Arabidopsis Chloroplast Mini-Ribonuclease III Participates in rRNA Maturation and Intron Recycling

Abstract

RNase III proteins recognize double-stranded RNA structures and catalyze endoribonucleolytic cleavages that often regulate gene expression. Here, we characterize the functions of RNC3 and RNC4, two Arabidopsis thaliana chloroplast Mini-RNase III-like enzymes sharing 75% amino acid sequence identity. Whereas rnc3 and rnc4 null mutants have no visible phenotype, rnc3/rnc4 (rnc3/4) double mutants are slightly smaller and chlorotic compared with the wild type. In Bacillus subtilis, the RNase Mini-III is integral to 23S rRNA maturation. In Arabidopsis, we observed imprecise maturation of 23S rRNA in the rnc3/4 double mutant, suggesting that exoribonucleases generated staggered ends in the absence of specific Mini-III-catalyzed cleavages. A similar phenotype was found at the 39 end of the 16S rRNA, and the primary 4.5S rRNA transcript contained 39 extensions, suggesting that Mini-III catalyzes several processing events of the polycistronic rRNA precursor. The rnc3/4 mutant showed overaccumulation of a noncoding RNA complementary to the 4.5S-5S rRNA intergenic region, and its presence correlated with that of the extended 4.5S rRNA precursor. Finally, we found rnc3/4-specific intron degradation intermediates that are probable substrates for Mini-III and show that B. subtilis Mini-III is also involved in intron regulation. Overall, this study extends our knowledge of the key role of Mini-III in intron and noncoding RNA regulation and provides important insight into plastid rRNA maturation.

PPR-SMR protein SOT1 has RNA endonuclease activity

“…These results suggest that the mutant harbors a defect in chloroplast translation. In vivo 35 S pulse-labeling experiments showed that the overall protein biosynthesis rate was dramatically lower in the mutant than in WT (SI Appendix, Fig. S2C), supporting the idea that chloroplast translation was impaired in the mutant.…”

“…Miniribonuclease III a_ppears to cleave the 23S-4.5S precursor to simultaneously produce the mature 5′ end of 23S and the 3′ end of 4.5S (35). The SOT1 cleavage site is located ∼40 nucleotides upstream of the miniribonuclease III cleavage site.…”

“…Chloroplast miniribonuclease III proteins RNC3 and RNC4 are known to cleave the 5′ and 3′ regions of 23S-4.5S rRNA precursor simultaneously and the loss of miniribonuclease III results in staggered 23S rRNA 5′ ends and less mature 4.5S rRNA (35). To investigate how SOT1 might function in the maturation of 23S and 4.5S rRNA, we compared the 5′ end of 23S rRNA and 3′ end of 4.5S rRNA in sot1-3 and miniribonuclease III mutant rnc3/4 using RACE assays.…”

“…SOT1 specifically binds the 5′ region of 23S-4.5S rRNA precursor with a 73-nt segment (denoted RNA73 hereafter) (34)(35)(36); thus, we reasoned that RNA73 should be an in vivo substrate for SOT1. To start, we aimed to investigate whether SOT1 could specifically cleave RNA73 using the recombinant SOT1 in vitro.…”

Correction

“…The cRT-PCR method was basically performed as described previously (Zimmer et al, 2012;Hotto et al, 2015). Two and a half micrograms of circularized wild-type, rap-1, and rbf1-1 RNAs were reverse transcribed using SuperScript III with a gene-specific oligo (16S 5# cRT-PCR F1, 5#-CACCCGTCCGC-CACTGGAAACACCA-3#).…”