AGFPsplicing reporter in acoilinmutant background reveals links between alternative splicing, siRNAs and coilin function inArabidopsis thaliana

Abstract

AbstractCoilin is a scaffold protein essential for the structure of Cajal bodies, which are nucleolar-associated, nonmembranous organelles that coordinate the assembly of nuclear ribonucleoproteins (RNPs) including spliceosomal snRNPs. To learn more about coilin functions and pathways in plants, we conducted a genetic suppressor screen using acoilin (coi1)mutant inArabidopsis thalianaand performed an immunoprecipitation-mass spectrometry analysis on coilin protein. Thecoi1mutations modify alternative splicing of aGFPreporter gene, resulting in a ‘hyper-GFP’ phenotype in youngcoi1seedlings relative to the intermediate wild-type level. As shown here, this hyper-GFP phenotype is extinguished in oldercoi1seedlings by posttranscriptional gene silencing triggered by siRNAs derived from aberrant splice variants ofGFPpre-mRNA. In thecoi1suppressor screen, we identified suppressor mutations in WRAP53, a putative coilin-interacting protein; SMU2, a predicted splicing factor; and ZC3HC1, an incompletely characterized zinc finger protein. These suppressor mutations return the hyper-GFP fluorescence of youngcoi1seedlings to the intermediate wild-type level. Additionally,zc3hc1 coi1mutants display more extensiveGFPsilencing and elevated levels ofGFPsiRNAs, suggesting the involvement of wild-type ZC3HC1 in siRNA biogenesis or stability. The immunoprecipitation-mass spectrometry analysis reinforced coilin’s roles in pre-mRNA splicing, nucleolar chromatin structure, and rRNA processing. Coilin’s participation in these processes, at least some of which incorporate small RNAs, supports the hypothesis that coilin acts as a chaperone for small noncoding RNAs. Our study demonstrates the usefulness of theGFPsplicing reporter for investigating alternative splicing, ribosome biogenesis, and siRNA-mediated silencing in the context of coilin function.