Transcriptional Slippage and RNA Editing Increase the Diversity of Transcripts in Chloroplasts: Insight from Deep Sequencing of Vigna radiata Genome and Transcriptome.

Ching-Ping Lin,Ching-I Kuo,Roland Schafleitner,Mao-Sen Liu,Chia-Yun Ko,Long-Fang Oliver Chen,Stefan Maas

doi:10.1371/journal.pone.0129396

Abstract

We performed deep sequencing of the nuclear and organellar genomes of three mungbean genotypes: Vigna radiata ssp. sublobata TC1966, V. radiata var. radiata NM92 and the recombinant inbred line RIL59 derived from a cross between TC1966 and NM92. Moreover, we performed deep sequencing of the RIL59 transcriptome to investigate transcript variability. The mungbean chloroplast genome has a quadripartite structure including a pair of inverted repeats separated by two single copy regions. A total of 213 simple sequence repeats were identified in the chloroplast genomes of NM92 and RIL59; 78 single nucleotide variants and nine indels were discovered in comparing the chloroplast genomes of TC1966 and NM92. Analysis of the mungbean chloroplast transcriptome revealed mRNAs that were affected by transcriptional slippage and RNA editing. Transcriptional slippage frequency was positively correlated with the length of simple sequence repeats of the mungbean chloroplast genome (R2=0.9911). In total, 41 C-to-U editing sites were found in 23 chloroplast genes and in one intergenic spacer. No editing site that swapped U to C was found. A combination of bioinformatics and experimental methods revealed that the plastid-encoded RNA polymerase-transcribed genes psbF and ndhA are affected by transcriptional slippage in mungbean and in main lineages of land plants, including three dicots (Glycine max, Brassica rapa, and Nicotiana tabacum), two monocots (Oryza sativa and Zea mays), two gymnosperms (Pinus taeda and Ginkgo biloba) and one moss (Physcomitrella patens). Transcript analysis of the rps2 gene showed that transcriptional slippage could affect transcripts at single sequence repeat regions with poly-A runs. It showed that transcriptional slippage together with incomplete RNA editing may cause sequence diversity of transcripts in chloroplasts of land plants.

Highlights

The chloroplast (CP) genome originated from the genome of endosymbiontic cyanobacterialike photosynthetic bacteria [1,2,3,4]
Through baiting and iterative mapping, CP sequence reads were extracted from the total read pools and assembled into whole CP genomes
The study demonstrated that deep RNA-seq is a powerful tool to obtain a detailed view of transcript variability and can detect sequence variations caused by transcriptional slippage (TS) and RNA editing (RE)

Summary

Introduction

The chloroplast (CP) genome originated from the genome of endosymbiontic cyanobacterialike photosynthetic bacteria [1,2,3,4]. The CP genes carry out functions in photosynthesis (e.g., atp, pet, ndh, psa and psb), or are involved with bacteria-like transcription (e.g., rpoA, B, C1, C2) and translation (e.g., rpl, rps, tRNAs, rRNAs). In bacteria such as Escherichia coli, transcriptional slippage (TS) occurs at simple sequence repeat (SSR) regions, especially at poly-A or poly-T runs [10,11]. If TS occurs in open reading frames, RNA transcripts of a gene are expected to encode different protein products Both plastid-encoded RNA polymerase (PEP) and nuclear-encoded RNA polymerases (NEP) are active in CPs [12]. Several RNA-seq-based papers showed the genome-wide view of transcript variability in Arabidopsis thaliana [16,17,18] and ferns [19], but these studies focused on only RNA editing (RE), not TS

Methods

Results

Conclusion