Arabidopsis DEAD-Box RNA Helicase UAP56 Interacts with Both RNA and DNA as well as with mRNA Export Factors

Abstract

The DEAD-box protein UAP56 (U2AF65-associcated protein) is an RNA helicase that in yeast and metazoa is critically involved in mRNA splicing and export. In Arabidopsis, two adjacent genes code for an identical UAP56 protein, and both genes are expressed. In case one of the genes is inactivated by a T-DNA insertion, wild type transcript level is maintained by the other intact gene. In contrast to other organisms that are severely affected by elevated UAP56 levels, Arabidopsis plants that overexpress UAP56 have wild type appearance. UAP56 localises predominantly to euchromatic regions of Arabidopsis nuclei, and associates with genes transcribed by RNA polymerase II independently from the presence of introns, while it is not detected at non-transcribed loci. Biochemical characterisation revealed that in addition to ssRNA and dsRNA, UAP56 interacts with dsDNA, but not with ssDNA. Moreover, the enzyme displays ATPase activity that is stimulated by RNA and dsDNA and it has ATP-dependent RNA helicase activity unwinding dsRNA, whereas it does not unwind dsDNA. Protein interaction studies showed that UAP56 directly interacts with the mRNA export factors ALY2 and MOS11, suggesting that it is involved in mRNA export from plant cell nuclei.

ALY RNA-Binding Proteins Are Required for Nucleocytosolic mRNA Transport and Modulate Plant Growth and Development

“…Arabidopsis cells (Sørensen et al 2017) and ALY2 interacts with the RNA helicase UAP56 in 183 yeast cells and in vitro (Kammel et al 2013 analyses (Kammel et al 2013), UAP56-GFP was detected exclusively in the cell nuclei ( Fig. …”

“…GFP-NLS (Antosch et al immunofluorescence microscopy analysis (Kammel et al 2013 (Table S1). In leaf cells, the nucleoplasmic distribution of 238 the ALY proteins appeared more heterogeneous than in root cells particularly for ALY4 that 239 partially localised to nucleoplasmic foci (Fig.…”

“…GFP-NLS (Antosch et al immunofluorescence microscopy analysis (Kammel et al 2013 with their amino acid sequence similarities (Supplemental Fig. S1), the subnuclear 244 distribution of ALY1/2 differs to some extent from that of ALY3/4.…”

“…Table S2) were expressed in E. coli using plasmid pET24b-GB1 (Hautbergue et al 2008) for 574 the expression of the ALY1 proteins fused to a 6xHis-tag for metal-chelate purification and a 575 GB1-tag increasing protein stability/solubility (Gronenborn et al 1991). The proteins were 576 isolated from E. coli lysates by metal-chelate affinity chromatography using Ni-NTA beads 577 (Qiagen) as previously described (Kammel et al 2013). Eluted ALY1 proteins were further 578 purified by FPLC ion exchange chromatography using a Resource Q column (GE Healthcare) 579 equilibrated in buffer D (10 mM sodium phosphate, pH 9.0, 1 mM EDTA, 1 mM DTT, 0.5 580 mM PMSF) (Grasser et al 1996).…”

DEAD-box RNA helicases and epigenetic control of abiotic stress-responsive gene expression

“…In plants, DEADbox RNA helicases have been associated with diverse molecular functions such as siRNA-mediated gene silencing, premRNA splicing, chloroplast/mitochondrial intron splicing, nonsense-mediated mRNA decay, mRNA export, ribosome biogenesis and exon-junction complex functions. [11][12][13][14][15][16][17][18][19][20][21][22] Furthermore, it has been shown that these helicases play roles in Keywords: abiotic stress, Arabidopsis thaliana, DEAD-box RNA helicases, epigenetic gene silencing, nucleolus, RNAdirected DNA methylation Abbreviations: ABA, abscisic acid; STRS, STRESS RESPONSE SUPPRESSOR; RdDM, RNA-directed DNA methylation. various biological processes including abiotic stress responses.…”