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The DEAD-box RNA helicase eIF4A regulates plant development and interacts with the hnRNP LIF2L1 in Physcomitrella patens

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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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Acknowledgements

VT and VP acknowledge financial assistance from Guru Gobind Singh Indraprastha University.

Funding

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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Authors and Affiliations

  1. University School of Biotechnology, Guru Gobind Singh Indraprastha University, Sector 16C, Dwarka, New Delhi, 110078, India

    Vidhi Tyagi, Vimala Parihar, Vaibhav Kalra & Meenu Kapoor

  2. Department of Botany, R.G. (P.G) College C.C.S. University, Meerut, Uttar Pradesh, 250001, India

    Garima Malik

  3. Interdisciplinary Centre for Plant Genomics and Department of Plant Molecular Biology, University of Delhi South, Campus Benito Juarez Road, New Delhi, 110021, India

    Sanjay Kapoor

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  1. Vidhi Tyagi
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  2. Vimala Parihar
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  4. Vaibhav Kalra
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  5. Sanjay Kapoor
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Contributions

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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Correspondence to Meenu Kapoor.

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VT, VP, GM, VK, SK declares that they have no conflict of interest.

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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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