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DEAD-Box RNA Helicases Are Among the Onstituents of the Tobacco

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DEAD-Box RNA Helicases Are Among the Onstituents of the Tobacco iochemis t B try n & la P P h f y o Hafidh et al., J Plant Biochem Physiol 2013, 1:3 s l i Journal of o a l n o DOI: 10.4172/2329-9029.1000114 r g u y o J ISSN: 2329-9029 Plant Biochemistry & Physiology Short Communication OpenOpen Access Access DEAD-Box RNA Helicases are among the Constituents of the Tobacco Pollen mRNA Storing Bodies Said Hafidh1, David Potěšil2,3, Zbyněk Zdráhal2,3 and David Honys1* 1Laboratory of Pollen Biology, Institute of Experimental Botany ASCR, Rozvojova 263, 165 02 Praha 6, Czech Republic 2CEITEC - Central European Institute of Technology, Masaryk University, Brno, Czech Republic 3National centre for Biomolecular Research, Faculty of Science, Masaryk University, Brno, Czech Republic Keywords: Translation; mRNA storage; RNA helicase; mRNP; Results and Discussion Ribonucleoprotein; Pollen Of the complex EPP proteome, we have identified three isoforms Introduction of the DEAD-box RNA helicase family eukaryotic translation initiation factor 4A (eIF4A)-8,9,13 as designated components of the EPP Male gametogenesis in flowering plants is attributed by RNA granules. eIF4A is required for binding of capped mRNA to substantial changes in cellular reorganization, cell fate specification the 40S ribosomal subunit via eIF3, whilst its ATP-dependent helicase and changes in cellular metabolism including RNA metabolism. activity functions in unwinding the secondary structure of the 5’ UTR Sequestration from immediate translation of protein coding mRNAs to facilitate ribosomal binding and translation. In synapses, eIF4A is has emerged as one aspect of mRNA post transcriptional regulation a target of smRNA (BC1) that blocks the eIF4A helicase activity and that is widespread during gametogenesis. Transcripts repressed from impose subsequent widespread translation repression [7]. We have translation are confiscated into mRNA storing bodies and function extended our analysis to tobacco Agilent microarray data [4,5] to search later during development and at early stages of embryogenesis. In for other helicases that could potentially be associated with EPP mRNA Arabidopsis, 88% and 12% of the zygotic transcriptome at 2-4 cells storing granules. Additional 49 helicases (including all three isoforms stage of development now known to originate from maternal and of eIF4A identified by orbitrap LC-MS/MS) were identified and were paternal gametes respectively [1]. Moreover, an activator of YODA expressed as late as 24 h of pollen tube growth (Table 1 and Figure 1A). (YDA) mitogen-activated protein kinase pathway in the zygote, the For majority, their transcripts were also detected in EPP RNA granules interleukin-1 receptor-associated kinase (IRAK)/Pelle-like kinase gene [5]. Analysis of their expression profiles show differential abundance but Short Suspensor (SSP) is specifically expressed in the Arabidopsis no apparent specificity in the male gametophyte (Figure 1A). Structural analysis classified majority as a DEAD-box motif helicases of SF2 male gametophyte but only translated upon gamete fusion to promote super family with two RecA-like domains and conserved C-terminal suspensor development [2]. Previously we have reported the proteome domain (Figure 1B and 1C). These helicases could potentially function of EPP complexes [3], the tobacco pollen large mRNPs granules alongside EPP-bound eIF4A-8,9,13. Among them, EMB2733/ESP3, a containing translationally sequestered mRNAs, non-coding smRNAs homologue of yeast PRP2 and related to the DEAH RNA helicases, is and a repertoire of associated protein complexes including factors for essential for embryogenesis and is extensively phosphorylated [8]. The initiation of translation [4]. Here, we have analysed our previous and role of these helicases in promoting gametogenesis and embryogenesis new EPP proteome data set obtained from orbitrap LC-MS/MS as well development is worth an investigation. as tobacco pollen Agilent microarray data [3-5] and highlighted that RNA helicases, predominantly DEAD-box domain containing helicases RNA helicases of the DEAD-box and the related DExD/H protein are among the constituents of the EPP RNA granules. Their potential families are involved in almost all aspects of RNA metabolism and function tied to local translation is explored. In addition, we discuss the contain characteristic twelve conserved sequence motifs (Figure 1C). The identified tobacco eIF4A isoforms show 70% identity with recent report of RNA helicases localization within peroxisomes bodies S. cerevisiae eIF4A (data not shown). Solved structural features of S. and speculate their likely role as an additional site for mRNA control cerevisiae eIF4A, domains of ATP binding, hydrolysis, RNA binding in the male gametophyte alongside EPP mRNA storing granules. Thus, and their configurations, are proposed to convey conformational mRNA homeostasis in the male gametophyte might be subjected to changes of bound transcripts as well as mRNP protein complexes multiple controls at diverse but likely interconnected cytoplasmic foci. including reshuffling of bound proteins to specify transcript fates EPP complexes were isolated as described previously [3]. New (Figure 1C and 1D). These activities could be associated with mRNP EPP proteome dataset was obtained by Orbitrap LC-MS/MS. Agilent localization once exported from the nucleus and most likely during microarray data were from Hafidh et al. [4,5]. Phylogenetic tree EPP complex assembly. Within domain II, the three motifs, motif IV- construction was performed with MUSCLE 3.7 and ClustalW 2.0.3 multiple protein alignment programs, Gblocks 0.91b for alignment curation, PhyML 3.0 aLRT with bootstrap for reconstructing *Corresponding author: David Honys, Laboratory of Pollen Biology, Institute of Experimental Botany ASCR, Rozvojova 263, 165 02 Praha 6, Czech Republic, Tel: phylogenetic tree and TreeDyn 198.3 for tree visualization. Final tree +420-225-106-450; Fax: +420-225-106-456; E-mail: [email protected] was manually curated in Inkscape software ver. 0.48.Protein domains and families were analysed according to SMART and pfam annotation. Received March 23, 2013; Accepted July 28, 2013; Published August 02, 2013 Arabidopsis pollen grains expressing pLAT52::CFP::PTS1 [6] in a qrt/ Citation: Hafidh S, Potěšil D, Zdráhal Z, Honys D (2013) DEAD-Box RNA qrt background were visualized with NIKON TE2000-E fluorescence Helicases are among the Constituents of the Tobacco Pollen mRNA Storing Bodies. J Plant Biochem Physiol 1: 114. doi:10.4172/2329-9029.1000114 microscope (Nikon, Japan) under Hoffman modulation contrast. Images were captured with ProgRes C5 camera (JENOPTIK Laser Copyright: © 2013 HafidhS, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted optic system, Germany) using NIS-Elements AR3.0 software (Nikon use, distribution, and reproduction in any medium, provided the original author and instruments, Melville, USA) and edited in Adobe Photoshop CS6. source are credited. J Plant Biochem Physiol ISSN: 2329-9029 JPBP, an open access journal Volume 1 • Issue 3 • 1000114 Citation: Hafidh S, Potěšil D, Zdráhal Z, Honys D (2013) DEAD-Box RNA Helicases are among the Constituents of the Tobacco Pollen mRNA Storing Bodies. J Plant Biochem Physiol 1: 114. doi:10.4172/2329-9029.1000114 Page 2 of 4 B DEAD2 50 HNH 40 A TIP49 30 MP PT4 PT24 Leaf Root 20 X79141 AAA CVO19592 # accessions DM003772 10 TA12960 DUF4371 TA14523 0 X79138 Families IBR S1 AF426837 MIT HSA R3H HA2 dsrm DEAD X79136 zf-CW F-BOX HIRAN DSHCT F5402411 Sec63 zf-RING2 zf-C3HC4 DV158454 rRNA_proc. E8440239 SNF2 Helicase_C_2 DV157904 OB_NTP_bind DV162553 DV159284 Helicase C DV161544 EB445607 EB8977889 0 10 20 30 40 50 TA20284 E8437962 Accessions TA16281 BP528577 DV158080 BP535495 Helicase core X79004 C DW002104 BP528980 Domain I Domain II BP129511 BP129511 EB431244 AF261026 Q-motif motif I Ia Ib Ic motif II motif III motif IV IVa motif V Va motif VI EB680135 BP131143 N -(23)-F -(16)-GFEEPSAIQ -(16)- AQSGTGKT-(25)- PTRELA -(42)- GG TPGRD -(19)- DEAD-(27)-SAT -(60)-VIFCNTRR HGQR -(45)- TDLLARGID RGDVV -(20)- HRIGRGGR -(42)- C BP137156 BP526162 BP130750 BP136507 AF260022 BQ842998 Domain I BP526804 E AT5G05120 AT5G50410 BP528617 AT5G05130 D AT3G09720 AT4G34910 BP527063 A54G60990 EB440204 AT5G07810 EB444389 CV019324 ATIG48310 AB120527 BP192642 PIE1 STRS1RH30 TA19989 CHR19/ETL1 AT2G47330 AF260025 Q-motif AT2G42520 BP165920 ATRAD54 AT5G63630 AT5G63630 BP135421 AT2G07750 DV161560 BP133020 EB682791 eIF4A BP130153 eIF4A-2eIF4A-1 EB427557 AT3G19760 BP128972 AT5G11200 EMB25 AT4G00660 EB428617 LOS4 ATRAD54 BP135576 AT4G09730 EMB1586 BP135198 AT3G46960 BP134707 AT1G59760 EB678943 Domain II AT1G27880 BP131085 AT5G61140 DV161529 BP529103 EMB1507 RecQ13 EB442655 RECQS1M BP129516 CV020205 FG639414 0.1 FG141341 ATSUV3 FG141341 CV507122 DV003354 CV016160 BP133061 BP532124 BP525724 F BP531978 CFP merge DV004445 BP525307 EB452141 i ii EB452141 BP532651 EH623377 EB433852 DV159964 DV159964 EB443347 FQ535434 X79135 BP131999 DV004917 BP135665 DV000669 CFP-PTS1 BP131435 EB28245 min max SPN Figure 1: Potential role of helicases and peroxisomes in RNA metabolism during male gametogenesis. (A) Heatmap of RNA helicases from log2 transformed microarray dataset at three time points of tobacco pollen development - mature pollen (MP), 4 h (PT4) and 24 h (PT24) in vitro cultivated pollen tubes, leaves (LF) and roots (RT). (B) Family and domain distribution of the RNA helicases expressed in tobacco pollen tube. Inset show domain distribution.
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