PAP8/pTAC6 Is Part of a Nuclear Protein Complex and Displays RNA Recognition Motifs of Viral Origin

Abstract

Chloroplast biogenesis depends on a complex transcriptional program involving coordinated expression of plastid and nuclear genes. In particular, photosynthesis-associated plastid genes are expressed by the plastid-encoded polymerase (PEP) that undergoes a structural rearrangement during chloroplast formation. The prokaryotic-type core enzyme is rebuilt into a larger complex by the addition of nuclear-encoded PEP-associated proteins (PAP1 to PAP12). Among the PAPs, some have been detected in the nucleus (PAP5 and PAP8), where they could serve a nuclear function required for efficient chloroplast biogenesis. Here, we detected PAP8 in a large nuclear subcomplex that may include other subunits of the plastid-encoded RNA polymerase. We have made use of PAP8 recombinant proteins in Arabidopsis thaliana to decouple its nucleus- and chloroplast-associated functions and found hypomorphic mutants pointing at essential amino acids. While the origin of the PAP8 gene remained elusive, we have found in its sequence a micro-homologous domain located within a large structural homology with a rhinoviral RNA-dependent RNA polymerase, highlighting potential RNA recognition motifs in PAP8. PAP8 in vitro RNA binding activity suggests that this domain is functional. Hence, we propose that the acquisition of PAPs may have occurred during evolution by different routes, including lateral gene transfer.

Keywords: Arabidopsis; PAP8/pTAC6; PEP/PAPs; RNA recognition motif; biogenesis; chloroplast.

Figure 1

PAP8 is detected within a nuclear subcomplex. (A) Organelle fractionation, purification scheme, and sample processing. BN-PAGE, blue native polyacrylamide gel electrophoresis. (B,D) BN-PAGE and corresponding Western blot analyses in (C,E) using the PAP8 antibody. P8r, recombinant PAP8 protein purified from E. coli; HS heparin Sepharose fraction produced with the intact chloroplast sample; GF, gel filtration sample following HS; N, N6×, and N1× different loading of the sonicated and soluble nuclear fraction (see Section 4). T, thylakoid fraction from broken CP. (C) arrowheads, blue for PEP-A, red for the Ncpx1 (large nuclear complex), yellow Ncpx2 (smaller complex), green for the thylakoid PAP8-containing complex, and αPAP8 for the primary anti-PAP8 antibody. (F–J) Bimolecular fluorescence complementation tests using in combination PAP8^ΔcTP-YC (8^ΔcTP-YC) with PAP5^ΔcTP-NY (5^ΔcTP-NY) in (F); 8^ΔcTP-YC with PAP7^ΔcTP-NY (7ΔcTP-NY) in (G); 8^ΔcTP-YC with PAP12^ΔcTP-NY (12^ΔcTP-NY) in (H); 7^ΔcTP-YC 5^ΔcTP-NY) in (I) and 7^ΔcTP-YC with (12^ΔcTP-NY) in (J); PAP10-RFP was used as internal positive control for transfection. Arrowheads indicate nuclei. Transgenes expressed under CaMV35S promoter (see [27] for published control experiments). Scale bars equal 20 µm.

Figure 2

A PAP8 N-terminal translational fusion is functional. (A) Structure of the recombinant DNA for the production of GFP translational fusions p35S, CaMV35S promoter; pP8, 1-kb PAP8 promoter; cTP8, predicted chloroplast transit peptide from PAP8. (B) Sample DNA gels for the PCR-based genotyping of the primary transformants (T1) according to [27]; wild-type PAP8 allele, (op8_rtp_F + op8i2R); mutant allele pap8-1, (op8_rtp_F + oLBb1.3) and transgene p8:cTP8GP8, (oGFP_Fmfe + oP8E3_R). Wt, wild-type; p8, pap8-1 genomic DNA and pLC23, plasmid DNA containing the transgene. The doubly heterozygous plants have all three bands. (C) Dot plot of the percentage of albinos obtained in the segregating offspring from the doubly heterozygous plants as independent T1 (yellow spots). Functional complementation corresponds to a ratio of 1/16 (6.25%). ε-test, α = 0.05; Fisher Yates: the 4 tested lines Z, ɣ, η, R in yellow circles gave a ratio not significantly different from 1/16, n[albino]/Ntotal: 27/353; 44/706; 22/284; 28/352, respectively (εZ = 0.989; εɣ = 0.019; εη = 0.943; εR = 1.181; all ε < 𝒰5% = 1.96). (D) Single T2 plant genotyping, primers as in (B); all plants positive for the transgene (p8::cTP8GP8, pPAP8::cTP8-GFP-PAP8), the status is given for the PAP8 locus as follow: wt, PAP8,PAP8, p8/+, PAP8/pap8-1 and p8, pap8-1/pap8-1. (E) Three-week-old plants corresponding to representative genotypes given in (D). (F–I) Transiently expressed pLC21 (p35S::cTP8-GFP-PAP8ΔcTP) in onion epidermal cells; dual localization in the nucleus (green arrowhead) and in plastids (yellow arrowheads); PAP10-RFP was used as internal positive control for transfection. (J) Confocal imaging on Arabidopsis cotyledons stably expressing pP8::cTP8-GFP-PAP8ΔcTP early after light exposure of skotomorphogenetic seedlings; the picture is a merge of two channels: GFP in green: protochlorophyllide in blue.

Figure 3

The region with the predicted NLS provides functions in addition to the nuclear localization signal. (A) Schematic illustration of the different PAP8 recombinant genes. cTP, chloroplast transit peptide; NLS, nuclear localization signal; m5, five substitutions leading to amino acid changes of K and R in G; dhc, double hemi-complementation test. (B) PCR-based genotyping of the different lines showing in particular that dhc possess the two constructions (Nm5 and ΔcTP). (C) Dot plot of the percentage of the delayed greening phenotype obtained in the segregating offspring from the doubly heterozygous plants from dhc (light orange dots) or independent T1 for BB647 (light green dots). A ratio of 1/4 (25%) corresponds to absence of genetic interaction between the transgene and the pap8-1 allele: no complementation. (ε-test, α = 0.05; Fisher Yates) dhc #04: 44/148 (ε = 1.26); #19: 115/543 (ε = 2.179); #22: 63/234 (ε = 0.663); #33: 83/292 (ε = 1.297); #36: 44/149 (ε = 1.212); #40: 15/47 (ε = 1.017); #46: 98/373 (ε = 0.558) sum of all is 462 [Albino] over 1324 [WT] ε_somme = 0.838 < 𝒰_5% = 1.96. BB647 #Na: 79/279 (ε = 1.229); #Za: 91/365(ε = 0.03); #Ka: 53/235 (ε = 0.897); #Ab: 91/366 (ε = 0.06); #Ec: 73/314 (ε = 0.734); #Lb: 84/340 (ε = 0.125); #Ob: 65/279 (ε = 0.672); #Ub: 43/212 (ε = 1.708); #Vb: 55/229 (ε = 0.348); #Gc: 56/269 (ε = 1.689); #41: 65/266 (ε = 0.214); sum: 755/3154 (ε_somme = 1.397 < 𝒰_5% = 1.96). (D) 7-day-old (7 DO) or (E) 30-day-old (30 DO) phenotype of the given genotypes.

Figure 4

SV40 NLS fused to PAP8 provides a robust nuclear epidermal marker. (A) Schematic illustration of the PAP8 recombinant gene: cTP4-GFP-NLS^SV40-PAP8^NLSm5; cTP4 corresponds to the chloroplast transit peptide of PAP4 and NLSSV40, the NLS from the simian virus 40 (SV40). The transgene is either under the control of CaMV35S promoter in pLC09 or the PAP8 promoter in pLC16. (B) Dot plot of the percentage of albinos obtained in the segregating offspring from the doubly heterozygous-independent T1 for LC16 (yellow dots). A ratio of 1/4 (25%) corresponds to absence of genetic interaction between LC16 and the pap8-1 allele: no complementation. LC16 #E: 137/566 (ε = 0.441); #X: 201/790 (ε = 0.285); #P: 118/616 (ε = 3.685); #k: 138/536 (ε = 0.395); #S: 199/682 (ε = 2.4); #c: 152/667 (ε = 1.361); sum: 945/3857 (ε_somme = 0.720 < 𝒰5% = 1.96). (C) 30-day-old phenotype of the genotype pap8-1, pap8-1; LC16. (D) Transiently expressed pLC09 (p35S::cTP8-GFP-PAP8^ΔcTP) in onion epidermal cells; dual localization in the nucleus (green arrowhead) and in plastids (yellow arrowheads); PAP10-RFP was used as internal positive control for transfection. DIC, differential interference contrast image. (E) Non-saturated image of a nucleus and its corresponding DIC image. (F) Confocal imaging of Arabidopsis cotyledons stably expressing pP8::cTP4-GFP-NLS^SV40-PAP8^ΔcTP during skotomorphogenesis; the picture is a merge of two channels: GFP in green: protochlorophyllide in blue; the blue channel has been electronically saturated to draw the cell contours in the epidermal layer; inset is the original image. White arrows show nuclei; the red arrow show reduced GFP signal.

Figure 5

PAP8 microhomology to RDR6 reveals RNA binding domains. (A) Domain mapping on the amino acid sequence of PAP8 according to the structural homology comparison with RDR6 (pdb:4K50) using T-coffee expresso. Highly conserved residues in the PAP8 orthologous family of proteins are represented as vertical blue lines; µ, amino acid microhomology between PAP8 and RDR6; RRM, RNA recognition motifs in RDR6. (B) Amino acid painting of the three-dimensional fold of RDR6 according to the homology with PAP8: red, good; pink, average; green, bad; blue, absent in PAP8 and yellow, double-stranded RNA sequence named rdr32 hereafter. (C) RNAfold server prediction for the topology of rdr32 RNA sequence used as a cy5-marked probe. (D) RNA electromobility shift assay rEMSA using rdr32cy5 as RNA probe and H6-PAP8 produced in E. coli; 8_{100 °C}, boiled PAP8 protein prior to setting the interaction assay with the probe; 16α, 16 µM of PAP8 + PAP8 antibody (5% of 10,000×). (E) Recombinant H6-PAP8 amino acid sequence; histidine tag in blue; homology regions with RDR6 in red; microhomology underlined and NLS in magenta.