The Arabidopsis U11/U12-65K is an indispensible component of minor spliceosome and plays a crucial role in U12 intron splicing and plant development

Abstract

The U12-dependent introns have been identified in a wide range of eukaryotes and are removed from precursor-mRNAs by U12 intron-specific minor spliceosome. Although several proteins unique to minor spliceosome have been identified, the nature of their effect on U12 intron splicing as well as plant growth and development remain largely unknown. Here, we characterized the functional role of an U12-type spliceosomal protein, U11/U12-65K in Arabidopsis thaliana. The transgenic knockdown plants generated by artificial miRNA-mediated silencing strategy exhibited severe defect in growth and development, such as severely arrested primary inflorescence stems, serrated leaves, and the formation of many rosette leaves after bolting. RNA sequencing and reverse transcription polymerase chain reaction (RT-PCR) analyses revealed that splicing of 198 out of the 234 previously predicted U12 intron-containing genes and 32 previously unidentified U12 introns was impaired in u11/u12-65k mutant. Moreover, the U11/U12-65K mutation affected alternative splicing, as well as U12 intron splicing, of many introns. Microarray analysis revealed that the genes involved in cell wall biogenesis and function, plant development, and metabolic processes are differentially expressed in the mutant plants. U11/U12-65K protein bound specifically to U12 small nuclear RNA (snRNA), which is necessary for branch-point site recognition. Taken together, these results provide clear evidence that U11/U12-65K is an indispensible component of minor spliceosome and involved in U12 intron splicing and alternative splicing of many introns, which is crucial for plant development.

Keywords: Arabidopsis; U11/U12-65K; U12 intron; minor spliceosome; plant development; splicing.