The DEAD-box RNA Helicase RH50 Is a 23S-4.5S rRNA Maturation Factor that Functionally Overlaps with the Plastid Signaling Factor GUN1

Abstract

DEAD-box RNA helicases (DBRHs) modulate RNA secondary structure, allowing RNA molecules to adopt the conformations required for interaction with their target proteins. RH50 is a chloroplast-located DBRH that colocalizes and is coexpressed with GUN1, a central factor in chloroplast-to-nucleus signaling. When combined with mutations that impair plastid gene expression (prors1-1, prpl11-1, prps1-1, prps21-1, prps17-1, and prpl24-1), rh50 and gun1 mutations evoke similar patterns of epistatic effects. These observations, together with the synergistic growth phenotype of the double mutant rh50-1 gun1-102, suggest that RH50 and GUN1 are functionally related and that this function is associated with plastid gene expression, in particular ribosome functioning. However, rh50-1 itself is not a gun mutant, although-like gun1-102-the rh50-1 mutation suppresses the downregulation of nuclear genes for photosynthesis induced by the prors1-1 mutation. The RH50 protein comigrates with ribosomal particles, and is required for efficient translation of plastid proteins. RH50 binds to transcripts of the 23S-4.5S intergenic region and, in its absence, levels of the corresponding rRNA processing intermediate are strongly increased, implying that RH50 is required for the maturation of the 23S and 4.5S rRNAs. This inference is supported by the finding that loss of RH50 renders chloroplast protein synthesis sensitive to erythromycin and exposure to cold. Based on these results, we conclude that RH50 is a plastid rRNA maturation factor.

GUN1 influences the accumulation of NEP-dependent transcripts and chloroplast protein import in Arabidopsis cotyledons upon perturbation of chloroplast protein homeostasis

“…A more drastic cotyledon phenotype was seen in gun1-9 ftsh2-3 seedlings, where highly vacuolated, irregularly shaped chloroplasts were rarely observed in mesophyll cells, and a considerable number of highly electron-dense structures – possibly the products of plastid degradation – were observed within vacuoles (Figure 8). Overall, it can be argued that the lack of the Δ- rpo compensatory response, together with the plastid-to-nucleus communication, in gun1 cotyledons add to the alteration of chloroplast protein homeostasis in ftsh and cphsc70-1 and sca3-1 mutants (see Figure 1, Figure 2, Figure S2, Figure S3 and Table S1), impairing chloroplast biogenesis as observed previously (Tadini et al, 2016 and Paieri et al, 2018).…”

“…As a member of the PPR protein family, a possible role for GUN1 as either a key regulator of plastid transcript synthesis or post-transcriptional processing can be envisaged to explain the additive phenotypic effects observed in gun1-102 prpl11-1 , gun1-102 prps17-1 and gun1-102 prpl24-1 double mutant seedlings reported in Tadini et al (2016) and Paieri et al (2018) and the similar patterns of epistatic effects evoked by rh50-1 and gun1-102 mutations (Paieri et al, 2018). To substantiate this idea, we used RNA gel-blot hybridization to measure (at 6 days after sowing, DAS) the levels of NEP- and PEP-dependent plastid transcripts in cotyledons of Col-0, gun1-102 and other mutants affected at different levels in chloroplast protein homeostasis, and grown in the absence or presence of lincomycin (Figure 1; Figure S1 and Table S1).…”

“…RNA gel-blot hybridizations confirmed the substantial increase in levels of mRNAs synthesized by the NEP in prps21-1 and prpl11-1 , but not in gun1-102 prps21-1 and gun1-102 prpl11-1 cotyledons (Table S1). The specific involvement of GUN1 protein in NEP-dependent transcript accumulation was further verified by using Arabidopsis mutants altered in chloroplast protein homeostasis such as ftsh5- 3, lacking the FtsH protease complex, cphsp70-1 , affected in plastid protein import and folding (Flores-Pérez and Jarvis, 2013; Sjuts et al, 2017), or rh50-1 , reported to be functionally related to GUN1 and characterized by altered plastid rRNA maturation (Paieri et al, 2018). No changes in the selected NEP-dependent mRNAs were observed in gun1-102 and gun1-102 ftsh5-3 cotyledons (relative to Col-0), while all of them exhibited a slight increase in level in ftsh5-3 seedlings (Figure 1c).…”

“…The plastid gene expression profile reported in Figure 1 (see also Table S1) suggests that GUN1 is required for the expression of genes transcribed by NEP upon depletion of PEP activity. To investigate if the increased steady-state levels of the NEP-dependent transcripts observed in Col-0 compared to gun1-102 plastids in presence of lincomycin was due to increased transcriptional activity rather than posttranscriptional effects such as decreased RNA stability, run-on transcription assays for the NEP-dependent transcripts rpoA and rps12 , and the PEP-dependent rbcL , psaA and psbA RNAs were performed in plastids isolated from Col-0, gun1-102 and plastids of gun1-102 in the presence of either the purified GUN1 recombinant protein (+ recGUN1) or RH50 recombinant protein (+ recRH50 - Dead-box RNA Helicase RH50; see also Paieri et al, 2018), the latter used as control (Figure 3a,b and Figure S2a,b). This assay demonstrated that the levels of newly synthesized NEP-dependent transcripts were significantly higher in Col-0 and gun1-102 + recGUN1 plastids compared to gun1-102 , whereas the PEP-dependent transcripts accumulated to similar levels (Figure 3a,b and Figure S2a,b), indicating that GUN1 is required for RopTp transcriptional activity rather than influencing RNA stability.…”

“…To further investigate the importance of GUN1 activity in chloroplast biogenesis upon alteration of plastid protein homeostasis (see also Tadini et. al., 2016 and Paieri et al, 2018), the gun1 ftsh , gun1-102 cphsc70-1 and gun1-102 sca3-1 double mutant seedlings were also analyzed at macroscopic level (Figure 7a and Figure S3a). Specifically, mutations in the nuclear genes for FtsH1 ( ftsh1-1 ), FtsH5 ( ftsh5-3 ) and FtsH8 ( ftsh8-1 ) proteins were introduced into the gun1-102 mutant background by manual crossing, while the CRISPR-Cas9 editing strategy was used to generate the ftsh2-3 mutant allele in the gun1-9 genetic background, since these two genes are closely linked on chromosome 2 (Figure S1).…”