GSE106459
Next Generation RNA Sequencing reveals defects in nuclear mRNA maturation of Arabidopsis thaliana lefko1 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 lefko1 mutant Methods: The lefko1 mutant was isolated from a genetic screen of an M2 EMS-mutagenized Arabidopsis thaliana Columbia (Col-0) background seed population. The lefko1 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 lefko1 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 SOAPnuke (version v.1.5.2) with parameters “-l 15 -q 0.2 -n 0.05” and the HISAT2 pipeline (version 2.0.4) with parameters “--phred64 --sensitive --no-discordant --no-mixed -I 1 -X 1000”. Differential alternative splicing events were detected for lefko1 and wild-type using rMATS and visualized using the Integrative Genomics Viewer (IGV) tool. Results: Splicing defects were observed in numerous nuclear genes of lefko1 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 lefko1 cotyledons. To less extend, exon skipping (ES) defects were also detected. Conclusions: Detailed nuclear splicing events were widely observed in lefko1 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). Conclusions: Detailed nuclear splicing events were widely observed in lefko1 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).