Identification of Coilin Mutants in a Screen for Enhanced Expression of an Alternatively Spliced GFP Reporter Gene in Arabidopsis thaliana

Abstract

Coilin is a marker protein for subnuclear organelles known as Cajal bodies, which are sites of various RNA metabolic processes including the biogenesis of spliceosomal small nuclear ribonucleoprotein particles. Through self-associations and interactions with other proteins and RNA, coilin provides a structural scaffold for Cajal body formation. However, despite a conspicuous presence in Cajal bodies, most coilin is dispersed in the nucleoplasm and expressed in cell types that lack these organelles. The molecular function of coilin, particularly of the substantial nucleoplasmic fraction, remains uncertain. We identified coilin loss-of-function mutations in a genetic screen for mutants showing either reduced or enhanced expression of an alternatively spliced GFP reporter gene in Arabidopsis thaliana. The coilin mutants feature enhanced GFP fluorescence and diminished Cajal bodies compared with wild-type plants. The amount of GFP protein is several-fold higher in the coilin mutants owing to elevated GFP transcript levels and more efficient splicing to produce a translatable GFP mRNA. Genome-wide RNA-sequencing data from two distinct coilin mutants revealed a small, shared subset of differentially expressed genes, many encoding stress-related proteins, and, unexpectedly, a trend toward increased splicing efficiency. These results suggest that coilin attenuates splicing and modulates transcription of a select group of genes. The transcriptional and splicing changes observed in coilin mutants are not accompanied by gross phenotypic abnormalities or dramatically altered stress responses, supporting a role for coilin in fine tuning gene expression. Our GFP reporter gene provides a sensitive monitor of coilin activity that will facilitate further investigations into the functions of this enigmatic protein.

A genetic screen implicates a CWC16/Yju2/CCDC130 protein and SMU1 in alternative splicing in Arabidopsis thaliana

“…The other two transcripts-a spliced GFP transcript resulting from removal of a canonical GT-AG intron and an unspliced GFP pre-mRNA-are not translatable owing to the presence of many premature termination codons after the initiating methionine ( Fig. 1; Kanno et al 2016). Our working hypothesis is that mutations in genes encoding splicing factors will change the ratio of the three transcripts and hence either increase or decrease GFP mRNA levels.…”

“…Our working hypothesis is that mutations in genes encoding splicing factors will change the ratio of the three transcripts and hence either increase or decrease GFP mRNA levels. Such changes will result, respectively, in either a "Hyper-GFP" or "GFP-weak" phenotype relative to the wild-type T line, which has an intermediate level of GFP fluorescence (Kanno et al 2016).…”

“…In addition to around a dozen GFP-weak (gfw) complementation groups, we recovered approximately 10 Hyper-GFP (hgf) complementation groups including hgf1, which comprises mutants defective in the Cajal body marker protein coilin (At1g13030) (Kanno et al 2016). …”

“…The downstream gray stippled area and ATG represent an unused promoter and initiation codon, respectively. The 3 ′ splice sites for the GT-AG and AT-AC introns are separated by only 3 nt with the noncanonical AC on the outside (Sasaki et al 2015;Kanno et al 2016).…”

“…To identify proteins that impact splicing efficiency and alternative splicing in plants, we are performing a forward genetic screen based on an alternatively spliced GFP reporter gene in Arabidopsis. The usefulness of this genetic system has been validated by the identification of a novel factor, RTF2 (Replication Termination Factor2), which may participate in ubiquitin-based regulation of the spliceosome (Sasaki et al 2015), and the finding of an unexpected role for the Cajal body marker protein coilin in attenuating splicing efficiency of a small subset of stress-related genes (Kanno et al 2016). Here we report three new mutants identified in the screen that are defective, respectively, in splicing factors SMU1, SmF, and CWC16, which are related to budding yeast first step factor Yju2 and human CCDC130.…”