Cooperative role of AtRsmD and AtRimM proteins in modification and maturation of 16S rRNA in plastids

Abstract

SUMMARY Chloroplast pre‐ribosomal RNA (rRNA) undergoes maturation, which is critical for ribosome assembly. While the central and auxiliary factors in rRNA maturation have been elucidated in bacteria, their mode of action remains largely unexplored in chloroplasts. We now reveal chloroplast‐specific factors involved in 16S rRNA maturation, Arabidopsis thaliana orthologs of bacterial RsmD methyltransferase (AtRsmD) and ribosome maturation factor RimM (AtRimM). A forward genetic screen aimed to find suppressors of the Arabidopsis yellow variegated 2 (var2) mutant defective in photosystem II quality control found a causal nonsense mutation in AtRsmD. The substantially impaired 16S rRNA maturation and translation due to the mutation rescued the leaf variegation phenotype by lowering the levels of chloroplast‐encoded proteins, including photosystem II core proteins, in var2. The subsequent co‐immunoprecipitation coupled with mass spectrometry analyses and bimolecular fluorescence complementation assay found that AtRsmD interacts with AtRimM. Consistent with their interaction, loss of AtRimM also considerably impairs 16S rRNA maturation with decelerated m2G915 modification in 16S rRNA catalyzed by AtRsmD. The atrimM mutation also rescued var2 mutant phenotypes, corroborating the functional interplay between AtRsmD and AtRimM towards modification and maturation of 16S rRNA and chloroplast proteostasis. The maturation and post‐transcriptional modifications of rRNA are critical to assembling ribosomes responsible for protein translation. Here, we revealed that the cooperative regulation of 16S rRNA m2G915 modifications by AtRsmD methyltransferase and ribosome assembly factor AtRimM contributes to 16S rRNA maturation, ribosome assembly, and proteostasis in chloroplasts.

Deciphering the RNA Modification Landscape in Arabidopsis Chloroplast tRNAs and rRNAs Reveals a Blend of Ancestral and Acquired Characteristics

“…RsmB incorporates the modification at the initial stages of SSU assembly by binding to free 16S rRNA, whereas the methylation by RsmD requires prior binding of ribosomal proteins, inducing a conformation change that results in accessible G966 for modification (116). Similarly, our data analysis suggests that chloroplast 16S rRNA undergoes analogous modifications at the equivalents in both of these positions - G915 and C916 (Figure 5a-d; (103, 109, 117). However, only the RsmD homologue (introducing m 2 G915) has been described so far.…”

“…The analysis of the Arabidopsis mutant defective in RsmD function confirmed its role in 16S rRNA maturation. The atrsmD mutants exhibit the accumulation of 16S rRNA precursors, reduced chloroplast-encoded protein levels, and show a cold stress-sensitive phenotype, indicating the role of RsmD in chloroplast translation (109, 117).…”

In vivoCRISPR screens identify novel virulence genes among proteins of unassigned subcellular localization inToxoplasma

“…RimM domain-containing protein was reported to regulate 16S rRNA processing in bacteria (39). It is reported that a RimM domain-containing protein in Arabidopsis thaliana (AtRimM) is located in the chloroplasts and contributes to rRNA maturation and proteostasis (40). Considering that the apicoplast evolved from secondary endosymbiosis (41) and that the zinc finger domain in TgRimM is essential for its function in our study, ApiRimM might facilitate rRNA maturation and proteostasis in the apicoplast.…”