two VcCD11 snoRNAs have the same antisense sequences as CrCD11e, which in C. reinhardtii can guide methylation at A419 of 18S rRNA and at C2645 of 26S rRNA (Figure 1B). This suggests that these two methylation sites already existed in the last common ancestor. In contrast, CrCD11c and CrCD11a have lost the 5′ and the 3′ antisense element, respectively. CrCD11b and CrCD11d have maintained the 5′ antisense element, but the 3′ antisense elements mutated so as to guide a new methylation site on C. reinhardtii 26S rRNA, at C1533.

Another snoRNA gene cluster with a quasi-identical sequence was found in a nearby intron of the same host gene (Figure 1A). A similar situation was encountered in 19 other host genes of C. reinhardtii. This suggests that snoRNAs and snoRNA clusters can be duplicated not only in the immediate vicinity, but also several hundred base pairs from their original site, into another intron of the same host gene. Cr189727 presents an interesting case of inversion of gene order (Figure 1A). The snoRNA clusters in the 9th and 37th introns are CD03-ACA01 dimers, while that in the 12th intron is made up of two CD03-ACA01 dimers. The intervening sequences between CD03 and ACA01 are well conserved in these clusters, suggesting that they were duplicated as a block. In contrast, the clusters in the 29th and 32nd introns are ACA01-CD03 dimers, i.e., showing the genes in the reverse order. The intervening sequences between ACA01 and CD03 in these dimers are highly similar to that lying between CrACA01b and CrCD03c. This suggests that the CD03b-ACA01b-CD03c-ACA01c tetramer was first formed by a local tandem duplication of a CD03-ACA01 dimer, and then was duplicated twice in the 29th and 32nd intron, after which loss of the outlying snoRNAs led to the ACA01-CD03 dimers that can now be observed.

DISCUSSION

High frequency of snoRNA gene duplication in the C. reinhardtii genome:

Owing to the similar sizes of the V. carteri and C. reinhardtii genomes (∼140 Mb and 120 Mb, respectively) and the short time of the divergence between the two species, we expected to detect similar numbers of snoRNA genes in the two organisms. However, among 93 snoRNA gene families that are conserved between C. reinhardtii and V. carteri, 46 contain more members in C. reinhardtii than V. carteri, 9 in the extreme case. In contrast, only 12 families contain more members in V. carteri than C. reinhardtii. Phylogenetic analyses were performed on the 39 families that harbor ≥2 members in both C. reinhardtii and V. carteri. In 30 families, the paralogous sequences of the same genome grouped with each other, distinct from their orthologs of the other genome. This, together with the large differences in the number of family members, suggests that most of the snoRNA genes in the C. reinhardtii and V. carteri genomes have been generated by recent duplication after the lineages diverged. The alternative hypothesis, namely that snoRNA duplicates are constantly homogenized within each organism by gene conversion, is unlikely because sequence conservation was uneven, with guiding sequences markedly more conserved that the rest of the snoRNAs.

The enrichment of snoRNAs in C. reinhardtii may be linked to its ability to survive in a variety of environmental conditions. Although mutants defective in one or two of most rRNA modifications of S. cerevisiae have no detectable phenotype (Qu et al. 1999; Piekna-Przybylska et al. 2007), loss of three to all five modifications in the helix 69 of S. cerevisiae 26S rRNA has been reported to alter its structure in the ribosome and causes the broadest defects including reduction of amino acid incorporation rate, reduction of rRNA level, and increase of stop codon read-through activity (Liang et al. 2007). Interestingly, different growth rates among yeast recombinants with deletion of modification guide snoRNAs to rRNAs indicated varied sensitivity of the strains to antibiotics at different temperatures (Li et al. 2005; Bi et al. 2007; Liang et al. 2007). Ribosomal modification has been suggested to incur to rRNA stability and influence ribosome function and may be required in extreme environments (Omer et al. 2000; Barneche et al. 2001; Chen et al. 2003). The abolition of a methylation of U6 snRNA by deleting the yeast mgU6-47 snoRNA may slightly affect the efficiency of mRNA splicing (Zhou et al. 2002). It has been reported that 90% of the ribosomal proteins are degraded when C. reinhardtii cells differentiate into gametes as a result of nitrogen starvation (Siersma and Chiang 1971; Rochaix 1995). As the gametes dedifferentiate, new ribosomes will assemble. During these transformations, the large number of snoRNAs in C. reinhardtii may provide for the rapid buildup of an efficient rRNA modification machinery.

The ancestral organization of intronic snoRNA gene clusters in plants:

Intronic snoRNA gene clusters, first reported in our study of rice Hsp70 (Qu et al. 1997), have since then been shown to be widespread in this organism (Liang et al. 2002; Chen et al. 2003). Their prevalence in C. reinhardtii suggests that they represent an ancestral characteristic of the plant lineage, distinct from the one-snoRNA-per-intron organization of vertebrates and yeasts. The small number of intronic snoRNA clusters characterized in A. thaliana probably relates to the smaller size of A. thaliana introns (∼170 nt on average) (Arabidopsis Genome Initiative 2000) compared to C. reinhardtii (∼373 nt) (Merchant et al. 2007) and O. sativa (∼360 nt) (Yu et al. 2002). In this respect, it might be interesting to examine the practically intronless Ostreococcus genomes (Palenik et al. 2007) or Physcomitrella patens and its long introns.

Still, intronic snoRNA clusters are not exclusive to the plant lineage. A total of 17 intronic clusters encoding 77 box H/ACA snoRNAs (70% of the whole gene set) have been identified in the Drosophila melanogaster genome (Huang et al. 2005). However, the 98 box C/D snoRNAs all obey the one-snoRNA-per-intron rule, which suggests that clustering is not an ancient feature. Intronic snoRNA clusters may have evolved independently in different lineages. Alternatively, they may have been present in ancestral eukaryotes and lost in some lineages. Further investigation of snoRNA genes in fully sequenced genomes of various taxa will help answer the question.

Whatever its origin, the intronic clustering of snoRNAs represents an economical way of producing these essential molecules. Bypassing the need for a dedicated transcription event, they make use of what is usually a discarded by-product of protein gene expression, the intron. At the same time, it may allow regulated and coordinated production of specific classes of snoRNAs, making use of the regulatory properties of the host gene. Unfortunately, nothing is known of the processing of intronic snoRNA gene clusters. Does it occur during or after intron splicing? Does it rely on endonucleolytic cleavage of the pre-mRNA or of the spliced circularized intron? Does the intron recircularize after a snoRNA is released, to allow processing of the other ones or is the maturation simultaneous for all members of the cluster? The S. cerevisiae polycistronic snoRNA that transcribe from a common promoter are processed by class II RNase III, Rnt1p (Qu et al. 1999; Ghazal et al. 2005). However, the AGNN stem-loop structure, the recognition signal of Rnt1p, could not be characterized in the interval of adjacent snoRNA genes in the C. reinhardtii snoRNA clusters. By using homology search of Rnt1p from yeast, a family with three members of RNase III genes, named AtRTL (RNase three-like) has been recently characterized in A. thaliana (Comella et al. 2007). Within this RNase III gene family, the AtRTL2 is the closest functional homolog of the S. cerevisiae Rnt1p and the only gene ubiquitously expressed in A. thaliana. While disruption of the AtRTL2 gene has no effect in the processing of polycistronic snoRNA in this organism, it seems that plant polycistronic snoRNAs are processed by a mechanism that does not involve RNase III-like activities. An interesting parallel can be made with the spliceosomal snRNA genes. In C. reinhardtii, the majority of U1, U2, and U4 snRNA genes also reside within introns of protein-coding genes (Merchant et al. 2007). However, their mechanism of expression is different: it has been proposed that they use a Pol II-dependent transcription start internal to the host gene, yielding transcripts that are polyadenylated and spliced and may serve as precursors for the mature snRNAs. Here also, the maturation pathway is largely unknown.

Thirty C. reinhardtii snoRNAs do not lie in predicted genes and have no EST nearby that could serve as evidence for a host gene. Twenty-seven of them that are grouped into six clusters were examined in greater detail; one of them could be found in the intron of a hemD pseudo gene, and two could be located in the intron of a hypothetical noncoding precursor. This suggests that they lie within specialized host genes that do not code for a protein, similar to the Drosophila and vertebrate dUhg genes (Tycowski and Steitz 2001; Huang et al. 2007). Comparison of the V. carteri and C. reinhardtii sequences will help elucidate the origin of these genes. One hypothesis is that they arise when a host gene loses its coding capacity while retaining its ability to splice because its introns harbor essential snoRNA genes.

Acknowledgments

We thank J.-H. Yang and L. Amar for valuable comments. This research is supported by the National Basic Research Program (2005CB724600), the National Natural Science Foundation of China (30470385 and 30570398), and the Ministry of Education of China and Guangdong province (IRT0447 and NSF-05200303). C.-L.C was a grant recipient of the French Embassy Ph.D. program and the Excellent Doctor Thesis program of Sun Yat-sen University. C.-J.C was the grant recipient of the Ph.D. program (China Scholarship Council) from the Chinese government.

Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession nos. EU410622–EU410943. Sequence data are also available as annotation tracks displayed on the JGI C. reinhardtii genome browser.

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