A study of alternative splicing, small RNA, long non-coding RNA, and RNA editing

Abstract

Chloroplasts and mitochondria are two major energy organelles in plant leaves. Chloroplasts convert light energy into chemical energy and mitochondria uses it to produce ATP. Arabidopsis thaliana Purple Acid Phosphatase 2 (AtPAP2) is a phosphatase anchored on the outer membranes of chloroplasts and mitochondria by its C-terminal transmembrane motif. In previous studies, overexpression of AtPAP2 was reported to accelerate plant growth, promote seed yield, and biomass. There are significant higher ATP and sucrose levels in plant leaves of the AtPAP2 overexpression (OE) lines.

Advances of high-throughput sequencing provides us a global view of plant transcriptome. By using this technology, I identified AS events and classified their spliced types in the leaves of 20-day-old wild-type (WT) and high energy OE plants at three timepoints (0, 1 and 8 hr after the light turned on). The differential AS (DAS) events between OE and WT were mostly related to photosynthesis, carbon and nitrogen assimilation processes. Extensive enrichment of AS events was discovered in photosystem genes. More than half of the photosystem genes contained DAS events.

Apart from coding transcripts, long non-coding RNAs are also an important component of transcriptome data. Using a bioinformatics pipeline, 851 novel lncRNAs and 396 TAIR 10 lncRNAs were identified, of which 46 novel lncRNAs and 33 TAIR10 lncRNAs were differentially expressed between OE and WT. Yet, lncRNA profile didn’t exhibit significant change under the high-energy status. Co-expression study showed that, unlike TAIR10 lncRNAs, more novel lncRNAs were positively co-expressed with nearby genes. Eight lncRNAs which could be targets of miRNA were also predicted.

Plant RNA editing is a specific cytidine (C) to uridine (U) RNA substitution in organellar transcripts. AtPAP2 was found to interact with seven MORF proteins which are an essential component of the editosome complex that mediates RNA editing. Then, 34 and 510 RNA editing sites from chloroplast and mitochondrial transcripts were discovered, respectively. The differential editing between OE and WT were clustered in the transcripts of several cytochrome c maturation (Ccm) genes. Western blotting of 2D BN-PAGE showed that the patterns of CcmFN1 polypeptides were different between the lines. It was proposed that AtPAP2 may influence cytochrome c biogenesis by modulating RNA editing through its interaction with MORF proteins.

Besides, I studied microRNAs (miRNA) and phased small interfering RNAs (phasiRNA) in soybean. Cultivated soybean, Glycine max, contributes a great deal to food production, but, compared to its wild kin, Glycine soja, it may lose some genetic information during domestication. In this work, the sRNA profiles of different tissues in both cultivated (C08) and wild soybeans (W05) were analyzed at three stages of development. A total of 443 known miRNAs and 15 novel miRNAs showed differential expression in different samples. Based on a sliding window workflow, 50 and 41 PHAS loci that generated 55 and 46 21-nucleotide phasiRNAs were identified in C08 and W05, respectively. Target prediction of miRNA was performed and 17 C08 miRNAs and 15 W05 miRNAs were predicted to trigger phasiRNAs biogenesis.