Springer 2006
Genetica (2006) 128:21–31 DOI 10.1007/s10709-005-2486-0
Diversity of endogenous small non-coding RNAs in Oryza sativa Zuyu Chen, Jingjing Zhang, Jin Kong, Shaoqing Li, Yan Fu, Shaobo Li, Hong Zhang, Yangsheng Li & Yingguo Zhu* Key Laboratory of MOE for Plant Developmental Biology, College of Life Science, Wuhan University, 430072, Wuhan, P. R. China; *Author for correspondence (Phone: +86-27-68756530; Fax: +86-2768756530; E-mail:
[email protected]) Received 12 March 2005 Accepted 25 August 2005
Key words: miRNA, Oryza sativa, rasiRNA, small non-coding RNA, tncRNA
Abstract Small non-coding RNAs play important roles in regulating cell functions by controlling mRNA turnover and translational repression in eukaryotic cells. Here we isolated 162 endogenous small RNA molecules from Oryza sativa, which ranged from 16 to 35 nt in length. Further analysis indicated that they represented a diversity of small RNA molecules, including 17 microRNAs (miRNAs), 30 tiny non-coding RNAs (tncRNAs) and 20 repeat-associated small interfering RNAs (rasiRNAs). Among 17 miRNAs, 13 were novel miRNA candidates and their potential targets were important regulatory genes in the rice genome. We also found that a cluster of small RNAs, including many rasiRNAs, matched to a nuclear DNA fragment that evolutionarily derived from chloroplast. These results demonstrate clearly the existence of distinct types of small RNAs in rice and further suggest that small RNAs may control gene regulation through diverse mechanisms. Introduction Small non-coding RNAs control eukaryotic gene expression by degrading mRNA, repressing translation, modifying chromatin, and defending against viruses and transposons through homologous interaction with target sequences (Finnegan & Matzke, 2003). The small RNAs mainly include siRNAs (short interfering RNAs) (Elbashir, Lendeckel & Tuschl, 2001), miRNAs (microRNAs) (Lagos-Quintana et al., 2001; Lau et al., 2001; Lee & Ambros, 2001), tncRNAs (tiny non-coding RNAs) (Ambros et al., 2003b), rasiRNAs (repeatassociated small interfering RNAs) (Aravin et al., 2003) and smRNAs (small modulatory dsRNAs) (Kuwabara et al., 2004). miRNAs are an regulatory RNAs about 22 nt in length, produced by Dicer-mediated cleavage of a single strand precursor RNA with hairpin structure (Ambros et al., 2003a). Previous studies suggest that miRNA genes represent nearly 1% of
predicted genes in the genomes of humans, nematodes and Drosophila (Bartel, 2004). A few miRNAs have been found to play roles in the growth and development of animals and plants (Olson & Ambros, 1999; Reinhart et al., 2000; Ambros, 2003c; Bartel & Bartel, 2003; Carrington & Ambros, 2003; Baker et al., 2005; Kidner & Martienssen, 2005). siRNAs are RNAs (20–30 nt) that are processed from complementary double-stranded RNAs. They are functionally linked to RNA silencing in animals (Fire et al., 1998), plants (Napoli, Lemieux & Jorgensen, 1990; van der Krol et al., 1990), and fungus (Romano & Macino, 1992). rasiRNAs are siRNAs that match to repetitive sequence elements including transposable elements, satellite and microsatellite repeats, stellate repeats and centromere heterochromatic repeats (Llave et al., 2002; Dykxhoorn & Lieberman, 2005), and are involved in modifying genome, rearranging chromatin structure and repressing transposons (Okamoto & Hirochika,
22 2001; Mochizuki et al., 2002). tncRNAs are small non-coding RNAs, similar in length to miRNA, but are not processed from miRNA-like hairpin precursors and are not phylogenetically conserved (Ambros et al., 2003b). In plants, there are at least four RNA silencing pathways based on the biogenesis and functions of small RNAs: the first pathway is cytoplasm siRNA silencing, which links to the degradation of exogenous RNA such as viruses or sense transgenic transcripts; the second pathway is miRNA dependent endogenous mRNA regulation; the third pathway is siRNA guided DNA methylation and transcription suppression (Baulcombe, 2004); and the last pathway is siRNA mediated endogenous mRNA degradation (Vazquez et al., 2004). These pathways involve diverse small RNA molecules. miRNAs have been identified from model plants Arabidopsis and Oryza sativa both by direct cDNA cloning (Park et al., 2002; Reinhart et al., 2002; Sunkar & Zhu, 2004; Wang et al., 2004a; Sunkar et al., 2005) and by computational predication and molecular verification (Bonnet et al., 2004; Wang et al., 2004b; Adai et al., 2005; Li, Li & Jin, 2005). Other short RNA species, like siRNAs, were also cloned from Arabidopsis (Llave et al., 2002; Gustafson et al., 2005). As a first step toward further understanding the roles of small RNAs in plant development, we sought to identify the small RNA populations from rice. To date, we have cloned and performed bioinformatics analysis of 162 endogenous small RNAs from rice leaves and anthers, suggesting that rice may employ diverse small RNAs to regulate gene expression. Our study should provide insights into further understanding of functional genomic elements in rice.
Materials and methods Plant materials The rice variety of 9311 (Oryza sativa L. ssp. indica) was cultivated in the experimental field on Wuhan University campus in summer 2003. Panicles after heading were harvested and placed in a light incubator for blossoming. Anthers (including pollen grains) were collected in mortar on ice by shaking the panicles and ground in liquid nitrogen, and then total RNA was extracted using TRIzol
reagent (Invitrogen). Total RNAs were similarly prepared from other rice tissues, including leaves, roots and stems.
Small RNAs isolation and analysis Small RNAs were isolated by RNA/DNA Midi Kit (Qiagen) according to the manufacturer’s instructions. Small RNAs gel purification and cloning were performed following the methods of Llave et al. (2002): Briefly, small RNAs were separated on a denaturing 15% polyacrylamide gel. A band between 18 and 32 nt (Decade Maker system; Ambio) was excised and then eluted into 0.3 M NaCl overnight at 4C. Those RNAs were dephosphorylated (30 ll reaction, 37C, 30 min, 10 U alkaline phosphatase; Fermentas). Then 3¢ adapter (pUUUaaccgcatccttctcx: uppercase, RNA; lowercase, DNA; p, phosphate; x, 4-hydroxymethylbenzyl; Dharmacon Research) was ligated to the dephosphorylated small RNAs (20 ll reaction, 37C 30 min, 2 lM 3¢ adapter, 25 U T4 RNA ligase; Fermentas). The ligation products were separated from 15% gel and 5¢ phosphorylated (20 ll reaction, 37C, 30 min, 10 U T4 polynucleotide kinase; Fermentas), and then the 5¢ adapter (tactaatacgactcactAAA: uppercase, RNA; lowercase, DNA; Dharmacon Research) was ligated as described above. The newly ligated products were gel purified and eluted from the gel slice, then reverse transcribed (15 ll reaction, 42C, 30 min, 200 U Superscript II reverse transcriptase; Invitrogen) with primer (GACTAGCTGGAATTCAAGGATGCGGTTAAA: bold, EcoRI site), and amplified using a 5¢ primer (CAGCCAACGGAATTCATACGACTCACTAAA: bold, EcoRI site) and the reverse transcription primer. The PCR products were purified by phenol/chloroform extraction and ethanol precipitation, and then digested with EcoRI (Takara) and concatamerized using T4 DNA ligase (Takara). Concatamers of a size ranging from 200 to 800 nt were separated on 3% agarose gel and recovered with DNA Extraction Kit (Fermentas). The recovered products were cloned into pGEM-T vector (Promega). Positive clones were screened by PCR with M13–48 (5AGCGGATAACAATTTCACACAG-3) and M13+47 (5-CGCCAGGGTTTTCCCAGTCAC3) sequencing primers and submitted for custom sequencing (United Gene Holdings, LTD, China).
23 Each of small RNA sequences was searched via BLAST against the NCBI database, indica (9311) database (http://rise.genomics.org.cn/rice2/index. jsp) and Oryza repeat database in TIGR (http:// tigrblast.tigr.org/euk-blast/index.cgi?project=osa1). Small RNA precursor structure was predicted with mfold (Zuker, 2003). Putative target sequences were further identified by BLASTing Rice Pseudomolecules database in TIGR, and rice cDNA and EST libraries in Oryzabase (http: // www.shigen.nig.ac.jp/rice/oryz abase/blast/blast. jsp). Northern blot analysis Northern blot was performed following the methods of Llave et al. (2002) and Park et al. (2002) with some modifications. Briefly, 50 lg total RNA was separated on a 15% polyacrylamide gel containing 8 M urea. RNA was electroblotted to Nytran Supercharge membranes (Schleicher & Schuell) using a Trans-Blot Electrophoretic Transfer cell (BioRad) and fixed to the membrane by baking in oven at 80C for 90 min. Antisense DNA oligonucleotides were end-labeled with c-32P-ATP using T4 polynucleotide kinase (Fermentas). Membranes were prehybridized using 6 SSPE/0.5% SDS/5 Denhardt’s reagent, hybridized using 7% SDS/0.25 M sodium phosphate, and washed in 6 SSPE/0.1% SDS buffer. DNA oligonucleotide probes used in Northern blot analysis are as follows: m1, 5-GGCAA TTATCCTAACCGTTGG-3; m29, 5-GCGCCG TAGCGCCTGGTATGA-3; m65, 5-ACGAATC CGTGCGACGCGGGG-3; m111, 5-TCCCG
ATCTGCACCAAGCGA-3; m174, 5-TTTT TAATCAATGTTTGACCACTC-3; m182, 5-ATGTGACCCCTTCTAGATTGATGT-3; 5S rRNA, 5-ACTACTCTCGCCCAAGCACG-3.
Results and discussion Small RNA populations in rice Using the methods of Llave et al. (2002), we obtained 169 clones that encoded potential endogenous small RNAs from rice anthers (114 clones) and leaves (55 clones). 162 unique sequences ranging from 16-35 nt in length were identified by DNA sequencing (Figure 1(a)). The majority of these small RNAs are 21–24 nt long, which are similar to those reported in Arabidopsis (Llave et al., 2002; Tang et al., 2003), suggesting that small RNAs in rice may play similar roles in posttranscriptional silencing regulation, systemic silencing or methylation of homologous DNA (Hamilton et al., 2002). siRNAs ranging from 15–29 nt can directly degrade target mRNAs, and the efficiency varies with their size (Caplen et al., 2001; Elbashir, Lendeckel & Tuschl, 2001; Martinez et al., 2002), implying that the newly identified small RNAs may be associated with various functions and targeting efficiency in rice. Comparison of these small RNA sequences to the genome sequences of rice (indica and japonica) revealed that 129 (79.6%) of the small RNA sequences completely matched to rice genomic DNA. These 129 targets included 47 known noncoding RNAs (10 tRNAs, 34 rRNAs, 1 snRNA
Figure 1. Small RNA populations in Oryza sativa. (a) Size distribution of small RNA fragments in rice. nt, nucleotides. (b) Three overlapped small RNAs. They were different in L (length, nt), but derived from the same precursor and each class shared the same base in 5¢ end.
24 and 2 snoRNAs), which were discarded for further analysis. In addition, 31 (19.2%) contained one or more internal mismatches (8 with one mismatches, 7 with two mismatches, 16 with even more mismatches). The remaining two small RNAs (1.2%) matched to sequences from other plant chloroplasts: m19 related to chloroplast Ile-tRNA-2 in spinach and maize; m188 matched to tobacco chloroplast tRNA-Trp. We chose to further analyze a subset of the small RNA sequences that perfectly matched to or had single-mismatch similarity to rice genome sequences and did not match to known non-coding RNAs in rice (82 perfect matching, 8 single mismatching) (Supplementary material). Among these 90 small RNAs, 44 matched to single locus, other 46 each exhibited multiple hits with a number of targets; collectively, they hit 535 loci of rice genome. Furthermore, 31 out of 90 small RNAs were similar or identical to the sense strands of matched cDNA, and some of those might not be the degradation products of mRNA; for example, m111 which was identified as miR168 matched to two cDNA (AK065610 and AK063802). There was only one fragment (m88) that matched to cDNA in antisense, which represented endogenous siRNA. Based on our analysis, these small RNAs represented 17 miRNAs, 20 rasiRNA and 30 tncRNAs with various sizes and possibly functions. Such a diversity of short RNAs in plants was consistent with the multiplicity of Dicer-like enzymes (Matzke et al., 2004). There are at least four Dicer-like genes in Arabidopsis (Tang et al., 2003), and each controls biogenesis of different types of small RNAs (Xie et al., 2004). Notably, we found there were seven Dicer-like genes in rice (our unpublished data), which may be partially responsible for the broad size distributions of small RNAs in rice. Additionally, some small RNAs of different lengths overlapped and shared the same base in 5¢ end (Figure 1(b)). m24, for example, represented seven overlapping fragments that all had uridine in 5¢ end. Those fragments could be classified into two classes: the short class (17–21 nt) and the long class (27–29 nt), the long ones overlapped with the short ones partially. It seemed that the shorter ones were not derived from the longer ones; they represented two different classes of small RNAs. It is possible that the length variation for each class resulted from subtle alterations in Dicer structure (Hannon, 2002).
miRNAs in Oryza sativa One important criterion for miRNA is that its precursor folds into hairpin structure and it displays phylogenetic conservation of sequences. We analyzed miRNA precursors up to 600 nt for stem-loop structures using mfold (Zuker, 2003). Out of 90 small RNAs, 17 were predicted as miRNA candidates according to their precursor structures (Figure 2(a) and Table 1). Four of them matched to previously validated families of miRNAs (miR156, miR167, miR168 and miR169). Five of these miRNAs were detected by Northern blot analysis (Figure 2(b)), whereas others were undetectable possibly due to low expression. Nonetheless, isolation of known miRNAs validated our experimental approaches. Lengths of precursors and miRNAs appeared to be more variable in plants than that in animals. Our results and others indicated that precursors are 60–720 nt in length and the sizes of miRNAs ranged from 18 to 32 nt in plants (Park et al., 2002; Reinhart et al., 2002; Sunkar & Zhu, 2004; Wang et al., 2004a; Sunkar et al., 2005). Whereas in animals, precursors are usually 60–80 nt long and the lengths of miRNAs is 21–24 nt (Ambros et al., 2003a). Those may be attributed to the presence of multiple Dicer-like proteins in plants. In contrast, there are only one or two Dicer proteins in animals (Matzke et al., 2004). Moreover, five small RNA fragments (m1, m29, m65, m69 and m240) were similar to Group II miRNAs since they corresponded to non-coding RNAs from other plants (Table 2) (Park et al., 2002). Northern blotting showed that m1, m29 and m65 had larger transcripts and expressed only in anthers (Figure 2(b)). The other 13 miRNA candidates matched to one or multiple genomic loci. Four of these (m2, m15, m38 and m150) hit a single locus; the rest nine each matched to multiple loci. The matched loci are located either in coding regions or in the c Figure 2. 13 candidate miRNAs from Oryza sativa. (a) Secondary structures of thirteen miRNAs from Oryza sativa. RNA structures predicted with mfold version 3.1 (Zuker, 2003). The miRNA sequences were underlined. The actual size of precursors may be longer or shorter than it represented. (b) Expression of miRNAs. Northern blots of total RNA isolated from root (R), stem (S), leaf (L) and anther (A) in Oryza sativa, probed for the indicated miRNA. tRNA and 5S rRNA were used as loading control.
25
UCCCGGCCCCGAACCCGU-18
CAGCCAAGGAUGACUUGCCGG-21
UGACAACGAGAGAGAGCACGC-21
UCGCUUGGUGCAGAUCGGGAC-21
UGAAGCUGCCAGCAUGAUCUGG-22
GCGUUUGUAGUCCAACGGUUAGGAUAAUUGCC-32
CACGAGGCUCAUACCAGGCGCUACGGCGC-29
CAGCCCCGCGUCGCACGGAUUCGU-24
UAAGGUAGCGGCGAGACGAGCCGUUU-26
CCGCCGCGUGCCGGCCGGGGGACGGACCGGG-31
m243
m44
m61
m111
m221
m1
m29
m65
m69
m240
Precursor
100
120
100
110
120
224
300
110
100
110
100
210 180
170
60
120
70
75
length
5¢
5¢
3¢
3¢
3¢
5¢
5¢
5¢
5¢
3¢
3¢ 3¢
5¢
3¢
3¢
5¢
5¢
arm
Foldback
miRNA
Group II
MIR167
MIR168
MIR157
MIR156
MIR169
family
miRNA
21 (1, 6, 7)
1 (10)
11 (3, 6, 7, 11)
10 (1, 2, 3, 3, 5)
1 (7)
2 (2, 6)
1 (2)
2 (11)
3 (4)
22 (1, 3, 6, 7)
5 (1, 3, 4) 1 (5)
1 (2)
1 (5)
1 (8)
19 (1, 3)
1 (9)
(Chra)
loci
dR26864,a 1088 nt dR181532
2071 nt uR40831, 1089 nt
611 nt upstream of OSJNAb0075K12.32
uR181531, 117 nt dR222561
987 nt uR76800, 117 nt uR26863, 117 nt
dR283762 4813 nt uR102311 Extron
Exon, 4841 nt uR102311, 1441 nt
83 nt uR129218
Exon in AP003635, exon in P0576F08.29
Non-coding transcript
1709 nt dR175078
Exon in AP005071,
1.4kb downstream SJNBa0060N03.1
none, 1165 nt dR26864 1164 nt dR181532
2008 nt uR40831,
Repeat65600, 86 nt dR87605, R347243 800 nt upstream of OSJNBa0017O06.7
Repeat147962 (antisense)
32 nt dRc309724
515 nt uR149495
599 nt uRb408321, 2496 nt uR76800
OsJRFA063980 and Repeat159180
Distance to the nearest gene
a: chr, chromosome; b: uR, upstream of Repeat; c: dR, downstream of Repeat. Lengths and secondary structures of precursors are similar for each miRNA family. Some loci were not known on chromosome by then nt, nucleotide.
AAUCAAUCUAAUAGGGGAUGUGAC-24
GAGUGGUCAAACAUUGAUUAAAAA-24 ACAUCAAUCUAGAAGGGGUCACAU-24
m150
m174 m182
CCCUGUCCGGCGCCGUCGCGCCC-23
CGGUGUUAGUUCUAGGGCGAA-21
m15
m38
AACUCUUCUCCUUUGCGACCGCGG-24
CCGUCGUCGGCGCAGCCGGU-20
m2
Sequences (5¢-3¢)
m14
name
Clone
Table 1. Sequences and genomic locations of 17 miRNAs from rice
26
27 Table 2. Five small RNAs corresponded to non-coding RNAs from other plants Clone name
Expression in other organism (full matching)
m1
Arabidopsis thaliana tRNA-Gly; Phaseolus vulgaris tRNA-Gly.
m29
Zea mays chloroplast rRNA operon; Triticum aestivum chloroplast DNA; Oryza sativa chloroplast; Maize chloroplast 4.5S and 23S rDNA.
m65
Panicum miliaceum 26S and 18S rRNA genes; Miscanthus transmorrisonensis
m69
Oryza sativa chloroplast; Zea mays, Poa pratensis, Bambusa multiplex, Triticum aestivum,
m240
Zea mays 25S rRNA gene.
17S and 25S rRNA genes; Zea mays 25S rRNA gene. C. communis and H.vulgare chloroplast; Zea mays strain NB mitochondrion.
intergenic regions. The flanking sequences of each locus were predicted with hairpin structure, representing a precursor of miRNA. Six miRNA candidates (m1, m2, m15, m38, m69 and m150) had only one precursor; the remaining seven had multiple precursors (Table 1). And the lengths and structures of precursors were conserved for each miRNA family. Five miRNA candidates (m2, m29, m69, m150 and m174) were homogenous to repeat sequences, implying that these miRNAs are possibly originated from the repeat sequences. m240 and m243 located on the two arms of the same hairpin structure, but they are likely differentially processed from the same precursor because they were different in length. Plant miRNAs are partly complementary to their potential target mRNAs (Rhoades et al., 2002). Either 5¢ or 3¢ end of miRNA is important for the target recognition and each miRNA could have multiple target loci (Brennecke et al., 2005). To predict potential targets of the miRNAs identified in this study, various databases for rice EST, cDNA and Pseudomolecules_CDS were searched. Our search allowed four mismatches with one gap and treated G:U pair as a match. When there was no predictable target for this criterion, then the best match was regarded as a putative target. Our analysis predicted 91 potential targets for 13 miRNA candidates (Table 3). The target sites were similar to those of other miRNAs in plants, which are located in ORF or UTR of mRNA. m243 had 35 fully complementary targets expressed almost in all the organisms. Four miRNAs (m14, m29, m65 and m243) were completely complementary to stress induced mRNA, indicating that those small RNAs may play a role in response to environmental stress. Interesting,
m150 transcribed in an antisense orientation to repeat147962, which was a LINE type retrotransposon. This is similar to an imprinted miRNA in mice that is expressed only maternally (Seitz et al., 2003), implying that miRNA participates in epigenetic control in rice. Repeat-associated small interfering RNAs (rasiRNAs) We used 90 small RNA fragments to BLAST against the 9311 database, NCBI Oryza sativa and the TIGR Oryza repeat databases, which include transposable elements, centromere related, telomere-related, rDNA and unclassified repetitive sequences. Results of the analysis showed that ten of the small RNAs matched to repetitive rDNA, eight matched to transposable elements sequences and two matched to simple repeat sequences (Table 4), suggesting that small RNAs arose more frequently from repetitive genome sequences in plants (Xie et al., 2004). The rDNA repetitive sequences that carry these rasiRNAs do not transcribe nuclear rRNA. In addition, the biogenesis of these rasiRNAs may be attributed to the activities of Dice-like 3, RNA-dependent RNA polymerase (RDR) 2 and HUA ENHANCER (HEN) 1 (Xie et al., 2004), implying that the small RNA derived from rDNA region are not the random degraded fragments of rRNA. Furthermore, we found that in the rice genome small RNAs were almost all near the repeat regions (Supplementary material); most of them were within a distance of 1000 nt from the repeat regions. Very few were 10000 nt away from the repeat regions; for example, m114 was 12647 nt downstream from Repeat233384, the farthest
28 Table 3. Potential regulatory targets of 13 miRNA candidates from Oryza sativa miRNANumber of miRNA target gene names (target region) clone
mismatching
m2
3 3, 4
9633.m02478; 9637.m01990; 9640.m00409: retrotransposon protein (ORF)
m14
0
AU164494: root mRNA
0
CA999190 S234N_B05; CA999119 S234N_E11: cold stress germination mRNA
m15
AK071508: hypothetical protein (ORFa)
0
BI798725 H114H03: Endosperm library mRNA
1
9636.m00396; 9636.m00393; hypothetical protein
3 3
9639.m00043; 9638.m03892; 9631.m00311: hypothetical protein AK068749: squamosa promoter-binding protein-like (ORF)
3
AK109758; AK100120: unknown (ORF)
m38
3
AK109960: possible SMC family protein (ORF)
4
AK069498: magnesium transporter CorA-like family protein
m150
4
AK067799: unknown (3¢ UTRb)
5
AK066571: unknown (ORF)
5, 6
AK100960: histon H3 protein (5¢ UTR)
m174
2 1, 3
AK066289: probable methionine aminopeptidase (3¢ UTR) AK109584; AK067569; AK069712: unknown (ORF)
m182
2
AK102332: putative RING zinc finger protein (3¢ UTR)
m243
2
9629.t06414: Fructose-bisphosphate aldolase class-I
0
AU198164; AU163193; AU163189; AU163153; AU162823; AU161696; AU161686; AU108436: root mRNA
0
AU197324; AU197323; AU197321; AU197319; AU197312; AU197311; AU164224; AU164199: panicle mRNA
0
AU181820; AU163306; AU163283; AU163258; AU161421; AU161314; AU100913; AU100865; AU100857;
0
AU092547; AU092546; AU069574; AU069574; AU069292; AU069197; AU069063; AU068721: callus mRNA BU673137: Drought stress
0
BI806990: stem mRNA
m1
8
AK104148; AK099038: putative Ubiquitin carrier protein UBC7
m29
0
BI306579; BI305844: Drought stress (leaf) mRNA sequence
0
BE230013; BE229926: Seedling Lambda ZAPII cDNA Library mRNA sequence
m65
0
BI305486: Drought stress (leaf) Oryza sativa cDNA clone mRNA sequence
0
AU069186: callus mRNA
m69 m240
0 0
CR286781 and CR291901: mRNA AU197527; AU163193; AU163136; AU163133; AU108436: root mRNA
0
AU197323; AU197321; AU197319; AU197312; AU197311; AU164224; AU164199: panicle mRNA
0
AU181820; AU100913; AU100865; AU092546; AU069574; AU069292; AU069175; AU069093; AU068721; AU067920: callus mRNA
a: ORF, open reading frame; b: UTR, untranslated region. Table 4. rasiRNAs and their derived repeat sequences Transposon class I
Transposon class II
m2 (Copia-like);
m42 (Stowaway-like);
m72 (CTCA)n;
m29; m32; m36;
m100 (Gypsy-like); m114 (unclassified);
m101 (TPA); m174 (MITE-adh or Tourist-like).
m209 (TTA)n.
m69; m144; m208; m215; m217; m242;
m133 (retroelement); m150 (LINE).
Simple repeat
rDNA repeat sequences
m245.
29
Figure 3. Cluster of small RNAs. Thirteen small RNA fragments scattered on 5894 nt genomic sequences and located mainly in chromosome X, IV, III, XII, and VIII. Numbers (nt) above the line indicated distances between two fragments.
Table 5. Thirty tncRNAs in Oryza sativa Clone Small RNA sequences 5¢-3¢
Length Clone Chromosome
name
(nt)
times
Genomic location
Organ
Anther
m16
CCACGGAGGAAUGUUCUA
18
1
6
4797 nt uRa9998
m17
GCGUCUGUAGUCCAACGG
18
1
4
138 nt dRb285544 Anther
m22
UUUAUUUUUUGUGGGGGAGGGCC
23
1
2 and 7
NRRc
m27
AUCAAGGCUGAGGCGUGAUGACGA
24
1
6
-
Anther
m31
UGGAGGAAAUUAAAGGGUCCG
21
1
1
389 nt dR47424
Anther
m34
UAGGGUGAAGCCAGAGGA
18
1
Unknown
-
m39
GUCUGGGUGGUGUAGUUGG
19
1
1, 3, 4, 8, 9, 11 and 12 NRR
Anther, leaf
m41 m49
AAUAGAGCGGUGCUAUUGCGG CAAGUUGCGUAGUUGGGCCGG
21 21
1 1
11 3
127 nt uR340997 657 nt dR287556
Anther Anther
4
1549 nt uR322963
m54
ACUACCAAAGAAGGAGAAGAACUA
24
1
12
284 nt uR203859
Anther
m79
CUAGGUCAUGAGAGCGAACGUGG
23
1
1, 2, 3, 4, 5, 6 and 11
NRR
Anther
m80
UUCUGUAACAGAAGGACAUGA
21
1
11
811 nt dR303964
Anther
m93
CUGUCGAGUGUUUUUUGAGAGGAA 24
1
12
49 nt uR10158
Anther
m114
CUACGCAAAAGAGAAGGUUGCAAC
24
1
2, 6, 8, 9 and 12
NRR
Anther
m117 m137
AAACCGUUUAGGAUGCCAGAUUUU UCAAAUCGUGGUAGUCAUAAAG
24 22
2 1
3 6
1247 nt dR99301 Anther 3615 nt dR163961 Anther
m140
UGGGAGGAGUGAUAGACGCGA
21
1
2
413 nt dR239899
Anther
m165
CUACUUCAGAUUGAACAGACU
21
1
11
589 nt dR188556
Anther
m167
UAUAUAGGUUCGGGCCAGUGAGA
23
1
3 and 7
23 nt uR104202
Anther
Anther
Anther
566 nt uR213466 m171
CGACGAGUCCGAGGAGGAGAAGC
23
1
3
52 nt dR108241
Anther
m189
CGCCAAAUAUAGGGGAGAUCGACC
24
1
1
822 nt dR328779
Anther
m192 m193
CUCAGUACGGUUCACAUGUGGGC AGAAUUGGUGAGUGACGCAAGA
23 22
1 1
10 11
NRR
Anther Anther
m205
AUCGACAAGGCGGCUGGGAGUGGG
24
1
7
270 nt dR61005
Anther
m206
CCCACUCGAUUUUGCAGCGAGU
22
1
1 and 10
NRR
Anther
m210
AGAUAAGGAGUCACUUGUGAGAGG
24
1
3
717 nt dR289210
Leaf
m232
AAGGUGGUGUUGGAUGCCUGGU
22
1
10
762 nt dR36390
Anther
m248
CUGCUAUUUCAAUGUGGAGGAU
22
1
1
781 nt uR290470
Leaf
m258
CGCCGCGCUGCAACGGCCUGCGGG
24
1
1 and 6
649 nt uR40831
Leaf
m259
AGACUCGUUGAGAAGAUGAGGAGG
24
1
9
222 nt dR95314
Leaf
a: uR, upstream of repeat; b: dR, downstream of repeat; c: NRR, near repeat region.
away from a repeat region. Thus our finding suggested that the small RNAs likely resulted in readthrough transcription from the promoter of flanking repeat sequences. Interestingly, we found most of these rasiRNAs that matched to rRNA-like repetitive sequences
were clustered on a 5894 nt nuclear DNA fragment (Figure 3), but this region was not annotated as rRNA in rice nuclear genome. This fragment resulted from chloroplast DNA fragments laterally transferred into nucleus (Yuan et al., 2002). It is likely that the chloroplast fragments were active in
30 the nucleus of rice, but they were negatively suppressed by the nuclear-encoded rasiRNAs. Tiny non-coding RNA We have also identified a larger portion of unique small RNAs that were structurally different from miRNAs but similar in length. Of the 90 small RNAs were isolated in this study, 30 fragments ranged from 18 to 24 nt in length. They locate in non-coding regions of rice genome, and do not meet the criterion for miRNAs (Table 5). Those small RNAs may represent the so-called tncRNAs in rice (Ambros et al., 2003b). Supplementary material is available to authorised users in the online version of this article at http:// dx.doi.org/10.1007/s10709-005-2486-0
Acknowledgements We thank Drs. Xueyan Duan and Xin-Hua Feng for their critical reading the manuscript. The work was supported by a grant from the State Project of 973 (2001CB108806).
References Adai, A., C. Johnson, S. Mlotshwa, S. Archer-Evans, V. Manocha, V. Vance & V. Sundaresan, 2005. Computational prediction of miRNAs in Arabidopsis thaliana. Genome Res. 15(1): 78–91. Ambros, V., B. Bartel, D. Bartel, C. Burge, J. Carrington, X. Chen, G. Dreyfuss, S. Eddy, S. Griffiths-Jones, M. Marshall, M. Matzke, G. Ruvkun & T. Tuschl, 2003a. A uniform system for microRNA annotation. RNA 9: 277– 279. Ambros, V., R. Lee, A. Lavanway, P. Williams & D. Jewell, 2003b. MicroRNAs and other tiny endogenous RNAs in C. elegans. Curr. Biol. 13: 807–818. Ambros, V., 2003c. MicroRNA pathways in flies and worms: growth, death, fat, stress, and timing. Cell 113: 673–676. Aravin, A., M. Lagos-Quintana, A. Yalcin, M. Zavolan, D. Marks, B. Snyder, T. Gaasterland, J. Meyer & T. Tuschl, 2003. The small RNA profile during Drosophila development. Dev. Cell 5: 337–350. Baker, C.C., P. Sieber, F. Wellmer & E.M. Meyerowitz, 2005. The early extra petals mutant uncovers a role for microRNA miR164c in regulating petal number in Arabidopsis. Curr. Biol. 15(4): 303–315. Bartel, B. & D. Bartel, 2003. MicroRNAs: at the root of plant development. Plant Physiol. 132: 709–717.
Bartel, D., 2004. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116: 281–297. Baulcombe, D., 2004. RNA silencing in plants. Nature 431: 356–362. Bonnet, E., J. Wuyts, P. Rouze & Y. Van de Peer, 2004. Detection of 91 potential conserved plant microRNAs in Arabidopsis thaliana and Oryza sativa identifies important target genes. Proc. Natl. Acad. Sci. USA. 101(31): 11511– 11516. Brennecke, J., A. Stark, R. Russell & S.M. Cohen, 2005. Principles of MicroRNA–Target Recognition. PLoS Biol. 3(3): e85. Caplen, N.J., S. Parrish, F. Imani, A. Fire & R.A. Morgan, 2001. Specific inhibition of gene expression by small doublestranded RNAs in invertebrate and vertebrate systems. Proc. Natl. Acad. Sci. USA. 98: 9742–9747. Carrington, J. & V. Ambros, 2003. Role of microRNAs in plant and animal development. Science 301: 336–338. Dykxhoorn, D.M. & J. Lieberman, 2005. The silent revolution: RNA interference as basic biology, research tool, and therapeutic. Ann. Rev. Med. 56: 401–423. Elbashir, S., W. Lendeckel & T. Tuschl, 2001. RNA interference is mediated by 21-and 22-nucleotide RNAs. Genes Dev. 15: 188–200. Finnegan, E.J. & M.A. Matzke, 2003. The small RNA world. J. Cell Sci. 116: 4689–4693. Fire, A., S. Xu, M.K. Montgomery, S.A. Kostas, S.E. Driver & C.C. Mello, 1998. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391: 806–811. Gustafson, A.M., E. Allen, S. Givan, D. Smith, J.C. Carrington & K.D. Kasschau, 2005. ASRP: the Arabidopsis Small RNA Project Database. Nucleic Acids Res. 33 (Database issue): D637–640. Hannon, G.J., 2002. RNA interference. Nature 418: 244–251. Hamilton, A., O. Voinnet, L. Chappell & D. Baulcombe, 2002. Two classes of short interfering RNA in RNA silencing. EMBO J. 21: 4671–4679. Kidner, C.A. & R.A. Martienssen, 2005. The developmental role of microRNA in plants. Curr Opin Plant Biol. 8(1): 38–44. Kuwabara, T., J. Hsieh, K. Nakashima, K. Taira & F.H. Gage, 2004. A small modulatory dsRNA specifies the fate of adult neural stem cells. Cell 116: 779–793. Lagos-Quintana, M., R. Rauhut, W. Lendeckel & T. Tuschl, 2001. Identification of novel genes coding for small expressed RNAs. Science 294: 853–858. Lau, N., L. Lim, E. Weinstein & D. Bartel, 2001. An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 294: 858–862. Lee, R. & V. Ambros, 2001. An extensive class of small RNAs in Caenorhabditis elegans. Science 294: 862–864. Li, Y., W. Li & Y.X. Jin, 2005. Computational identification of novel family members of microRNA genes in Arabidopsis thaliana and Oryza sativa. Acta Biochim. Biophys. Sin. (Shanghai). 37(2): 75–87. Llave, C., K. Kasschau, M. Rector & J. Carrington, 2002. Endogenous and silencing-associated small RNAs in plants. Plant Cell 14: 1605–1619.
31 Martinez, J., A. Patkaniowska, H. Urlaub, R. Luhrmann & T. Tuschl, 2002. Single-stranded antisense siRNAs guide target RNA cleavage in RNAi. Cell 110: 563–574. Matzke, M., W. Aufsatz, T. Kanno, L. Daxinger, I. Papp, M.F. Mette & A.J.M. Matzke, 2004. Genetic analysis of RNA-mediated transcriptional gene silencing. Biochimica et Biophysica Acta 1677: 129–141. Mochizuki, K., N.A. Fine, T. Fujisawa & M. Gorovsky, 2002. Analysis of a piwi-related gene implicates small RNAs in genome rearrangement in Tetrahymena. Cell 110: 689–699. Napoli, C., C. Lemieux & R.A. Jorgensen, 1990. Introduction of a chimeric chalcone synthase gene into Petunia results in reversible co-suppression of homologous genes in trans. Plant Cell 2: 279–289. Okamoto, H. & H. Hirochika, 2001. Silencing of transposable elements in plants. TRENDS in Plant Sci. 6: 527–534. Olson, P. & V. Ambros, 1999. The lin-4 regulator RNA controls developmental timing in Caenorhabditis elegans by blocking LIN-14 protein synthesis after the initiation of translation. Dev. Biol. 216: 671–680. Park, W., J. Li, R. Song, J. Messing & X. Chen, 2002. CARPEL FACTORY, a Dicer homolog, and HEN1, a novel protein, act in microRNA metabolism. Curr. Biol. 12: 1484–1495. Reinhart, B.J., F.J. Slack, M. Basson, J.C. Bettinger, A.E. Pasquinelli, A.E. Rougvie, H.R. Horvitz & G. Ruvkun, 2000. The 21 nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 403: 901–906. Reinhart, B., E. Weinstein, M. Rhoades, B. Bartel & D. Bartel, 2002. MicroRNAs in plants. Genes Dev. 16: 1616–1626. Rhoades, M., B. Reinhart, L. Lim, C. Burge, B. Bartel & D. Bartel, 2002. Prediction of plant microRNA targets. Cell 110: 513–520. Romano, N. & G. Macino, 1992. Quelling: transient inactivation of gene expression in Neurospora crassa by transformation with homologous sequences. Mol. Microbiol. 6: 3343–3353. Seitz, H., N. Youngson, S.P. Lin, S. Dalbert, M. Paulsen, J.P. Bachellerie, A.C. Ferguson-Smith & J. Cavaille, 2003. Imprinted microRNA genes transcribed antisense to a
reciprocally imprinted retrotransposon-like gene. Nature Genet. 34(3): 261–262. Sunkar, R. & J.K. Zhu, 2004. Novel and stress-regulated MicroRNAs and other small RNAs from Arabidopsis. Plant Cell 16: 2001–2019. Sunkar, R., T. Girke, P.K. Jain & J.K. Zhu, 2005. Cloning and characterization of microRNAs from rice. Plant Cell 17: 1397–1411. Tang, G., B. Reinhart, D. Bartel & P. Zamore, 2003. A biochemical framework for RNA silencing in plants. Genes Dev. 17: 49–63. Van der Krol, A.R., L.A. Mur, M. Beld, J.N. Mol & A.R. Stuitje, 1990. Flavonoid genes in Petunia: addition of a limited number of gene copies may lead to a suppression of gene expression. Plant Cell 2: 291–299. Vazquez, F, H. Vaucheret, R. Rajagopalan, C. Lepers, V. Gasciolli, A.C. Mallory, J.L. Hilbert, D.P. Bartel & P. Crete, 2004. Endogenous trans-acting siRNAs regulate the accumulation of Arabidopsis mRNAs. Mol. Cell 16(1): 69–79. Wang, J., H. Zhou, Y. Chen, Q. Luo & L. Qu, 2004a. Identification of 20 microRNAs from Oryza sativa. Nucleic Acids Res. 32: 1688–1695. Wang, X.J., J.L. Reyes, N.H. Chua & T. Gaasterland, 2004b. Prediction and identification of Arabidopsis thaliana microRNAs and their mRNA targets. Genome Biol. 5(9): R65. Xie, Z., L.K. Johansen, A.M. Gustafson, K.D. Kasschau, A.D. Lellis, D. Zilberman, S.E. Jacobsen & J.C. Carrington, 2004. Genetic and functional diversification of small RNA pathways in plants. PLoS biology 2: 642–652. Yuan, Q., J. Hill, J. Hsiao, K. Moffat, S. Ouyang, Z. Cheng, J. Jiang & C.R. Buell, 2002. Genome sequencing of a 239-kb region of rice chromosome 10L reveals a high frequency of gene duplication and a large chloroplast DNA insertion. Mol. Genet. Genom. 267: 713–720. Zuker, M., 2003. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31: 3406–3415.
