A Genetic Screen Identifies PRP18a, a Putative Second Step Splicing Factor Important for Alternative Splicing and a Normal Phenotype inArabidopsis thaliana

Abstract

Splicing of pre-mRNA involves two consecutive trans-esterification steps that take place in the spliceosome, a large dynamic ribonucleoprotein complex situated in the nucleus. In addition to core spliceosomal proteins, each catalytic step requires step-specific factors. Although the Arabidopsis thaliana genome encodes around 430 predicted splicing factors, functional information about these proteins is limited. In a forward genetic screen based on an alternatively-spliced GFP reporter gene in Arabidopsis thaliana, we identified a mutant impaired in putative step II factor PRP18a, which has not yet been investigated for its role in pre-mRNA splicing in plants. Step II entails cleavage at the 3′ splice site accompanied by ligation of the 5′ and 3′ exons and intron removal. In the prp18 mutant, splicing of a U2-type intron with non-canonical AT-AC splice sites in GFP pre-mRNA is reduced while splicing of a canonical GT-AG intron is enhanced, resulting in decreased levels of translatable GFP mRNA and GFP protein. These findings suggest that wild-type PRP18a may in some cases promote splicing at weak, non-canonical splice sites. Analysis of genome-wide changes in alternative splicing in the prp18a mutant identified numerous cases of intron retention and a preponderance of altered 3′ splice sites, suggesting an influence of PRP18a on 3′ splice site selection. The prp18a mutant featured short roots on synthetic medium and small siliques, illustrating that wild-type PRP18a function is needed for a normal phenotype. Our study expands knowledge of plant splicing factors and provides foundational information and resources for further functional studies of PRP18 proteins in plants.

PRP4KA, a Putative Spliceosomal Protein Kinase, Is Important for Alternative Splicing and Development in Arabidopsis thaliana

“…The 3′ AT splice site is only 3 nt downstream of the 3′ AG splice site (Kanno et al 2008, 2016, 2017a,b). Figure adapted from figure 1 in Kanno et al (2018).…”

Highly ABA-Induced 1 (HAI1)-Interacting protein HIN1 and drought acclimation-enhanced splicing efficiency at intron retention sites

“…The effect of low ψ w acclimation on pre-mRNA splicing patterns was examined using an established workflow to identify splice variants that had a stress-induced change in abundance which could not be accounted for by change in total transcript level (37, 38). Among the splicing events significantly (Q < 0.05) affected by low ψ w in WT, IR was most prevalent (521 sites from 400 genes; Fig.…”

Differential Alternative Splicing Genes and Isoform Regulation Networks of Rapeseed (Brassica napus L.) Infected with Sclerotinia sclerotiorum

“…The AS of SR genes can increase plant tolerance to hormones and stress treatments in rice and B. napus [ 51 , 67 , 68 ]. It was interesting to discover that the gene encoding the splicing factor PRP18 protein ( BnaA09g50970D ) is an overlapping DAS gene, as the homologous mutant in A. thaliana produces short roots and small siliques following its induction of large genome-wide IR events [ 45 ]. Additionally, BnaCnn1978 , encoding an RNA binding protein, was identified as an infection-induced DAS gene.…”

“…Based on the annotated functions of the homologous genes in A. thaliana, four of the 79 DAS genes were putatively related to RNA splicing, including BnaCnn1978 (RNA-binding protein), BnaC08g21130D (SR protein 34a), and BnaC07g01360D (RNA-binding protein). One gene (BnaA09g50970D, splicing factor PRP18 protein), which was previously shown to be involved in promoting splicing at weak splice sites [45], was identified in both the infection-induced and cultivar-related DAS gene groups. Three of the 79 DAS genes were known to be related to the defense response, including BnaA05g20710D (immune-associated nucleotide binding 9), which encodes a plasma membrane-localized protein that functions as a negative regulator of basal immunity in A. thaliana [46]; BnaC09g07670D (LRR [Leucine-rich repeat] protein), which is reported likely as a pseudogene with its two paralogs transducing the defense signal downstream of TNL(TIR -type sensor NLR) in A. thaliana [47]; and BnaC04g37090D (immunoglobulin E-set superfamily protein).…”