Professional Documents
Culture Documents
com
Abstract
MicroRNAs (miRNAs) are a class of non-coding RNAs that have important gene regulation roles in various organisms. To date, a total of 1279
plant miRNAs have been deposited in the miRNA miRBase database (Release 10.1). Many of them are conserved during the evolution of land
plants suggesting that the well-conserved miRNAs may also retain homologous target interactions. Recently, little is known about the
experimental or computational identification of conserved miRNAs and their target genes in tomato. Here, using a computational homology search
approach, 21 conserved miRNAs were detected in the Expressed Sequence Tags (EST) and Genomic Survey Sequence (GSS) databases.
Following this, 57 potential target genes were predicted by searching the mRNA database. Most of the target mRNAs appeared to be involved in
plant growth and development. Our findings verified that the well-conserved tomato miRNAs have retained homologous target interactions
amongst divergent plant species. Some miRNAs express diverse combinations in different cell types and have been shown to regulate cell-specific
target genes coordinately. We believe that the targeting propensity for genes in different biological processes can be explained largely by their
protein connectivity.
2008 Elsevier B.V. All rights reserved.
Keywords: Conserved microRNAs; Tomato; Target genes; MFEI; Homology
1. Introduction
MicroRNAs (miRNAs) are single-stranded non-coding
RNAs that ranging in length from 19 nucleotides (nt) to 25 nt
Abbreviations: 3 UTR, 3 untranslated region; 5RLM-RACE, 5 RNA
ligase mediated rapid amplification of cDNA ends; G, folding free energies;
AGO1, Argonaute-1; Ap2, APETALA2; ARF, auxin response transcription
factor; DCL1, Dicer-like protein; EREBPs, ethylene-responsive element binding
proteins; EST, expressed sequence tag; GH3, Grim helix 3; GSS, genomic
survey sequence; MFE, minimal folding free energy; MFEI, minimal folding
free energy index; miRNA, microRNA; miRNA, opposite miRNA sequence;
nt, nucleotide(s); pre-miRNA, microRNA precursor; pri-miRNAs, microRNA
primary; Pro, proline; RISC, RNA-induced silencing complex; SBP, Squamosa
promoter Binding Proteins; Ser, serine; SPL, SBP-like proteins; Thr, threonine.
Corresponding author. State Key Laboratory of Crop Biology, Shandong
Agricultural University, Shandong, 271018, PR China. Tel.: +86 538 8242903;
fax: +86 538 8242226.
E-mail address: ffshen@sdau.edu.cn (F. Shen).
0378-1119/$ - see front matter 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.gene.2008.02.007
61
62
Fig. 1. Schematic representation of the miRNA gene search procedure used to identify tomato homology to known miRNAs.
(http://www.ncbi.nlm.nih.gov/BLAST/) was used for the analysis of potential targets. The criteria were as follows: (a) four or
fewer mismatched nucleotides at complementary sites between
miRNA sequences and potential mRNA targets; (b) one mismatch in the complementary region of the miRNA at nucleotide
positions 212, but not at positions 10 or 11, which is a predicted cleavage site; and (c) up to three additional mismatches
between 12 nt and 23 nt but with no more than two continuous
mismatches within this region.
where MFE denotes the negative folding free energies (G); (d) the
miRNA has less than six mismatches with the opposite miRNA
sequence (miRNA*) on the other arm; (e) potential miRNAs are
located on the same arm of the stem-loop structure as their known
homologs; and (f) there is no large loop or break in the miRNA
sequence. Predicted miRNAs and their related information were
recorded. Closely related EST and GSS sequences were blasted
against each other and analyzed. If a high degree of similarity
(N 98%) was observed, the sequences were deemed to have been
created from the same sequences and considered as one miRNA.
2.3. Prediction of potential target genes and their functions
Fig. 2 summarizes the major steps employed in predicting potential target genes and their functions. BLASTX
Fig. 2. Schematic representation of the potential target genes search procedure by blasting mRNA databases of tomato with newly identified miRNA
sequences.
63
Table 1
List of the newly identified miRNAs in tomato
New miRNAs
miRNA sequences
Gene ID
Gene source
Location
NM (nt)
LM (nt)
LP (nt)
A+U (%)
G (kal/mol)
MFEIs
156a
156b
157a
157b
157c
159
160
162
167a
167b
168
169
172a
172b
399a
399b
403
437
830
869.1
1030
ugaaagauagagcagugagcac
ugacagaagagagagagcac
uugacagaagauagagagcac
uugacagaagauagagagcac
uugacagaagauagagagcac
uuuggauugaagggagcucua
ugccuggcuccuuguaugcca
ucgauaaaccucugcauccag
ugaagcugccagcaugaucua
uaaagcugccagcaugaucugg
ucgcuuggugcaggucgggac
Uagccaaaaaugacuugccag
agaaucuugaugaugcugcau
agaaucuugaugaugcugcau
ugccaaaggagaguugcccua
ugccaagggagaauugcccua
cuagauucacgcacaagcucg
aaaguuagagaaguuugaaau
ugacuauuaugagaagaagug
auugguuuaauuuugguguug
Aucugcaugugcaccugcacc
84260136
72526205
117704089
84273338
84236627
116645971
72471353
72276781
117695324
72348884
9505413
84234106
117691535
72400226
117723706
72479804
72312989
84192580
84049570
117684944
84291882
GSS
GSS
EST
GSS
GSS
EST
GSS
GSS
EST
GSS
EST
GSS
EST
GSS
EST
GSS
GSS
GSS
GSS
EST
GSS
5
5
5
5
5
3
5
3
5
5
5
5
3
3
3
3
3
5
3
5
5
3
0
0
0
0
0
0
0
0
2
1
3
0
0
0
1
2
3
3
2
2
22
20
21
21
21
21
21
21
21
22
21
21
21
21
21
21
21
21
21
21
21
85
355
100
84
83
178
80
98
73
237
145
68
106
135
71
72
76
54
115
61
139
60.00
49.56
64.00
60.71
61.45
62.92
53.75
53.06
64.38
75.53
45.52
51.47
66.04
72.59
57.75
58.33
60.52
83.33
71.30
63.93
58.27
22.56
95.70
43.40
39.10
40.20
73.61
33.30
33.70
27.30
60.90
59.80
27.50
39.80
74.80
30.24
31.30
28.90
8.30
22.50
15.40
35.40
0.71
0.70
1.20
1.18
1.26
1.11
0.90
0.73
1.05
1.05
0.76
0.83
1.11
2.02
1.03
1.04
0.96
1.30
0.74
0.70
0.73
NM: number of mismatch; LM: length of mature miRNAs; LP: length of precursor; G: folding free energies; MFEIs: minimal folding free energy indexes.
64
Table 2
Potential targets of the identified miRNAs in tomato
miRNA
Targeted protein
Target function
156/157
Squamosa promoter
binding protein
(SBP)/ SBPL
Tospovirus
resistance protein
Lateral suppressor
protein
AUX/IAA protein
160
167
168
172
869.1
DNA (cytosine-5)methyltransferase
Auxin-responsive
factor
Serine/threonine
protein kinase
Auxin-responsive
factor
Putative phosphatase
Sucrose transporter
mRNA binding
protein precursor
Ethylene-responsive
element binding
protein
Putative protease/
hydrolase
Phospholipase family
Insulin degrading
enzyme
Polyphenol oxidase
Eukaryotic translation
initiation factor
Threonine deaminase
Target genes
BE436147
BI925272
Auxin-responsive BP880486
factor
Transcription factor AW038480
Auxin-responsive
factor
BI925927 BP902363
BE434602 CD003107
DB715336 AW735838
AW223741
Transcription factor DB697961 DB706552
BF097936
Transcription factor BE461110
Transcription factor BE462736
Transcription factor BM413247 AI486618
BI208808 BM410833
BM408872
Transcription factor AI484737
Transcription factor BP893968 BI935838
Metabolism
BG627220
Metabolism
AI774952
Transcription factor BE436427 BE432648
Transcription factor BI935333 BI9353116
BI933914 BI933284
BI933270 BI932869
BI932857 BI935963
BG134948 BM413156
BG127250 BG129951
BG126331 BG126303
BG123821
Transcription factor BG626979
Ethylene-responsive
nuclear protein
P69C protein
Transcription factor BI932424
Subtilisin-like protease Metabolism
BG421482 BI935794
NBS-LRR resistance Transcription factor BG132455 BM410377
protein-like protein
degradation of ARF8 but not ARF6 (Ru et al., 2006); this was
explained by the fact that the fewer base-pairs between ARF6 and
miR167 may result in inefficient cleavage of the ARF6 transcripts.
Mallory et al. (2005) suggested that miR160 and miR167
coordinately modulate GH3-like mRNA expression by regulating
expression of repressing and activating ARF proteins encoded by
ARF17 and ARF8. Thus, in the future, it will be fascinating to
experimentally determine the subtle correlations among miR160,
miR167and ARFs.
Ethylene-responsive element binding proteins (EREBPs) and
APETALA2 (AP2) are prototypic members of a plant-specific
family of AP2/EREBP transcription factors (Nole-Wilson and
Krizek, 2000; Riechmann and Meyerowitz, 1998; Theissen and
Saedler, 1999). Recent studies using A. thaliana show that
miR172 and its AP2-Like target genes regulate flowering time and
floral organ identity (Aukerman and Sakai, 2003; Chen, 2004). In
addition, Glossy15, an AP2-like gene from maize that regulates
leaf epidermal cell identity, is also detected as a target of miR172
(Lauter et al., 2005). In this research, five predicted targets of
miR172 were found to be members of the AP2 gene family (Table
2). Aukerman and Sakai (2003) suggested that the progressive
accumulation of miR172 results in the complete absence of AP2
and other AP2-like proteins; as a consequence, plants set flowers
early with disrupted specification of floral organ identity.
Recently, another study demonstrated that flower development
is regulated by at least four miRNAs: miR156, miR159, miR164
and miR172 (Gandikota et al., 2007). In fact, miR156 and miR172
are expressed in opposite temporal patterns, suggesting that the
miR156 module may regulate the expression of miR172 (Willmann and Poethig, 2007). In addition, Nilsson et al. (2007)
suggested that the AP2 gene in spruce has the capacity to
substitute an A class gene required in flower development.
Additional studies are now required to determine the basis for this
regulation and the complicated regulating network.
As the above data suggest, the molecular identities of the
miRNAs and their targets seem to have remained constant, providing additional evidence for the real existence of these miRNAs.
Presently, however, it is not possible to determine whether this
conservation is the result of shared ancestry or functional convergence from an independent origin during evolution.
Different combinations of miRNAs are expressed in different
cell types and may regulate cell-specific target genes coordinately (Cimmino et al., 2005). For example, Vaucheret et al.
(2004) confirmed that AGO1 is controlled by miR168 in
A. thaliana. However, in this study, the predicted targets of
miR168 did not encode AGO family proteins, but rather
putative phosphatase and sucrose transporter (Table 2). Maybe,
it is some small changes in the temporal, spatial or environmental regulation of these modules over long periods of time
that result in large different effects in developmental processes
of other species. Moreover, among the detected targets of
miR156/157, miR160, miR167, miR172 and miR869.1, there
were some atypical target genes that have yet to be validated by
an experimental approach. Interestingly, serine/threonine protein kinase was among the targeted proteins of miR160.
Combined with the fact that transcriptional repressor ARFs all
have Pro-Ser-Thr-rich middle regions (Tiwari et al., 2004), this
65
66
Carrington, J.C., Ambros, V., 2003. Role of microRNAs in plant and animal
development. Science 301, 336338.
Chen, X., 2004. A microRNA as a translational repressor of APETALA2 in
Arabidopsis flower development. Science 303, 20222025.
Chen, X., 2005. MicroRNA biogenesis and function in plants. FEBS Lett. 579,
59235931.
Cimmino, A., et al., 2005. miR-15 and miR-16 induce apoptosis by targeting
BCL2. Proc. Natl. Acad. Sci. U. S. A 102, 1394413949.
Dharmasiri, S., Estelle, M., 2002. The role of regulated protein degradation in
auxin response. Plant Mol. Biol 49, 401409.
Fahlgren, N., et al., 2007. High-throughput sequencing of Arabidopsis
microRNAs: evidence for frequent birth and death of MIRNA genes.
PLoS. ONE. 2, e219.
Gandikota, M., Birkenbihl, R.P., Hohmann, S., Cardon, G.H., Saedler, H.,
Huijser, P., 2007. The miRNA156/157 recognition element in the 3' UTR of
the Arabidopsis SBP box gene SPL3 prevents early flowering by translational inhibition in seedlings. Plant J. 49, 683693.
Gleave, A.P., et al., 2008. Identification and characterisation of primary
microRNAs from apple (Malus domestica cv. Royal Gala) expressed
sequence tags. Tree Genetics & Genomes 4, 343358.
Guilfoyle, T.J., Ulmasov, T., Hagen, G., 1998. The ARF family of transcription
factors and their role in plant hormone-responsive transcription. Cell. Mol.
Life Sci 54, 619627.
Guo, H.S., Xie, Q., Fei, J.F., Chua, N.H., 2005. MicroRNA directs mRNA
cleavage of the transcription factor NAC1 to downregulate auxin signals for
Arabidopsis lateral root development. Plant Cell 17, 13761386.
Ioshikhes, I., Roy, S., Sen, C.K., 2007. Algorithms for mapping of mRNA
targets for microRNA. DNA Cell Biol. 26, 265272.
Itaya, A., et al., 2008. Small RNAs in tomato fruit and leaf development.
Biochim. Biophys. Acta. 1779, 99107.
Jones-Rhoades, M.W., Bartel, D.P., 2004. Computational identification of plant
microRNAs and their targets, including a stress-induced miRNA. Mol. Cell
14, 787799.
Kurihara, Y., Watanabe, Y., 2004. Arabidopsis micro-RNA biogenesis through
Dicer-like 1 protein functions. Proc. Natl. Acad. Sci. U. S. A 101, 1275312758.
Lai, E.C., Tomancak, P., Williams, R.W., Rubin, G.M., 2003. Computational
identification of Drosophila microRNA genes. Genome Biol. 4, R42.
Lall, S., et al., 2006. A genome-wide map of conserved microRNA targets in
C. elegans. Curr. Biol. 16, 460471.
Laufs, P., Peaucelle, A., Morin, H., Traas, J., 2004. MicroRNA regulation of the
CUC genes is required for boundary size control in Arabidopsis meristems.
Development 131, 43114322.
Lauter, N., Kampani, A., Carlson, S., Goebel, M., Moose, S.P., 2005. microRNA172
down-regulates glossy15 to promote vegetative phase change in maize. Proc.
Natl. Acad. Sci. U. S. A 102, 94129417.
Leonardi, C., Ambrosino, P., Esposito, F., Fogliano, V., 2000. Antioxidative
activity and carotenoid and tomatine contents in different typologies of fresh
consumption tomatoes. J. Agric. Food Chem. 48, 47234727.
Li, Y., Li, W., Jin, Y.X., 2005. Computational identification of novel family
members of microRNA genes in Arabidopsis thaliana and Oryza sativa.
Acta Biochim. Biophys. Sin. (Shanghai) 37, 7587.
Liang, C.Y., et al., 2004. Construction of a BAC library of Physcomitrella
patens and isolation of a LEA gene. Plant Sci. 167, 491498.
Lin, S.L., Chang, D., Ying, S.Y., 2005. Asymmetry of intronic pre-miRNA
structures in functional RISC assembly. Gene 356, 3238.
Liscum, E., Reed, J.W., 2002. Genetics of Aux/IAA and ARF action in plant
growth and development. Plant Mol. Biol 49, 387400.
Mallory, A.C., Bartel, D.P., Bartel, B., 2005. MicroRNA-directed regulation of
Arabidopsis AUXIN RESPONSE FACTOR17 is essential for proper development and modulates expression of early auxin response genes. Plant Cell
17, 13601375.
Mallory, A.C., Dugas, D.V., Bartel, D.P., Bartel, B., 2004. MicroRNA regulation
of NAC-domain targets is required for proper formation and separation of
adjacent embryonic, vegetative, and floral organs. Curr. Biol. 14,
10351046.
Megraw, M., Baev, V., Rusinov, V., Jensen, S.T., Kalantidis, K., Hatzigeorgiou,
A.G., 2006. MicroRNA promoter element discovery in Arabidopsis. RNA.
12, 16121619.
Nilsson, L., Carlsbecker, A., Sundas-Larsson, A., Vahala, T., 2007. APETALA2
like genes from Picea abies show functional similarities to their Arabidopsis
homologues. Planta 225, 589602.
Nole-Wilson, S., Krizek, B.A., 2000. DNA binding properties of the Arabidopsis floral development protein AINTEGUMENTA. Nucleic Acids Res.
28, 40764082.
Pan, X., Zhang, B., San, F.M., Cobb, G.P., 2007. Characterizing viral
microRNAs and its application on identifying new microRNAs in viruses.
J. Cell Physiol. 211, 1018.
Park, M.Y., Wu, G., Gonzalez-Sulser, A., Vaucheret, H., Poethig, R.S., 2005.
Nuclear processing and export of microRNAs in Arabidopsis. Proc. Natl.
Acad. Sci. U. S. A 102, 36913696.
Pilcher, R.L., et al., 2007. Identification of novel small RNAs in tomato
(Solanum lycopersicum). Planta 226, 709717.
Qiu, C.X., et al., 2007. Computational identification of microRNAs and their
targets in Gossypium hirsutum expressed sequence tags. Gene 395, 4961.
Rhoades, M.W., Reinhart, B.J., Lim, L.P., Burge, C.B., Bartel, B., Bartel, D.P.,
2002. Prediction of plant microRNA targets. Cell 110, 513520.
Riechmann, J.L., Meyerowitz, E.M., 1998. The AP2/EREBP family of plant
transcription factors. Biol. Chem. 379, 633646.
Riese, M., Hohmann, S., Saedler, H., Munster, T., Huijser, P., 2007. Comparative analysis of the SBP-box gene families in P. patens and seed plants.
Gene 401, 2837.
Ru, P., Xu, L., Ma, H., Huang, H., 2006. Plant fertility defects induced by the
enhanced expression of microRNA167. Cell Res. 16, 457465.
Schwab, R., Palatnik, J.F., Riester, M., Schommer, C., Schmid, M., Weigel, D.,
2005. Specific effects of microRNAs on the plant transcriptome. Dev. Cell 8,
517527.
Sunkar, R., Zhu, J.K., 2004. Novel and stress-regulated microRNAs and other
small RNAs from Arabidopsis. Plant Cell 16, 20012019.
Theissen, G., Saedler, H., 1999. The golden decade of molecular floral development (19901999): A cheerful obituary. Dev Genet. 25, 181193.
Tiwari, S.B., Hagen, G., Guilfoyle, T.J., 2004. Aux/IAA proteins contain a
potent transcriptional repression domain. Plant Cell 16, 533543.
Vaucheret, H., Vazquez, F., Crete, P., Bartel, D.P., 2004. The action of
ARGONAUTE1 in the miRNA pathway and its regulation by the miRNA
pathway are crucial for plant development. Genes Dev. 18, 11871197.
Wang, J.W., Wang, L.J., Mao, Y.B., Cai, W.J., Xue, H.W., Chen, X.Y., 2005.
Control of root cap formation by MicroRNA-targeted auxin response factors
in Arabidopsis. Plant Cell 17, 22042216.
Willmann, M.R., Poethig, R.S., 2007. Conservation and evolution of miRNA
regulatory programs in plant development. Curr. Opin. Plant Biol. 10, 503511.
Wu, G., Poethig, R.S., 2006. Temporal regulation of shoot development in
Arabidopsis thaliana by miR156 and its target SPL3. Development 133,
35393547.
Wu, M.F., Tian, Q., Reed, J.W., 2006. Arabidopsis microRNA167 controls
patterns of ARF6 and ARF8 expression, and regulates both female and male
reproduction. Development 133, 42114218.
Zhang, B.H., Pan, X.P., Wang, Q.L., Cobb, G.P., Anderson, T.A., 2005.
Identification and characterization of new plant microRNAs using EST
analysis. Cell Res. 15, 336360.
Zhang, B.H., Pan, X.P., Cannon, C.H., Cobb, G.P., Anderson, T.A., 2006a.
Conservation and divergence of plant microRNA genes. Plant J. 46, 243259.
Zhang, B.H., Pan, X.P., Cobb, G.P., Anderson, T.A., 2006b. Plant microRNA: a
small regulatory molecule with big impact. Dev. Biol. 289, 316.
Zhang, B.H., Pan, X.P., Cox, S.B., Cobb, G.P., Anderson, T.A., 2006c. Evidence
that miRNAs are different from other RNAs. Cell Mol. Life Sci. 63,
246254.
Zhang, B.H., Pan, X.P., Wang, Q.L., Cobb, G.P., Anderson, T.A., 2006d.
Computational identification of microRNAs and their targets. Comput. Biol.
Chem. 30, 395407.
Zhang, B.H., et al., 2007. Identification of cotton microRNAs and their targets.
Gene 397, 2637.
Zhang, Y., 2005. miRU: an automated plant miRNA target prediction server.
Nucleic Acids Res. 33, W701W704.