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RIPseq of AtMKIP1-YFP and AtBE3-YFP in Arabidopsis thaliana

ABSTRACT: Chloroplast group IIA introns derive from bacterial ribozymes. Their splicing likely requires Maturase K (MatK), which has been largely inaccessible to functional analyses being itself a chloroplast intron-encoded protein. We show that MatK physically interacts with a conserved, essential plastid-localized homolog of starch branching enzymes (BEs), dubbed MATURASE K INTERACTING PROTEIN1 (MKIP1). We demonstrate that MKIP1 proteins have lost BE activity and acquired an insertion enabling direct interaction with the N-terminal region of MatK. Arabidopsis MKIP1 specifically co-precipitates all known intron targets of MatK. Induced MKIP1 silencing results in pale newly emerging leaves, in which the splicing of these intron targets is strongly reduced. Our data suggest that MKIP1 functionally diverged from canonical BEs to facilitate splicing in conjunction with MatK. We propose that the N-terminus of MatK, in turn, has evolved from an RNA-binding domain into a platform for protein interaction, helping its transition towards a general splicing factor.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE284378 | GEO | 2026/02/11

REPOSITORIES: GEO

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Project description: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 is represented by the Salk T-DNA collection unicode SALK_044699/emb1692 and was obtained from the Nottingham Arabidopsis Stock Centre (NASC, Nottingham, UK). Embryo development in homozygous lefko2 plants is ceased abruptly arresting embryogenesis upon globular-to-heart stage transition. Total RNA was extracted using plant RNA kit spin columns with an on-column DNase treatment from heart-stage lefko2 mutant and wild-type Arabidopsis embryos. 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: RNA-seq uncovered an outstanding number of coding or non-coding novel transcripts in lefko2 mutant embryos. Splicing defects were observed in numerous nuclear and plastid genes of lefko2 embryos 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 highly disturbed in lefko2 embryos. To less extend, exon skipping (ES) defects were also detected. Conclusions: Detailed nuclear and chloroplast splicing events were widely observed in lefko2 aborted mutant embryos demonstrating a prevalent role of LEFKOTHEA in the splicing of nuclear pre-mRNA introns and chloroplast group II introns.

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RIPseq of AtMKIP1-YFP and AtBE3-YFP in Arabidopsis thaliana

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