Unexpected Diversity of Chloroplast Noncoding RNAs as Revealed by Deep Sequencing of the Arabidopsis Transcriptome

Abstract

Noncoding RNAs (ncRNA) are widely expressed in both prokaryotes and eukaryotes. Eukaryotic ncRNAs are commonly micro- and small-interfering RNAs (18–25 nt) involved in posttranscriptional gene silencing, whereas prokaryotic ncRNAs vary in size and are involved in various aspects of gene regulation. Given the prokaryotic origin of organelles, the presence of ncRNAs might be expected; however, the full spectrum of organellar ncRNAs has not been determined systematically. Here, strand-specific RNA-Seq analysis was used to identify 107 candidate ncRNAs from Arabidopsis thaliana chloroplasts, primarily encoded opposite protein-coding and tRNA genes. Forty-eight ncRNAs were shown to accumulate by RNA gel blot as discrete transcripts in wild-type (WT) plants and/or the pnp1-1 mutant, which lacks the chloroplast ribonuclease polynucleotide phosphorylase (cpPNPase). Ninety-eight percent of the ncRNAs detected by RNA gel blot had different transcript patterns between WT and pnp1-1, suggesting cpPNPase has a significant role in chloroplast ncRNA biogenesis and accumulation. Analysis of materials deficient for other major chloroplast ribonucleases, RNase R, RNase E, and RNase J, showed differential effects on ncRNA accumulation and/or form, suggesting specificity in RNase-ncRNA interactions. 5′ end mapping demonstrates that some ncRNAs are transcribed from dedicated promoters, whereas others result from transcriptional read-through. Finally, correlations between accumulation of some ncRNAs and the symmetrically transcribed sense RNA are consistent with a role in RNA stability. Overall, our data suggest that this extensive population of ncRNAs has the potential to underpin a previously underappreciated regulatory mode in the chloroplast.

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

“…More than 10 7 reads were obtained from each sample, of which ;75% aligned to the chloroplast genome (Supplemental Table 1), an increase over the 30 to 50% of chloroplast-specific reads obtained from total leaf RNA (Hotto et al, 2011). Reads per kilobase of transcript per million mapped reads (RPKM) values for exons and introns showed that expression levels between replicates were highly reproducible (Supplemental Figure 7; r $ 0.93).…”

“…These enzymes and factors include a variety of RNases (Stoppel and Meurer, 2012) and large families of sequencespecific RNA binding proteins, such as those of the pentatricopeptide repeat (PPR) and RNA recognition motif families (Ruwe et al, 2011;Nakamura et al, 2012). Evidence from high-throughput RNA sequencing (RNA-Seq) and microarrays suggests that both strands of the ;150-kb chloroplast genome are fully transcribed (Hotto et al, 2011;Zmie nko et al, 2011;Zhelyazkova et al, 2012), emphasizing the critical role of posttranscriptional processes in defining the steady state RNA population.…”

Arabidopsis Chloroplast RNA Binding Proteins CP31A and CP29A Associate with Large Transcript Pools and Confer Cold Stress Tolerance by Influencing Multiple Chloroplast RNA Processing Steps

“…Recently, RNAs antisense to ycf1 were detected in a deep-sequencing study of the Arabidopsis chloroplast transcriptome and in mutants of the chloroplast PNPase, which is a 39-to-59 exonuclease (Hotto et al, 2011). RNA gel blot analyses demonstrated that this PNPase is needed for the accumulation of processed as-ycf1 transcripts (Hotto et al, 2011). In the future, studies on the interplay among CP31A, PNPase, and other factors will allow us to understand how chloroplast 39-degradation of ndhF (and potentially other mRNAs) is regulated.…”

The Small Regulatory RNA SyR1/PsrR1 Controls Photosynthetic Functions in Cyanobacteria

“…However, the functions of other classes of potential RNA regulators, such as long noncoding RNAs and cis-natural antisense transcripts, are scarcely understood (De Lucia and Dean, 2011). Noncoding RNAs are not only transcribed in the plant nucleus but are also abundant in plant chloroplasts (Hotto et al, 2011;Zhelyazkova et al, 2012). With regard to the possible involvement of noncoding RNAs in plastidrelated regulatory networks, it is unclear at present whether these have evolved from their cyanobacterial ancestors and to what extent such regulators play a role in cyanobacteria.…”