ISSN 1021�4437, Russian Journal of Plant Physiology, 2014, Vol. 61, No. 6, pp. 807–810. © Pleiades Publishing, Ltd., 2014.Original Russian Text © A.A. Bari, A.I. Sagaidak, I.V. Pinskii, S.B. Orazova, A.T. Ivashchenko, 2014, published in Fiziologiya Rastenii, 2014, Vol. 61, No. 6, pp. 850–854.

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INTRODUCTION

Plant microRNAs (miRNAs) play an important roleby regulating the expression of genes that determine theprocesses related to growth, development, stressresponse, etc. [1–5]. The action of some miRNAs onthe expression of genes encoding the plant transcriptionfactors was established [6–9]. The increased concentra�tions of miR396 suppressed the synthesis of growth�reg�ulating factors (GRF), whereas the decreased expres�sion of miR396 stimulated the expression of these genes[10, 11]. The action of miRNAs on the expression ofgenes encoding transcription factors in the genomes ofmany plants was studied insufficiently [12, 13]. There�fore, it was important to reveal miRNAs that bind tomRNA of GRF genes playing a key role in plant growthand development.

MATERIALS AND METHODS

Nucleotide sequences of GRF genes of Arabidopsisthaliana, Glycine max, Hordeum vulgare, Medicagotruncatula, Oryza sativa, Sorghum bicolor, Solanumlycopersicum, Triticum aestivum, Vitis vinifera, andZea mays were obtained from GenBank(http://www.ncbi.nlm.nih.gov/). Nucleotide sequencesof miRNAs of the genomes of studied plantswere obtained from the database miRBase(http://mirbase.org/). Since the current prediction

programs for miRNA acting on target genes of plantsreveal a lot of false binding sites of miRNAs tomRNAs, we have developed a program MirTarget thatlacks these shortcomings. It calculates the free energy(ΔG, kJ/mol) of miRNA binding, the relative value offree energy (ΔG/ΔGm, %), as well as the position andschemes of potential binding sites. The ΔGm for miRNAbinding was defined as the free energy of miRNA bind�ing to the fully complementary nucleotide sequence.The relative quantity of free energy (ΔG/ΔGm) was usedas a comparative criterion for evaluating the degree ofinteraction of miRNA and mRNA.

Nucleotide sequences of genes of the followingtranscription factors were used in this work:A. thaliana: AT2G22840, AT4G37740, AT2G36400,AT3G52910, AT5G53660, AT4G24150, AT2G45480;G. max: Gma001682, Gma011587, Gma021030,Gma021805, Gma033196, Gma035899, Gma045435,Gma005521, Gma011182, Gma013188, Gma018091,Gma019479, Gma022734, Gma024536, Gma025321,Gma042617, Gma042774, Gma046226; H. vulgare:Hvu003170, Hvu008248, Hvu011812; M. truncatula:Mtr003954, Mtr019402, Mtr028218, Mtr028986;O. sativa: Os06g0116200, Os02g0678800,Os02g0701300, Os02g0776900, Os03g0674700,Os04g0600900, Os03g0729500, Os11g0551900,Os12g0484900; S. bicolor: Sbi007115, Sbi009008,Sbi023539, Sbi031583, Sbi010080, Sbi018012,Sbi019465, Sbi025078; S. lycopersicum: Sly011359,Sly013222, Sly014377; T. aestivum: Tae005214,Tae003589, Tae007720; V. vinifera: Vvi000913,Vvi001038, Vvi006461, Vvi017355, Vvi032173,Vvi036239, Vvi037877, Vvi040456, Vvi044157,

RESEARCH PAPERS

Binding of miR396 to mRNA of Genes Encoding Growth�Regulating Transcription Factors of Plants

A. A. Bari, A. I. Sagaidak, I. V. Pinskii, S. B. Orazova, and A. T. IvashchenkoResearch Institute of Biology and Biotechnology Problems, prosp. Al�Farabi 71, Almaty, 050038 Kazakhstan;

e�mail: a_ivashchenko@mail.ruReceived December 20, 2013

Abstract—The sites of miR396 binding to mRNA of genes encoding growth�regulating transcription factors(GRF) were identified for Arabidopsis thaliana, Glycine max, Hordeum vulgare, Medicago truncatula, Oryzasativa, Sorghum bicolor, Solanum lycopersicum, Triticum aestivum, Vitis vinifera, and Zea mays. The freeenergy of interaction of miR396 with mRNA was calculated, and the schemes of nucleotide linking in thebinding sites were determined. In mRNA of GRF genes of all studied plants, miR396�binding sites werelocated in the protein�coding part and encoded oligopeptides RSRKPVE or RSRKHVE. The regulation ofthe expression of plant GRF genes by miR396 family is discussed.

Keywords: higher plants, miRNA, mRNA, miR396, transcription factor

DOI: 10.1134/S1021443714050033

Abbreviations: GRF—growth�regulating transcription factors;miRNA—micro RNA; ΔG—free energy of miRNA binding;ΔGm—free energy for binding of miRNA to its completely com�plementary nucleotide sequence; ΔG/ΔGm—relative value of thefree energy.

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Vvi002405, Vvi020113, Vvi035036, Vvi002507,Vvi004254, Vvi006110, Vvi018177, Vvi022350,Vvi042923; Z. mays: Zma000362, Zma002853,Zma002963, Zma003319, Zma004217, Zma004793,Zma013797, Zma013999, Zma015997, Zma023472,Zma024988, Zma027510, Zma027808, Zma040855,Zma033368, Zma035070, Zma042909, Zma050402,Zma050878, Zma051502, Zma051860, Zma052142,Zma052445, Zma054079, Zma058313, Zma058936,Zma007079, Zma007506, Zma011215, Zma011730,Zma030100, Zma030201, Zma030802, Zma031491,Zma031965, and Zma033300.

RESULTS AND DISCUSSION

Genomes of completely sequenced plants are yetinsufficiently annotated, and many of their miRNAsremain undetermined. The identified orthologousmiRNAs have highly homologous nucleotidesequences despite the many millions of years passedafter the divergence of plant species studied in thiswork. Particularly, the nucleotide sequences ofmiR396a of rice and A. thaliana are identical. Thisproperty of miRNA allows a reliable means to find thetarget genes for the supposed miRNAs in differentplant species.

We found that among 338 miRNAs of A. thaliana,only one of them, ath�miR396a,b was able to bindeffectively to GRF mRNA. Among nine GRF genes,mRNAs of genes GRF1–GRF4 and GRF7–GRF9 wereidentified as targets for miR396, these genes possessthe miRNA�binding sites characterized by theΔG/ΔGm ratio of 85.0 to 90.0% (table). In mRNA ofarabidopsis GRF genes, the binding sites were locatedin the protein�coding part; they featured the highhomology of nucleotide sequences and encoded hep�tapeptides RSRKPVE or RSRKHVE (figure).

We found that, among 661 miRNAs of rice, all ninemembers of osa�miR396a�i family bind with highspecificity to mRNA of nine growth�regulating factors(table). Based on the degree of binding to mRNA ofGRF genes, these miRNAs were distributed into fivegroups: osa�miR396a,b, osa�miR396c, osa�miR396d,e, osa�miR396f, and osa�miR396g,h,i. Therelative free energy of binding (ΔG/ΔGm) for mRNAsof six GRF rice genes ranged from 85.4 to 100%.Judging from the ratio ΔG/ΔGm, these genes werearranged in a row: osa�miR396a,b < osa�miR396c <osa�miR396d,e < osa�miR396f < osa�miR396g,h,i.The relative free energy of binding of osa�miR396a–ifamily members with mRNA of three GRF rice genesranged from 75.9 to 88.1%; the ΔG/ΔGm ratioincreased in the following order: osa�miR396f < osa�miR396d,e < osa�miR396a,b < osa�miR396g,h,i <osa�miR396c. The binding sites of osa�miR396a–i inmRNA of all GRF rice genes were located in the pro�tein�coding part; they were homologous and encodedthe heptapeptides RSRKPVE or RSRKHVE (figure).

All eight members of zma�miR396a–h family,which are present among 321 maize miRNAs, werefound to bind mRNA of 36 growth�regulating maizefactors with a high specificity (table). These miRNAsbind to various extents to mRNA of GRF genes. Theywere distributed into five groups: zma�miR396a,b,zma�miR396c, zma�miR396d,e, zma�miR396f, andzma�miR396g,h. The relative binding energy ofzma�miR396a–i family members with mRNA of16 GRF maize genes ranged from 82.1 to 100%; theΔG/ΔGm ratio increased in the following order:zma�miR396f < zma�miR396d,e < zma�miR396a,b <zma�miR396g,h < zma�miR396c. In the case ofmRNA of ten maize genes, the relative binding energyof the members of zma�miR396a–h family rangedfrom 80.9 to 88.8% (table); the ΔG/ΔGm ratioincreased in the same order. In all cases, the freeenergy of binding of the members of zma�miR396a–hfamily to mRNA of 36 GRF maize genes constitutedmore than 80.9% of the maximum free energy, whichis an indicator of their strong interaction. The dataobtained show that all paralogous GRF maize genesbind to all members of zma�miR396 family. Conse�quently, synthesis of all growth�regulating factors thatdetermine many traits of maize productivity is exclu�sively under the control of zma�miR396a–h familymembers. In mRNA of 36 genes encoding growth�regulating factors of maize, the binding sites of zma�miR396 family were located in the protein�codingpart; they were highly homologous and encoded theheptapeptides RSRKPVE or RSRKHVE (figure). Wefound that eight GRF maize genes listed in the interna�tional database of transcription factors are the iso�forms of products of the respective miRNA targetgenes. They have also zma�miR396�binding sites sim�ilar to the longest respective isoform.

According to miRBase database, only a fewmiRNAs were found in the genomes of wheat and bar�

Relative free energy of interaction of miR396 family withmRNA of GRF genes in various plant species

Species miRNA ΔG/ΔGm, %

A. thaliana ath�miR396a,b 85.2–90.4

G. max gma�miR396a–c,h,i 91.8–96.2

H. vulgare osa�miR396a–i 80.4–100

M. truncatula mtr�miR396a,b 92.0–96.1

O. sativa osa�miR396a–i 75.9–100

S. bicolor sbi�miR396a–e 87.0–96.4

S. lycopersicum osa�miR396a–i 83.0–100

T. aestivum osa�miR396a–i 80.8–100

V. vinifera vvi�miR396a–d 90.2–96.1

Z. mays zma�miR396a–h 80.0–100

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BINDING OF miR396 TO mRNA OF GENES ENCODING 809

ley. In the genome of wheat, tae�miR160,tae�miR1122, tae�miR156, tae�miR398, and tae�miR408 are known, which have the ΔG/ΔGm ratio ofbinding to mRNA of growth�regulating factors of lessthan 80%. In the genome of barley, hvu�miR6196,hvu�miR6180, hvu�miR5053, and hvu�miR181 areknown, whose ΔG/ΔGm ratio is also less than 80%. Weused nine rice miRNAs of osa�miR396a–i family toidentify the sites of binding of miRNAs to mRNAs ofgrowth�regulating factors of wheat and barley. Theratio ΔG/ΔGm of binding of osa�miR396a–i tomRNAs of Tae003589, Tae005214, and Tae007720growth�regulating factors of wheat ranged from 80.8 to100% (table). The ratio ΔG/ΔGm of binding ofosa�miR396a–i to mRNAs of Hvu003170,Hvu006219, and Hvu008248 growth�regulating fac�

tors of barley ranged from 80.4 to 100% (table).Hence, the expression of GRF genes of wheat and bar�ley, determining many traits of productivity of theseplants, is under control of the members of miR396family. In mRNAs of all genes encoding growth�regu�lating factors of wheat and barley, the miR396�bindingsites were located in the protein�coding part; they werehighly homologous and encoded heptapeptidesRSRKPVE or RSRKHVE (figure).

In the genome of V. vinifera, 18 GRF genes werefound. The mRNA of all these genes binds effectivelyto vvi�miR396a–d (table). According to the degree ofaffinity, GRF genes of V. vinifera were distributed inthree groups, for each of which a specific interval ofchanges in ΔG/ΔGm was found.

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The mtr�miR396a,b of M. truncatula were found tobind to mRNA of three GRF genes with ΔG/ΔGm ratioranging from 92.0 to 96.1% (table). Thus, the expres�sion of all GRF genes of M. truncatula can be sup�pressed very effectively by means of two miRNAs ofmiR396 family.

Eight GRF genes of S. bicolor were distributed inthree groups according to the degree of binding tosbi�miR396a–e (table). The ΔG/ΔGm ratios forinteraction of osa�miR396a–i family with mRNAs oftwo groups of GRF genes of S. bicolor were almostidentical, and they varied from 87.0 to 92.2% formRNA of Sbi025078 gene. The binding of mRNAencoded by Sbi025078 gene to sbi�miR396a–e did notexceed 80%. Thus, the expression of this transcriptionfactor is weakly regulated by miRNAs.

Since miRNAs of miR396 family have not yet beenfound in the genome of tomato, we used osa�miR396a–i of rice for our calculations. Only threeGRF genes are currently known in the genome ofS. lycopersicum (table). The ratio ΔG/ΔGm for interac�tion of mRNAs of tomato GRF genes withosa�miR396a–i ranged from 83.3 to 100%.

In the genome of G. max 18 GRF genes are known(table). Binding of mRNAs of these genes to gma�miR396a–c,h,i was characterized by the ratioΔG/ΔGm varying from 91.0 to 96.2%.

In mRNAs of GRF genes of the plant species exam�ined, all sites of binding to miR396 were located in theprotein�coding part; they were homologous andencoded heptapeptides RSRKPVE or RSRKHVE(figure). It should be noted that the binding of miRNAto the protein�coding region of the mRNA encoded bythe transcription factor genes is not accidental. Suchlocalization of miRNA�binding sites in mRNAs deter�mines a highly conserved relationship between them inthe process of plant evolution [14].

Analysis of interactions of miR396 with theorthologs of GRF in A. thaliana, G. max, M. truncatula,O. sativa, S. lycopersicum, S. bicolor, V. vinifera, andZ. mays indicates the conserved structure ofmiRNA�binding sites. The results of this study showthat miR396�binding sites are located within the cod�ing region of mRNA and that their nucleotidesequence is conservative in evolutionary distant spe�cies. A high ΔG/ΔGm ratio of miRNA binding tomRNA indicates that the expression of genes ofGRF family can be suppressed strongly by miR396.

ACKNOWLEDGMENTS

This work was supported by the Ministry of Educa�tion and Science of the Republic of Kazakhstan (grantno. 0143/GF).

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Translated by M. Shulskaya