Next Generation RNA Sequencing reveals defects in nuclear mRNA maturation of Arabidopsis thaliana lefko2 mutant

Introduction: The Plant Organelle RNA Recognition (PORR) domain proteins are nucleus-encoded RNA-binding proteins that have acquired specific roles in organelle RNA metabolism as splicing factors of chloroplast group II introns. LEFKOTHEA (At5g62990) is a nuclear gene encoding a PORR domain protein that carries a transit peptide (TP) and monopartite or bipartite nuclear localization signals (NLS). These motifs result in dual-targeting of LEFKOTHEA to the nucleus and chloroplasts implying a role in the splicing of chloroplast group II introns and nuclear pre-mRNA introns. Therefore, we examined the splicing efficiency of plastid and nuclear genes in lefko2 mutant.Methods: The lefko2 mutant was isolated from a genetic screen of an M2 EMS-mutagenized Arabidopsis thaliana Columbia (Col-0) background seed population. The lefko2 mutant allele has a white cotyledon phenotype caused by a G to A mutation in the coding region resulting in a Glycine (G) 373 to Aspartic acid (D) conversion. Total RNA was extracted using plant RNA kit spin columns with an on-column DNase treatment from lefko2 mutant and wild-type Arabidopsis cotyledons. The quantity and integrity of the RNA was assessed using a NanoDrop 1000 spectrophotometer and agarose gel electrophoresis. RNA-seq libraries were generated using the TruSeq Low Input kit according to the manufacturer's instructions (Illumina). Sequencing was performed on an Illumina HiSeq 2000 instrument at BGI (Beijing Genomics Institute). Raw reads were filtered into clean reads and aligned to the Arabidopsis genome (TAIR10). RNA-seq data were analyzed using the SOAP (Release 2.121) with parameters “-s 40 -l 32 -v 3 -r 2” and the TopHat/Cufflinks pipeline (version 2) with parameters “-p 16 --solexa1.3-quals --segment-length 30 --segment-mismatches 2 -r 20 --mate-std-dev 20 --library-type fr-unstranded”. We generated 480 million paired-end reads (101 bp in length) for each sample. On average, about 90% of these reads could be unambiguously aligned to the TAIR10 reference genome sequence. Alternative splicing events were detected for lefko2 and wild-type using Spladder. Intron retention events detected in wild-type were filtered out from lefko2 or vice-versa, and visualized using the Integrative Genomics Viewer (IGV) tool.Results: Splicing defects were observed in numerous nuclear genes of lefko2 cotyledons compared to wild type. Among them, intron retention (IR) events were the most prominent. Further, the fidelity of 5' splice site (5'SS) donor and 3'SS acceptor splicing was disturbed in lefko2 cotyledons. To less extend, exon skipping (ES) defects were also detected.Conclusions: Detailed nuclear splicing events were widely observed in lefko2 cotyledons demonstrating a prevalent role of LEFKOTHEA in the splicing of nuclear pre-mRNA introns.Overall design: RNA-seq libraries were generated using the TruSeq Low Input kit according to the manufacturer's instructions (Illumina). Sequencing was performed on an Illumina HiSeq 2500 instrument at BGI (Beijing Genomics Institute).

2