Developmentally-specific transcripts from the ccmFN-rps1 locus in wheat mitochondria

Abstract

We have examined precursor and processed transcripts arising from the wheat mitochondrial ccmFN-rps1 region, which encodes a cytochrome c biogenesis component and S1 ribosomal protein, for the embryo-to-seedling stages of development. Northern analysis revealed 3.2-kb ccmFN-rps1 precursors, 2.6-kb bicistronic mRNA and 0.7-kb monocistronic rps1 transcripts, although their relative abundances were seen to shift during development. The 3.2-kb transcript levels peak during the 12-h to 2-day period, whereas 2.6-kb transcripts continue to increase during seedling growth, consistent with the newly-synthesized RNAs being more efficiently processed in later developmental stages. The 3.2-kb ccmFN-rps1 precursors consist of primary transcripts and 5'-processed RNAs based on pyrophosphatase-treated circular-RT-PCR analysis, whereas the 5' termini of 2.6-kb transcripts appear to be generated by endonucleolytic cleavage. The 0.7-kb rps1 transcripts are abundant during early germination but not in the seedlings; their 5' ends are heterogeneous and most of them lack the expected initiation codon. Notably all three size classes of RNAs share similar 3' termini. The 2.6-kb ccmFN-rps1 mRNAs exhibited full C-to-U editing at the sites examined, whereas the other two categories were slightly under-edited. A subset of all three-sized transcripts possessed short stretches of non-encoded adenosines, thus adding another layer of complexity to RNA level events in plant mitochondria.

Multiple splicing pathways of group II trans-splicing introns in wheat mitochondria

“…S4). Our observations are in keeping with the view that generation of the mRNA 3'terminus is an early and simple RNA processing event compared to more complex 5′ end maturation steps (Forner et al, 2007;Calixte and Bonen, 2008) and that these 3′ termini appear to be protected from degradation.…”

Deep sequencing of the tobacco mitochondrial transcriptome reveals expressed ORFs and numerous editing sites outside coding regions

“…Orf216 encodes a mitochondrial RPS1 protein. RPS1 has not been defined in tobacco, but has been identified in the mitogenome other plants such as wheat and primrose [ 45 , 46 ]. Five ORFS — 160, 115 , 166b, 177 , and 222 do not encode identifiable proteins but are transcribed and polysome associated.…”

Impact of genomic environment on mitochondrial rps7 mRNA features in grasses

“…The 5Ј terminal heterogeneity seen in certain grasses may reXect a combination of the use of multiple promoters, discrete endonucleolytic cleavage events, and random exonucleolytic attack, the latter being mitigated if there is protection by secondary structure and/or RNA stability proteins. It is worth noting that the 5Ј heterogeneity seen for rps7 mRNAs is less than we observed for rps1 monocistronic transcripts in wheat embryos, where many molecules lacked the expected initiation codon, even though they all possessed full-length 3Ј UTRs (Calixte and Bonen 2008). Although little is known about the nature of factors which specify end-cleavage, an RNA processing factor RPF2 has recently been shown to be required for 5Ј-end maturation of nad9 and cox3 mRNAs in Arabidopsis (Jonietz et al 2010).…”

“…This study has focused on mitochondrial RNA events in germinating embryos, and because mitochondrial ribosomal protein transcripts appear to be particularly sensitive to developmental regulation (cf. Li-Pook-Than et al 2004;Calixte and Bonen 2008;Naydenov et al 2008), it will also be of interest to examine the behavior of mitochondrial RNA maturation/stability pathways during other stages of plant development. Our preliminary examination of rps7 transcript proWles for other grasses in the seedling stage suggests that they too have relatively lower steadystate levels of precursors and mature mRNAs than in germinating embryos (unpublished observations).…”

“…Kuhn and Binder 2002;Calixte and Bonen 2008) was used. To circularize the transcripts, approximately 5 g of mitochondrial RNA was incubated overnight with T4 RNA ligase (New England Biolabs) in the presence of RNAsin (Promega).…”