The critical function of the plastid rRNA methyltransferase, CMAL, in ribosome biogenesis and plant development

Abstract

Methylation of nucleotides in ribosomal RNAs (rRNAs) is a ubiquitous feature that occurs in all living organisms. The formation of methylated nucleotides is performed by a variety of RNA-methyltransferases. Chloroplasts of plant cells result from an endosymbiotic event and possess their own genome and ribosomes. However, enzymes responsible for rRNA methylation and the function of modified nucleotides in chloroplasts remain to be determined. Here, we identified an rRNA methyltransferase, CMAL (Chloroplast MraW-Like), in the Arabidopsis chloroplast and investigated its function. CMAL is the Arabidopsis ortholog of bacterial MraW/ RsmH proteins and accounts to the N4-methylation of C1352 in chloroplast 16S rRNA, indicating that CMAL orthologs and this methyl-modification nucleotide is conserved between bacteria and the endosymbiont-derived eukaryotic organelle. The knockout of CMAL in Arabidopsis impairs the chloroplast ribosome accumulation and accordingly reduced the efficiency of mRNA translation. Interestingly, the loss of CMAL leads not only to defects in chloroplast function, but also to abnormal leaf and root development and overall plant morphology. Further investigation showed that CMAL is involved in the plant development probably by modulating auxin derived signaling pathways. This study uncovered the important role of 16S rRNA methylation mediated by CMAL in chloroplast ribosome biogenesis and plant development.

Arabidopsis mTERF9 protein promotes chloroplast ribosomal assembly and translation by establishing ribonucleoprotein interactions in vivo

“…To firmly establish the in vivo function of mTERF9, we performed a comprehensive analysis of the in vivo RNA and protein interaction networks of mTERF9. Our co-immunoprecipitation results demonstrated that mTERF9 binds to the 16S rRNA in chloroplasts as well as ribosomal proteins, CPN60 chaperonins and known auxiliary ribosomal factors involved in rRNA processing such as the MraW-like 16S rRNA methyltransferase (CMAL) ( 68 ), YbeY endoribonuclease ( 67 ), RNase J ( 50 ), RNase E-like protein (RHON1) ( 51 ) or DEAD/DEAH-box RNA helicases (RH3 and ISE2) ( 47–49 ). The fractionation of chloroplast high-molecular-weight protein complexes combined with the comparative mTERF9 protein interactome in presence or absence of RNase together with the 16S rRNA mTERF9 co-immunoprecipitation indicate that mTERF9 preferentially associates with the 30S small ribosome subunit in vivo .…”

N4‐methylcytidine ribosomal RNA methylation in chloroplasts is crucial for chloroplast function, development, and abscisic acid response in Arabidopsis

“…CMAL contains a 41 amino acid‐long putative chloroplast transit peptide at the N‐terminus, and the chloroplast localization of CMAL has recently been demonstrated (Zou et al, 2020). The transient expression of the CMAL‐GFP (green fluorescent protein) fusion protein in tobacco leaves further confirmed that the nucleus‐encoded CMAL protein is localized to the chloroplasts (Figure 2A).…”

“…These growth and developmental defects of the cmal mutant might be attributed to deficiencies in chloroplast biogenesis and function. A recent study demonstrated that the structures of the cmal mutant chloroplasts are abnormal and the photosynthetic activity of the cmal mutant is lower than that of the wild type (Zou et al, 2020). Moreover, our data indicate that the cmal mutant does not develop true green leaves on sucrose‐deficient medium, and this defect can be recovered by adding sucrose to the medium (Figure 2D).…”

“…Despite the increasing understanding of the role of RNA methyltransferases in the nucleus, the proteins responsible for the methylation of chloroplast RNAs are still elusive. Recently, an Arabidopsis ortholog of RsmH in E. coli , named CMAL for chloroplast mraW‐like (At5g10910), has been characterized as an rRNA methyltransferase that installs m 4 C modifications at the C 1352 position in chloroplast 16S rRNA (Zou et al, 2020). Previous analysis of the cmal mutant demonstrated that CMAL is indispensable for chloroplast biogenesis and function, which is crucial for normal plant growth (Zou et al, 2020).…”

Arabidopsis mTERF9 protein promotes chloroplast ribosomal assembly and translation by establishing ribonucleoprotein interactionsin vivo

“…Consistent with that, the stability of several isoforms of the 23S rRNA resulting from the hidden breaks processing were impaired in mterf9. Several auxiliary factors involved in chloroplast ribosomal assembly have been recently characterized and the majority of these are bacterial homologs or harbor RNA binding domains that are conserved in bacteria (47)(48)(49)(50)(51)55,(68)(69)(70). Without any surprise, these protein homologs perform conserved functions in rRNA processing and therefore, ribosome assembly in chloroplasts.…”