PALE CRESS binds to plastid RNAs and facilitates the biogenesis of the 50S ribosomal subunit

Abstract

The plant-specific PALE CRESS (PAC) protein has previously been shown to be essential for photoautotrophic growth. Here we further investigated the molecular function of the PAC protein. PAC localizes to plastid nucleoids and forms large proteinaceous and RNA-containing megadalton complexes. It co-immunoprecipitates with a specific subset of chloroplast RNAs including psbK-psbI, ndhF, ndhD, and 23S ribosomal RNA (rRNA), as demonstrated by RNA immunoprecipitation in combination with high throughput RNA sequencing (RIP-seq) analyses. Furthermore, it co-migrates with premature 50S ribosomal particles and specifically binds to 23S rRNA in vitro. This coincides with severely reduced levels of 23S rRNA in pac leading to translational deficiencies and related alterations of plastid transcript patterns and abundance similar to plants treated with the translation inhibitor lincomycin. Thus, we conclude that deficiency in plastid ribosomes accounts for the pac phenotype. Moreover, the absence or reduction of PAC levels in the corresponding mutants induces structural changes of the 23S rRNA, as demonstrated by in vivo RNA structure probing. Our results indicate that PAC binds to the 23S rRNA to promote the biogenesis of the 50S subunit.

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

“…Note that PRPL11 and PRPS5 accumulate in fractions 3 and 4 when extracts are pretreated with RNase. This might be due to conformational changes induced by partial digestion of the rRNA in mature ribosomes, which makes them less compact, such that they emerge from the column earlier (Jenkins and Barkan, 2001;Meurer et al, 2017). Equal loading is demonstrated by Coomassie Brilliant Blue staining of the membrane.…”

“…S3). We chose to examine psaA mRNA, which is efficiently loaded onto polysomes and hence migrates deep into the Suc gradient (Amann et al, 2004;Meurer et al, 2017). Under nonstressed conditions, loading of psaA mRNA onto rh50 polysomes was indistinguishable from the loading onto wild-type polysomes with the peak fractions 9 to 11 (Supplemental Fig.…”

PUMPKIN, the Sole Plastid UMP Kinase, Associates with Group II Introns and Alters Their Metabolism

“…genes, while transcription of nucleus-encoded RNA polymerase (NEP)-dependent genes increases (Meurer et al, 2017). To check whether this notion is also true for pumpkin, we performed transcriptome-wide expression analysis by RNA deep sequencing (-Seq) of rRNAdepleted total leaf RNA obtained from the pumpkin mutant and the wild type.…”

“…3A). Interestingly, the signals within chloroplasts appeared spotty and prompted us to check for chloroplast nucleoid localization using the nucleoid marker protein RAP Meurer et al, 2017). Merging the fluorescence of the PUMPKIN-YFP and RAP-CFP in cotransformed protoplasts of N. benthamiana led to only partial overlap of the signals, making the localization of PUMPKIN in chloroplast nucleoids ambiguous ( Fig.…”

“…A variety of different nuclear encoded, newly evolved RNA-binding proteins, predominantly numerous helical-repeat proteins, bind these specific sites to safeguard transcript stability by preventing endonucleolytic attacks and progression of exonucleolytic degradation (Stern et al, 2010;Jacobs and Kück, 2011;Meurer, 2012, 2013;Germain et al, 2013). Depending on the number of RNA targets and secondary effects, corresponding nuclear mutants display more or less pleiotropic effects related to plastid mRNA patterns and abundance (Pfalz et al, 2009;Belcher et al, 2015;Hammani et al, 2016;Zoschke et al, 2016;Meurer et al, 2017;Teubner et al, 2017).…”