Short non-coding RNA fragments accumulating in chloroplasts: footprints of RNA binding proteins?

Abstract

Chloroplast RNA metabolism is controlled and excecuted by hundreds of nuclear-encoded, chloroplast-localized RNA binding proteins. Contrary to the nucleo-cytosolic compartment or bacteria, there is little evidence for non-coding RNAs that play a role as riboregulators of chloroplasts. We mined deep-sequencing datasets to identify short (16–28 nt) RNAs in the chloroplast genome and found 50 abundant small RNAs (sRNAs) represented by multiple, in some cases, thousands of sequencing reads, whereas reads are in general absent from the surrounding sequence space. Other than sRNAs representing the most highly abundant mRNAs, tRNAs and rRNAs, most sRNAs are located in non-coding regions and many are found a short distance upstream of start codons. By transcript end mapping we show that the 5′ and 3′ termini of chloroplast RNAs coincide with the ends of sRNAs. Sequences of sRNAs identified in Arabidopsis are conserved between different angiosperm species and in several cases, we identified putative orthologs in rice deep sequencing datasets. Recently, it was suggested that small chloroplast RNA fragments could result from the protective action of pentatricopeptide repeat (PPR) proteins against exonucleases, i.e. footprints of RNA binding proteins. Our data support this scenario on a transcriptome-wide level and suggest that a large number of sRNAs are in fact remnants of PPR protein targets.

Effects of Reduced Chloroplast Gene Copy Number on Chloroplast Gene Expression in Maize

“…The authors proposed that proteins that stabilize chloroplast mRNAs are in limiting concentrations such that only a fraction of newly synthesized transcripts escape rapid degradation. Several nucleus-encoded proteins that stabilize specific chloroplast RNAs have been discovered in maize and Arabidopsis (Barkan et al, 1994;Felder et al, 2001;Meierhoff et al, 2003;Lezhneva and Meurer, 2004;Yamazaki et al, 2004;Beick et al, 2008;Pfalz et al, 2009;Johnson et al, 2010;Stoppel et al, 2011;Hammani et al, 2012), and there is evidence that the majority of chloroplast mRNAs in angiosperms are stabilized by proteins that block exoribonucleases (Ruwe and Schmitz-Linneweber, 2012;Zhelyazkova et al, 2012a). Our results imply that such proteins may typically be in limiting concentrations in mature chloroplasts in angiosperms, as in C. reinhardtii chloroplasts.…”

RNA binding and RNA remodeling activities of the half-a-tetratricopeptide (HAT) protein HCF107 underlie its effects on gene expression

“…Additional evidence that the native binding site of HCF107 resides within the 5′-most 21 nt of HCF107-dependent psbH mRNA isoforms comes from the observation that this region is represented by abundant small RNAs (sRNAs) in Arabidopsis, rice, barley, and maize (19,20). The binding sites of several PPR proteins accumulate as sRNAs in vivo, and there is evidence that these sRNAs are in vivo protein footprints that accumulate because they are protected by the cognate protein from endogenous ribonucleases (17,19,20).…”

“…The binding sites of several PPR proteins accumulate as sRNAs in vivo, and there is evidence that these sRNAs are in vivo protein footprints that accumulate because they are protected by the cognate protein from endogenous ribonucleases (17,19,20). To evaluate whether the sRNA mapping to the 5′ end of processed psbH RNA is the HCF107 footprint, we determined whether it fails to accumulate in a maize hcf107 (Zm-hcf107) mutant ( Fig.…”

A Nucleus-Encoded Chloroplast Phosphoprotein Governs Expression of the Photosystem I Subunit PsaC in Chlamydomonas reinhardtii

“…Interestingly, HCF107, the ortholog of Mbb1 in Arabidopsis thaliana and maize (Zea mays), can bind psbH RNA in vitro and forms a sRNA footprint in vivo . Mac1 is only found in Chlorophyta, whereas in maize, the pentatricopeptide repeat (PPR) protein CRP1 binds the 59UTR of psaC, where it generates a sRNA footprint and is required for its efficient translation (Fisk et al, 1999;Schmitz-Linneweber et al, 2005b;Ruwe and Schmitz-Linneweber, 2012).…”

“…This mechanism can lead to the accumulation of RNA footprints, small RNA fragments that are protected by the respective RNA binding proteins (Pfalz et al, 2009;Ruwe and Schmitz-Linneweber, 2012;Zhelyazkova et al, 2012;Loizeau et al, 2014). Footprints that map to the 59end of the psaC transcripts could be identified in Chlamydomonas small RNA databases ( Figure 5; Supplemental Figure 5).…”