Abstract
eIF4A is a RNA-stimulated ATPase and helicase. Besides its key role in regulating cap-dependent translation initiation in eukaryotes, it also performs specific functions in regulating cell cycle progression, plant growth and abiotic stress tolerance. Flowering plants encode three eIF4A paralogues, eIF4A1, eIF4A2 and eIF4A3 that share conserved sequence motifs but differ in functions. To date, however, no information is available on eIF4A in basal land plants. In this study we report that genome of the moss Physcomitrella patens encodes multiple eIF4A genes. The encoded proteins possess the highly conserved motifs characteristic of the DEAD box helicases. Spatial expression analysis shows these genes to be ubiquitously expressed in all tissue types with Pp3c6_1080V3.1 showing high expression in filamentous protonemata. Targeted deletion of conserved core motifs in Pp3c6_1080V3.1 slowed protonemata growth and resulted in dwarfing of leafy gametophores suggesting a role for Pp3c6_1080V3.1 in regulating cell division/elongation. Rapid and strong induction of Pp3c6_1080V3.1 under salt stress and slow recovery of knockout plants upon exposure to high salt further suggest Pp3c6_1080V3.1 to be involved in stress management in P. patens. Protein–protein interaction studies that show Pp3c6_1080V3.1 to interact with the Physcomitrella heterogenous ribonucleoprotein, LIF2L1, a transcriptional regulator of stress-responsive genes in Arabidopsis. The results presented in this study provide insight into evolutionary conserved functions of eIF4A and shed light on the novel link between eIF4A activities and stress mitigation pathways/RNA metabolic processes in P. patens.
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References
Andreou AZ, Klostermeier D (2013) The DEAD-box helicase eIF4A: paradigm or the odd one out? RNA Biol 10:19–32
Arya D, Kapoor S, Kapoor M (2016) Physcomitrella patens DNA methyltransferase 2 is required for recovery from salt and osmotic stress. FEBS J 283:556–570
Ashraf M, Harris PJC (2004) Potential biochemical indicators of salinity tolerance in plants. Plant Sci 166:3–16
Aubourg S, Kreis M, Lecharny A (1999) The DEAD box RNA helicase family in Arabidopsis thaliana. Nucl Acids Res 27:628–636
Ballut L, Marchadier B, Baguet A, Tomasetto C, Séraphin B, Le Hir H (2005) The exon junction core complex is locked onto RNA by inhibition of eIF4AIII ATPase activity. Nat Struct Mol Biol 12:861–869
Bush MS, Hutchins AP, Jones AM, Naldrett MJ, Jarmolowski A, Lloyd CW, Doonan JH (2009) Selective recruitment of proteins to 5′ cap complexes during the growth cycle in Arabidopsis. Plant J 59:400–412
Bush MS, Crowe N, Zheng T, Doonan JH (2015) The RNA helicase, eIF4A-1, is required for ovule development and cell size homeostasis in Arabidopsis. Plant J 84:989–1004
Bush MS, Pierrat O, Nibau C, Mikitova V, Zheng T, Corke FM, Vlachonasios K, Mayberry LK, Browning KS, Doonan JH (2016) eIF4A RNA helicase associates with cyclin-dependent protein kinase A in proliferating cells and is modulated by phosphorylation. Plant Physiol 172:128–140
Caruthers JM, Johnson ER, McKay DB (2000) Crystal structure of yeast initiation factor 4A, a DEAD-box RNA helicase. Proc Natl Acad Sci USA 97:13080–13085
Chan CC, Dostie J, Diem MD, Feng W, Mann M, Rappsilber J, Dreyfuss G (2004) eIF4A3 is a novel component of the exon junction complex. RNA 10:200–209
Charron AJ, Quatrano RS (2009) Between a rock and a dry place: the water-stressed moss. Mol. Plant 2:478–486
Conroy SC, Dever TE, Owens CL, Merrick WC (1990) Characterization of the 46,000-dalton subunit of eIF-4F. Arch Biochem Biophys 282:363–371
Dangwal M, Kapoor S, Kapoor M (2014) The PpCMT chromomethylase affects cell growth and interacts with the homolog of LIKE HETEROCHROMATIN PROTEIN 1 in the moss Physcomitrella patens. Plant J 77:589–603
Frank W, Decker EL, Reski R (2005) Molecular tools to study Physcomitrella patens. Plant Biol 7:220–227
Galicia-Vazquez G, Cencic R, Robert F, Agenor AQ, Pelletier J (2012) A cellular response linking eIF4A1 activity to eIF4AII transcription. RNA 18:1373–1384
Gorbalenya AE, Koonin EV (1993) Helicases: amino acid sequence comparisons and structure-function relationships. Curr Opin Struct Biol 3:419–429
Grifo JA, Abramson RD, Satler CA, Merrick WC (1984) RNA-stimulated ATPase activity of eukaryotic initiation factors. J Biol Chem 259:8648–8654
Hori K, Maruyama F, Fujisawa T et al (2014) Klebsormidium flaccidum genome reveals primary factors for plant terrestrial adaptation. Nat Commun 5:3978
Hutchins AP, Roberts GR, Lloyd CW, Doonan JH (2004) In vivo interaction between CDKA and eIF4A: a possible mechanism linking translation and cell proliferation. FEBS Lett 556:91–94
Jiang CJ, Shoji K, Matsuki R, Baba A, Inagaki N, Ban H, Iwasaki T, Imamoto N, Yoneda Y, Deng XW, Yamamoto N (2001) Molecular cloning of a novel importin alpha homologue from rice, by which constitutive photomorphogenic 1 (COP1) nuclear localization signal (NLS)-protein is preferentially nuclear imported. J Biol Chem 276:9322–9329
Koroleva OA, Calder G, Pendle AF, Kim SH, Lewandowska D, Simpson CG, Jones IM, Brown JW, Shaw PJ (2009a) Dynamic behavior of Arabidopsis eIF4A-III, putative core protein of exon junction complex: fast relocation to nucleolus and splicing speckles under hypoxia. Plant Cell 21:1592–1606
Koroleva OA, Brown JW, Shaw PJ (2009b) Localization of eIF4A-III in the nucleolus and splicing speckles is an indicator of plant stress. Plant Signal Behav 4:1148–1151
Latrasse D, Germann S, Houba-Hérin N, Dubois E, Bui-Prodhomme D, Hourcade D, Juul-Jensen T, Le Roux C, Majira A, Simoncello N, Granier F, Taconnat L, Renou JP, Gaudin V (2011) Control of flowering and cell fate by LIF2, an RNA binding partner of the polycomb complex component LHP1. PLoS One 6(1):e16592. https://doi.org/10.1371/journal.pone.0016592
Li Q, Imataka H, Morino S, Rogers GW Jr, Richter-Cook NJ, Merrick WC, Sonenberg N (1999) Eukaryotic translation initiation factor 4AIII (eIF4AIII) is functionally distinct from eIF4AI and eIF4AII. Mol Cell Biol 19:7336–7346
Linder P, Lasko PF, Ashburner M, Leroy P, Nielsen PJ, Nishi K, Schnier J, Slonimski PP (1989) Birth of the D-E-A-D box. Nature 337:121–122
Liu YC, Vidali L (2011) Efficient polyethylene glycol (PEG) mediated transformation of the moss Physcomitrella patens. J Vis Exp 19:2560
Lu WT, Wilczynska A, Smith E, Bushell M (2014) The diverse roles of the eIF4A family: you are the company you keep. Biochem Soc Trans 42:166–172
Manjulatha M, Sreevathsa R, Kumar AM, Sudhakar C, Prasad TG, Tuteja N, Udayakumar M (2014) Overexpression of a pea DNA helicase (PDH45) in peanut (Arachis hypogaea L.) confers improvement of cellular level tolerance and productivity under drought stress. Mol Biotechnol 56:111–125
Molitor AM, Latrasse D, Zytnicki M, Andrey P, Houba-Hérin N, Hachet M, Battail C, Del Prete S, Alberti A, Quesneville H, Gaudin V (2016) The Arabidopsis hnRNP-Q Protein LIF2 and the PRC1 subunit LHP1 function in concert to regulate the transcription of stress-responsive genes. Plant Cell 28:2197–2211
Nath M, Garg B, Sahoo RK, Tuteja N (2015) PDH45 overexpressing transgenic tobacco and rice plants provide salinity stress tolerance via less sodium accumulation. Plant Signal Behav 10:e992289
Nielsen PJ, Trachsel H (1988) The mouse protein synthesis initiation factor 4A gene family includes two related functional genes which are differentially expressed. EMBO J 7:2097–2105
Ortiz-Ramirez C, Hernandez-Coronado M, Thamm A, Bruno C, Wang M, Dolan L, Feijo JA, Becker JD (2016) A transcriptome atlas of Physcomitrella patens provides insights into the evolution and development of land plants. Mol Plant 9:205–220
Parihar V, Arya D, Walia A, Tyagi V, Dangwal M, Verma V, Khurana R, Boora N, Kapoor S, Kapoor M (2019) Functional characterization of LIKE HETROCHROMATIN PROTEIN 1 in the moss Physcomitrella patens: its conserved protein interactions in land plants. Plant J 97(2):221–239
Pause A, Sonenberg N (1992) Mutational analysis of a DEAD box RNA helicase: the mammalian translation initiation factor eIF-4A. EMBO J 11:2643–2654
Rao SRBT, Vijaya Naresh J, Sudhakar Reddy P, Reddy MK, Mallikarjuna G (2017) Expression of Pennisetum glaucum eukaryotic translational initiation factor 4A (PgeIF4A) confers improved drought, salinity, and oxidative stress tolerance in groundnut. Front Plant Sci 8:453
Ray BK, Lawson TG, Kramer JC, Cladaras MH, Grifo JA, Abramson RD, Merrick WC, Thach RE (1985) ATP-dependent unwinding of messenger RNA structure by eukaryotic initiation factors. J Biol Chem 260:7651–7658
Rhodes D, Hanson AD (1993) Quaternary ammonium and tertiary sulfonium compounds in higher plants. Annu Rev Plant Physiol Plant Mol Biol 44:357–384
Rogers GW Jr, Richter NJ, Merrick WC (1999) Biochemical and kinetic characterization of the RNA helicase activity of eukaryotic initiation factor 4A. J Biol Chem 274:12236–12244
Rozen F, Pelletier J, Trachsel H, Sonenberg N (1989) A lysine substitution in the ATP-binding site of eucaryotic initiation factor 4A abrogates nucleotide-binding activity. Mol Cell Biol 9:4061–4063
Rozen F, Edery I, Meerovitch K, Dever TE, Merrick WC, Sonenberg N (1990) Bidirectional RNA helicase activity of eucaryotic translation initiation factors 4A and 4F. Mol Cell Biol 10:1134–1144
Saavedra L, Svensson J, Carballo V, Izmendi D, Welin B, Vidal S (2006) A dehydrin gene in Physcomitrella patens is required for salt and osmotic stress tolerance. Plant J 45:237–249
Sahoo RK, Gill SS, Tuteja N (2012) Pea DNA helicase 45 promotes salinity stress tolerance in IR64 rice with improved yield. Plant Signal Behav 7:1042–1046
Sanan-Mishra N, Pham XH, Sopory SK, Tuteja N (2005) Pea DNA helicase 45 overexpression in tobacco confers high salinity tolerance without affecting yield. Proc Natl Acad Sci USA 102:509–514
Sarkar G, Edery I, Sonenberg N (1985) Photoaffinity labeling of the cap-binding protein complex with ATP/dATP. Differential labeling of free eukaryotic initiation factor 4A and the eukaryotic initiation factor 4A component of the cap-binding protein complex with [alpha-32P] ATP/dATP. J Biol Chem 260:13831–13837
Schmid SR, Linder P (1991) Translation initiation factor 4A from Saccharomyces cerevisiae: analysis of residues conserved in the D-E-A-D family of RNA helicases. Mol Cell Biol 11:3463–3471
Shibuya T, Tange TO, Sonenberg N, Moore MJ (2004) eIF4AIII binds spliced mRNA in the exon junction complex and is essential for nonsense-mediated decay. Nat Struct Mol Biol 11:346–351
Story RM, Li H, Abelson JN (2001) Crystal structure of a DEAD box protein from the hyperthermophile Methanococcus jannaschii. Proc Natl Acad Sci USA 98:1465–1470
Tanner NK, Cordin O, Banroques J, Doere M, Linder P (2003) The Q motif: a newly identified motif in DEAD box helicases may regulate ATP binding and hydrolysis. Mol Cell 11:127–138
Tuteja N, Vashisht AA, Tuteja R (2008) Translation initiation factor 4A: a prototype member of dead-box protein family. Physiol Mol Biol Plants 14:101–107
Tuteja N, Banu MS, Huda KM, Gill SS, Jain P, Pham XH, Tuteja R (2014) Pea p68, a DEAD-box helicase, provides salinity stress tolerance in transgenic tobacco by reducing oxidative stress and improving photosynthesis machinery. PLoS One 9(5):e98287
Van Breusegem F, Dat JF (2006) Reactive oxygen species in plant cell death. Plant Physiol 141(2):384–390
Vain P, Thole V, Worland B, Opanowicz M, Bush MS, Doonan JH (2011) A T-DNA mutation in the RNA helicase eIF4A confers a dose-dependent dwarfing phenotype in Brachypodium distachyon. Plant J 66:929–940
Weinstein DC, Honoré E, Hemmati-Brivanlou A (1997) Epidermal induction and inhibition of neural fate by translation initiation factor 4AIII. Development 124:4235–4242
Zhong S, Lin Z, Fray RG, Grierson D (2008) Improved plant transformation vectors for fluorescent protein tagging. Transgenic Res 17:985–989
Acknowledgements
VT and VP acknowledge financial assistance from Guru Gobind Singh Indraprastha University.
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This study was funded by Council of Scientific and Industrial Research (CSIR), Government of India (Grant # 38(1380)/14/EMR-II).
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VT, VP and VK conducted the experiments. MK, GM and SK conceptualised the work, acquired funds and resources. VT and MK wrote the manuscript. All authors read and approved the manuscript.
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ESM_3
(a) Diagram showing core eIF4A motifs and description of their involvement in different biochemical activities based on published literature. (b) Alignment of amino acid residues of eIF4A from Arabidopsis thaliana, Oryza sativa, Homo sapiens, P. patens and Klebsormidium nitens. Consensus sequences in each conserved motif have been shown in red while differences between PpeIF4A1/PpeIF4A2 and PpeIF4A3 have been indicated in blue. The DEAD box in motif II is shown in purple colour. The highly conserved phenylalanine (F) 17 amino acids upstream of the Q motif is shown in bold. Mutation of either F or the invariant Glutamine (Q) in this motif is known to be lethal in yeast (Tanner et al. 2003). (PDF 216 kb)
ESM_4:
Representation of conserved motif sequences and spacings between the motifs of P. patens and K. nitens eIF4A from amino terminal end (NH2) to carboxy terminal end (COOH). The nine conserved motifs with consensus sequences are boxed while the number of non-conserved residues between them are represented by a line. (PDF 444 kb)
ESM_5
: Gene knockout construct preparation and identification of positive transformants (a) (Top) Schematic representation of protein structure showing the conserved motifs in domains1 (green) and 2 (blue). (Below) Genomic organisation of exons and introns in Pp3c6_1080V3.1 and the organisation in the disrupted locus Pp3c6_1080V3.1 KO showing deletion of exon 3 encoding motifs II–IV and insertion of 2009 bp NPTII cassette. Position of primers used for amplification of genomic fragments and screening of positive recombinants for insertion of transgene by homologous recombination events at 5′ (HR) and 3′ (HR) ends, for identification of full-length transgene and for RNA analysis in gene knockout (KO) lines #1 and #6 are marked. Sizes of amplicons obtained using each primer pair is mentioned in base pairs (bp). (b) Genomic DNA analysis of wild-type (WT) and knockout lines #1 and #6 to characterise transgene insertion at 5′ and 3′ ends and identification of full-length transgene. M refers to the 1-kb DNA marker (c) RNA analysis for checking loss of Pp3c6_1080V3.1 transcripts in knockout lines#1 and #6 by RT-qPCR. PpHistone3 was used as endogenous control. (d) Analysis of PpeIF4A1/2 expression in wild-type and Pp3c6_1080V3.1 knockout lines by RT-qPCR. Error bars denote standard deviation between the technical triplicates. Statistical analysis was done using t-tests with ***p > 0.001; ** p > 0.01. (PDF 795 kb)
ESM_6:
Quantitative and statistical analysis of cells in internode regions of wild-type and mutant gametophores and the expanded regions of phyllids. (PDF 118 kb)
ESM_7
: Estimation of total protein content in wild-type and Pp3c6_1080V3.1 Knock out line#6 gametophores by Bradford method. Biological replicates (B1, B2) of each sample type are shown along with the average (Av) of the values used for estimating protein concentration. Statistical analysis (t test) showed p > 0.01 and correlation coefficient to be -1. (PDF 217 kb)
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Tyagi, V., Parihar, V., Malik, G. et al. The DEAD-box RNA helicase eIF4A regulates plant development and interacts with the hnRNP LIF2L1 in Physcomitrella patens. Mol Genet Genomics 295, 373–389 (2020). https://doi.org/10.1007/s00438-019-01628-x
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DOI: https://doi.org/10.1007/s00438-019-01628-x