64To investigate factors influencing pre-mRNA splicing in plants, we conducted a forward 65 genetic screen using an alternatively-spliced GFP reporter gene in Arabidopsis thaliana. This 66 effort generated a collection of sixteen mutants impaired in various splicing-related proteins, 67 many of which had not been recovered in any prior genetic screen or implicated in splicing in 68 plants. The factors are predicted to act at different steps of the spliceosomal cycle, snRNP 69 biogenesis pathway, transcription, and mRNA transport. We have described eleven of the 70 mutants in recent publications. Here we present the final five mutants, which are defective, 71 respectively, in RNA-BINDING PROTEIN 45D (rbp45d), DIGEORGE SYNDROME 72WITH SPT6 (iws1) and CAP BINDING PROTEIN 80 (cbp80). We provide RNA-sequencing 74 data and analyses of differential gene expression and alternative splicing patterns for the cbp80 75 mutant and for several previously published mutants, including smfa and new alleles of cwc16a, 76 for which such information was not yet available. Sequencing of small RNAs from the cbp80 77 mutant highlighted the necessity of wild-type CBP80 for processing of microRNA (miRNA) 78 precursors into mature miRNAs. Redundancy tests of paralogs encoding several of the splicing 79 factors revealed their functional non-equivalence in the GFP reporter gene system. We discuss 80 the cumulative findings and their implications for the regulation of pre-mRNA splicing 81 efficiency and alternative splicing in plants. The mutant collection provides a unique resource for 82 further studies on a coherent set of splicing factors and their roles in gene expression, alternative 83 splicing and plant development. 84 85 86 87 88 89 90 91 92 93 94 95Splicing of pre-mRNAs by the excision of introns and ligation of flanking exons is a 96 prerequisite for the expression of most eukaryotic genes. Splicing entails two transesterification 97 reactions carried out by the spliceosome, a large and dynamic ribonucleoprotein (RNP) machine 98 located in the nucleus. At least six structurally and functionally distinct spliceosomal complexes 99 containing core spliceosomal proteins, transiently-associated factors and different combinations 100 of five different small nuclear (sn) RNAs -U1, U2, U4, U5 and U6 -act sequentially to execute 101 the two catalytic steps of the splicing process (Matera and Wang, 2014;Yan et al., 2017). The 102 spliceosome is able to carry out constitutive splicing, in which the same splice sites are always 103 used for a given intron, and alternative splicing, in which splice site usage for a given intron is 104 variable. Alternative splicing increases transcriptome and proteome diversity (Nilsen and 105 Graveley, 2010;Syed et al., 2012;Reddy et al., 2013) and is important for development and 106 stress adaptation in plants (Staiger and Brown, 2013; Filichkin et al., 2015; Szakonyi and Duque, 107 2018). 108Most information on spliceosome composition and the splicing mechanism has been derived 109 from genetic, bioc...

Prp4 kinase (Prp4k) is the first spliceosome-associated kinase shown to regulate splicing in fungi and metazoans, but nothing is yet known about its functions in plants. Here, Kanno and Venhuizen et al. report...

Coilin is a scaffold protein essential for the structure of Cajal bodies, which are nucleolar-associated, nonmembranous organelles that coordinate the assembly of nuclear ribonucleoproteins including spliceosomal snRNPs. To learn more about coilin functions and pathways in plants, we conducted a genetic suppressor screen using a coilin mutant in Arabidopsis thaliana and performed an immunoprecipitation-mass spectrometry analysis on coilin protein. The coi1 mutations modify alternative splicing of a GFP reporter gene, resulting in a hyper-GFP phenotype in young coi1 seedlings relative to the intermediate wild-type level. As shown here, this hyper-GFP phenotype is extinguished in older coi1 seedlings by posttranscriptional gene silencing triggered by siRNAs derived from aberrant splice variants of GFP pre-mRNA. In the coi1 suppressor 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 young coi1 seedlings to the intermediate wild-type level. Additionally, zc3hc1 coi1 mutants display more extensive GFP silencing and elevated levels of GFP siRNAs, suggesting the involvement of wild-type ZC3HC1 in siRNA biogenesis or stability. The immunoprecipitation-mass spectrometry analysis reinforced the roles of coilin in pre-mRNA splicing, nucleolar chromatin structure, and rRNA processing. The participation of coilin 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 the GFP splicing reporter for investigating alternative splicing, ribosome biogenesis, and siRNA-mediated silencing in the context of coilin function.

Coilin 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 study coilin function in plants, we conducted a genetic suppressor screen using a coilin (coi1) mutant in Arabidopsis thaliana and performed an immunoprecipitation-mass spectrometry analysis on coilin protein. The coi1 mutations modify alternative splicing of a GFP reporter gene, resulting in a hyper-GFP phenotype in young coi1 seedlings relative to the intermediate wild-type level. As shown here, this hyper-GFP phenotype is extinguished in older coi1 seedlings by posttranscriptional gene silencing triggered by siRNAs derived from aberrant splice variants of GFP pre-mRNA. In the coi1 suppressor screen, we identified suppressor mutations in WRAP53, a putative coilin-interacting protein; SMU2, a predicted splicing factor; and ZCH1, an incompletely characterized zinc finger protein. These suppressor mutations return the hyper-GFP fluorescence of young coi1 seedlings to the intermediate wild-type level. Additionally, coi1 zch1 mutants display more extensive GFP silencing and elevated levels of GFP siRNAs, suggesting the involvement of wild-type ZCH1 in siRNA biogenesis or stability. The immunoprecipitation-mass spectrometry analysis reinforced the roles of coilin in pre-mRNA splicing, nucleolar chromatin structure, and rRNA processing. The participation of coilin in these processes, at least some of which incorporate small RNAs, supports the hypothesis that coilin provides a chaperone for small RNA trafficking. Our study demonstrates the usefulness of the GFP splicing reporter for investigating alternative splicing, ribosome biogenesis, and siRNA-mediated silencing in the context of coilin function.