Ribonuclease II preserves chloroplast RNA homeostasis by increasing mRNA decay rates, and cooperates with polynucleotide phosphorylase in 3′ end maturation

Abstract

SUMMARYRibonuclease R (RNR1) and polynucleotide phosphorylase (cpPNPase) are the two known 3¢ fi 5¢ exoribonucleases in Arabidopsis chloroplasts, and are involved in several aspects of rRNA and mRNA metabolism. In this work, we show that mutants lacking both RNR1 and cpPNPase exhibit embryo lethality, akin to the non-viability of the analogous double mutant in Escherichia coli. We were successful, however, in combining an rnr1 null mutation with weak pnp mutant alleles, and show that the resulting chlorotic plants display a global reduction in RNA abundance. Such a counterintuitive outcome following the loss of RNA degradation activity suggests a major importance of RNA maturation as a determinant of RNA stability. Detailed analysis of the double mutant demonstrates that the enzymes catalyze a two-step maturation of mRNA 3¢ ends, with RNR1 polishing 3¢ termini created by cpPNPase. The bulky quaternary structure of cpPNPase compared with RNR1 could explain this activity split between the two enzymes. In contrast to the double mutants, the rnr1 single mutant overaccumulates most mRNA species when compared with the wild type. The excess mRNAs in rnr1 are often present in non-polysomal fractions, and half-life measurements demonstrate a substantial increase in the stability of most mRNA species tested. Together, our data reveal the cooperative activity of two 3¢ fi 5¢ exoribonucleases in chloroplast mRNA 3¢ end maturation, and support the hypothesis that RNR1 plays a significant role in the destabilization of mRNAs unprotected by ribosomes.

The Conserved Endoribonuclease YbeY Is Required for Chloroplast Ribosomal RNA Processing in Arabidopsis

“…In the extreme thermophile Thermus thermophilus HB8, a gram-negative eubacterium, disruption of the YbeY gene had a greater impact on mRNA abundance (Ohyama et al, 2014). In Arabidopsis, RNase E-or PNPase-deficient mutants also exhibited defects in chloroplast mRNA or tRNA metabolism (Walter et al, 2010;Germain et al, 2012). It is unlikely that the higher steady-state levels of mRNAs and tRNAs result from an increased specific transcription, because the accumulation of mRNA of accD transcribed by nucleus-encoded RNA polymerase and mRNA of rbcL and psbA by plastidencoded RNA polymerase in atybeY mutant was the same as in the wild type (Fig.…”

“…In Arabidopsis, the absence of PNPase from chloroplasts caused defects in rRNA maturation and overaccumulation of tRNAs (Walter et al, 2002). Depletion of RNase R or RNase E in chloroplasts also leads to multiple abnormalities in RNA metabolism, including rRNA processing defects and overaccumulation of some precursor transcripts of mRNAs and tRNAs (Kishine et al, 2004;Walter et al, 2010;Germain et al, 2012). Whether the similar defects of chloroplast rRNA processing in atybeY mutants and other RNase mutants indicate partially overlapping functions is currently unclear.…”

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

“…For example, the absence of the endoribonuclease RNase E leads to severe chlorosis and the accumulation of long mRNA precursors (Walter et al, 2010), and deficiency of the endoribonuclease/exoribonuclease RNase J results in massive accumulation of antisense RNA (Sharwood et al, 2011a). Absence of either of the two prominent exoribonucleases, polynucleotide phosphorylase and RNase II, leads to defects in 39 end maturation of mRNAs and rRNAs, and depletion of both causes embryo lethality in Arabidopsis thaliana (Germain et al, 2011(Germain et al, , 2012. Clearly, accurate RNA metabolism and processing is essential for plastid biogenesis and function.…”

Analysis of the role of RNS2 and autophagy in the turnover of ribosomal RNA in Arabidopsis thaliana

“…The physiological importance and mechanistic insights of RNA turnover has been studied extensively in both prokaryotes and eukaryotes (Deutscher 2006;Barrangou et al 2007;Buchan and Parker 2009;Cooper et al 2009;Huang et al 2011;Germain et al 2012;Kelly and Bedwell 2015). The loss of functional RNA turnover machinery eliminates a posttranscriptional layer of gene regulation and can also lead to mis-regulated expression and accumulation of aberrant transcripts and peptides.…”

“…RNA surveillance machinery helps to minimize ER stress by removing aberrant transcripts before peptides can be synthesized (Sakaki et al 2012). In plants, the loss of some ribonucleases involved in RNA degradation also results in severe or lethal phenotypes Germain et al 2012). …”