A DEAD box protein is required for formation of a hidden break in Arabidopsis chloroplast 23S rRNA

Abstract

SUMMARYIn plant chloroplasts, the ribosomal RNA (rRNA) of the large subunit of the ribosome undergoes postmaturation fragmentation processing. This processing consists of site-specific cleavage that generates gapped, discontinuous rRNA molecules. However, the molecular mechanism underlying introduction of the gap structure (the 'hidden break') is poorly understood. Here, we found that the DEAD box protein RH39 plays a key role in introduction of the hidden break into the 23S rRNA in Arabidopsis chloroplasts. Genetic screening for an Arabidopsis plant with a drastically reduced level of ribulose-1,5-bisphosphate carboxylase/oxygenase identified an RH39 mutant. The levels of other chloroplast-encoded photosynthetic proteins were also severely reduced. The reductions were not due to a failure of transcription, but rather inefficiency in translation. RNA gel blotting revealed incomplete fragmentation of 23S rRNA in chloroplasts during maturation. In vitro analysis with recombinant RH39 suggested that the protein binds to the adjacent sequence upstream of the hidden break site to exert its function. We propose a molecular mechanism for the RH39-mediated fragmentation processing of 23S rRNA in chloroplasts.

Nucleoid-Enriched Proteomes in Developing Plastids and Chloroplasts from Maize Leaves: A New Conceptual Framework for Nucleoid Functions

“…Williams-Carrier, A. Vichas, G. Friso, A. Barkan, and K.J. van Wijk, unpublished data), and RH39 (Nishimura et al, 2010): the precise function of VDL is unknown, whereas both RH3 and RH39 are involved in rRNA maturation; RH3, in addition, promotes the splicing of some group II introns (Y. Asakura, E. Galarneau, K.P. Watkins, R.E.…”

Plant Temperature Acclimation and Growth Rely on Cytosolic Ribosome Biogenesis Factor Homologs

“…5S and 5.8S rRNAs were detectable but not clearly separated. We analyzed 23S 300 rRNA by summation of the two previously described post-maturation fragments 23S´ and 301 23S´´ that result from hidden breaks of the mature 23S rRNA (Nishimura et al, 2010;Tiller 302 et al, 2012) Fragments 23S´ and 23S´´ were abundant in all analyzed samples. A third small 303 23S´´´ hidden break product and the potential full size rRNA precursor (Tiller et al, 2012) 304 were detectable only at very low intensity and disregarded in this study.…”

“…rRNA containing fractions were chosen for microfluidic 1046 electrophoresis (Salvo-Chirnside et al, 2011) and qualitative analysis of rRNA species (e.g. 1047 Figure 5, Supplemental Figure S3), with cytoplasmic 25S and 18S rRNA and chloroplast 23S 1048 and 16S rRNA annotations according to Nishimura et al (2010) and Tiller et al (2012). 1049…”

Characterization of silk gland ribosomes from a bivoltine caddisfly, Stenopsyche marmorata : translational suppression of a silk protein in cold conditions

“…Azpurua et al recently revealed that 28S rRNA of naked mole-rat, a subterranean rodent, is cleaved into two fragments within ES15L (D6) region; naked mole-rat cells have a 4-10-fold higher global translation fidelity relative to mouse cells, despite equivalent translation rates [30]. Additionally, the chloroplast ribosomes of an Arabidopsis thaliana nara12-1 mutant, which exhibits defective chloroplast 23S rRNA fragmentation processing, have dysfunctional translation elongation [31], thereby suggesting 28S/23S rRNA fragmentation is a translation control strategy in various organisms, regardless of its variable effects. Therefore, it is a leading hypothesis that the central hidden break involves splitting of 60S subunits in S. marmorata silk glands exposed to cold stress and, thus, diminished translation of Smsp-1 mRNA.…”

“…Besides the central hidden break, large subunit rRNA (28S/23S) fragmentation has been detected in various living cells and organelles [30][31][32][33][34][35][36][37][38][39]. Azpurua et al recently revealed that 28S rRNA of naked mole-rat, a subterranean rodent, is cleaved into two fragments within ES15L (D6) region; naked mole-rat cells have a 4-10-fold higher global translation fidelity relative to mouse cells, despite equivalent translation rates [30].…”