Complex Processing Patterns of mRNAs of the Large ATP Synthase Operon in Arabidopsis Chloroplasts

Abstract

Chloroplasts are photosynthetic cell organelles which have evolved from endosymbiosis of the cyanobacterial ancestor. In chloroplasts, genes are still organized into transcriptional units as in bacteria but the corresponding poly-cistronic mRNAs undergo complex processing events, including inter-genic cleavage and 5′ and 3′ end-definition. The current model for processing proposes that the 3′ end of the upstream cistron transcripts and the 5′ end of the downstream cistron transcripts are defined by the same RNA-binding protein and overlap at the level of the protein-binding site.We have investigated the processing mechanisms that operate within the large ATP synthase (atp) operon, in Arabidopsis thaliana chloroplasts. This operon is transcribed by the plastid-encoded RNA polymerase starting from two promoters, which are upstream and within the operon, respectively, and harbors four potential sites for RNA-binding proteins. In order to study the functional significance of the promoters and the protein-binding sites for the maturation processes, we have performed a detailed mapping of the atp transcript ends. Our data indicate that in contrast to maize, atpI and atpH transcripts with overlapping ends are very rare in Arabidopsis. In addition, atpA mRNAs, which overlap with atpF mRNAs, are even truncated at the 3′ end, thus representing degradation products. We observe, instead, that the 5′ ends of nascent poly-cistronic atp transcripts are defined at the first protein-binding site which follows either one of the two transcription initiation sites, while the 3′ ends are defined at the subsequent protein-binding sites or at hairpin structures that are encountered by the progressing RNA polymerase. We conclude that the overlapping mechanisms of mRNA protection have only a limited role in obtaining stable processed atp mRNAs in Arabidopsis. Our findings suggest that during evolution of different plant species as maize and Arabidopsis, chloroplasts have evolved multiple strategies to produce mature transcripts suitable for translation.

Systematic sequencing of chloroplast transcript termini fromArabidopsis thalianareveals >200 transcription initiation sites and the extensive imprints of RNA-binding proteins and secondary structures

“…Five TSS were identified in the atpI cluster, including what might be specific promoters for atpH and atpA, and 15 TSS in the ndhH cluster, which were located predominantly toward the distal end. Most of these 3' and 5' termini were previously identified in a thorough investigation of the atpI cluster processing (53). The existence of an accumulating monocistronic atpI transcript in Arabidopsis has been debated (53,66) and our data suggest that the main atpI 3' end is 584 nt downstream of the stop codon, inside atpH.…”

“…The existence of an accumulating monocistronic atpI transcript in Arabidopsis has been debated (53,66) and our data suggest that the main atpI 3' end is 584 nt downstream of the stop codon, inside atpH. This 3' end is more abundant than the one at position +493, whose processing depends on PPR10 (53,67). 27 termini in addition to TSS were found for the ndhH cluster.…”

“…Although we attempted to obtain an exhaustive catalog of TSS, the data filters we used to reduce the false discovery rate eliminated a few known initiation sites (Supplementary Figure S3B). For instance, the PpsbN -32 promoter (51) and PatpE -430 (52,53) are absent from our list because they have a +TAP:-TAP ratio of <10 (9.9 for PatpE and 2.5 for PpsbN). On the other hand, our data shed light on the uncertainty of whether PpsbD -186 (position 32525) is a genuine promoter or processed end (54)(55)(56), with our results being strongly diagnostic of a posttranscriptional processing site.…”

“…The major Terminome-Seq 3' end is located further downstream, inside the atpH coding region and does not correlate with a smRNA. Systematic mapping of the atp operon termini already revealed that transcripts with overlapping ends are a minority (53) and co-immunoprecipitation showed that PPR10 is preferentially associated with processed atpH mRNA rather than processed atpI mRNA (67). At a genomewide level, only ~10 footprints (including PPR38 and HCF152) can be linked to simultaneous stabilization of 5' and 3' ends, suggesting that this is the exception rather than the rule.…”

Relative expression of rRNA transcripts and 45S rDNA promoter methylation status are dysregulated in tumors in comparison with matched-normal tissues in breast cancer

“…Some forms of polycistronic mRNAs and miRNAs that are expressed from the same promoter have been shown to be post-transcriptionally regulated and exist at different levels in plants (53,54). The SNRPN-SNuRF gene, possessing a biscistronic structure and sharing a common promoter, for example, is differentially expressed, as identified by both northern blot and microarray analysis in mammalian cells (55,56).…”