Global RNA association with the transcriptionally active chromosome of chloroplasts

Abstract

Processed chloroplast RNAs are co-enriched with preparations of the chloroplast transcriptionally active chromosome. Chloroplast genomes are organized as a polyploid DNA-protein structure called the nucleoid. Transcriptionally active chloroplast DNA together with tightly bound protein factors can be purified by gel filtration as a functional entity called the transcriptionally active chromosome (TAC). Previous proteomics analyses of nucleoids and of TACs demonstrated a considerable overlap in protein composition including RNA binding proteins. Therefore the RNA content of TAC preparations from Nicotiana tabacum was determined using whole genome tiling arrays. A large number of chloroplast RNAs was found to be associated with the TAC. The pattern of RNAs attached to the TAC consists of RNAs produced by different chloroplast RNA polymerases and differs from the pattern of RNA found in input controls. An analysis of RNA splicing and RNA editing of selected RNA species demonstrated that TAC-associated RNAs are processed to a similar extent as the RNA in input controls. Thus, TAC fractions contain a specific subset of the processed chloroplast transcriptome.

Chloroplast Translation: Structural and Functional Organization, Operational Control, and Regulation

“…There is no obvious spatial separation that would compartmentalize RNA metabolism and translation. Interestingly, many of the factors known to be involved in transcript processing (e.g., RNA binding proteins), stabilization, and translation colocalize with processed and unprocessed transcripts in nucleoids and transcription complexes (Pfalz et al, 2006;Majeran et al, 2012;Lehniger et al, 2017). This raises the question whether unprocessed mRNAs, often referred to as "precursors" or "immature transcripts," are utilized as templates for translation or whether any partitioning (e.g., temporal separation) exists between RNA processing and translation in plastids.…”

Transcription-directed membrane association organizes the chloroplast nucleoid structure

“…The in vivo relevance of the central body remains unclear due to the limitations of sample fixation for electron microscopy and insufficient resolution of light microscopy. The function of the central body is also unclear with some evidence favoring this structure as the site of active transcription 11,15 while others suggesting that it may be associated with lower levels of transcription 16 .…”