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.

Keywords: Arabidopsis thaliana; Cajal body; alternative splicing; coilin; stress.

Figure 1

GFP reporter gene system and GFP phenotypes in mutants. (A) The GFP reporter gene in the T line and alternative splicing of GFP pre-mRNA. The GFP coding region (green bar) is under the control of virus-derived transcriptional regulatory elements: a truncated 35S promoter (TATA) and the endogenous pararetrovirus (EPRV) enhancer (∼1.2 kb, black bar), which contains a tandem repeat (three copies of a 41–42-bp monomer, arrowheads) in the 5′ distal portion. Transcription of GPF pre-mRNA begins around the tandem repeat region. Two opposing arrows above the diagram indicate the positions of primers used for RT-PCR to detect three major GFP transcripts: the untranslatable “long” and “middle” transcripts, and a “short” translatable mRNA. The middle and short transcripts result from alternative splicing of U2-type introns containing canonical (GT-AG) and noncanonical (AT-AC) splice sites, respectively (Sasaki et al., 2015). (B) GFP phenotypes in seedlings. The wild-type T line shows an intermediate level of GFP fluorescence visible mainly in the stem (hypocotyl) and shoot and root apices of young seedlings. Mutants obtained following EMS mutagenesis of the T line could display either reduced (“Weak”) or stronger (“Hyper”) GFP fluorescence. Cotyledons (the first set of leaves sprouting from the seed) appear red owing to auto-fluorescence of chlorophyll at the excitation wavelength for GFP.

Figure 2

Domain organization of Arabidopsis coilin and the positions of hgf loss-of-function mutations. Arabidopsis coilin consists of 608 amino acids. The N-terminal self-association domain and C-terminal Tudor-like domain are the most highly conserved regions of coilin proteins. Analysis of the secondary structure of Arabidopsis coilin predicted three domains: the N-terminal globular domain (NOD), the internal disordered domain (IDD), and the C-terminal domain (CTD) (Makarov et al., 2013). Also shown are two nuclear localization (NLS) signals and one cryptic nucleolar localization signal (NoLS) as well as E-rich and K-rich domains (Makarov et al., 2013). The eight hgf mutations retrieved in our forward genetic screen are indicated. Some alleles (hfg1-1, hgf1-3, hgf1-6, and hgf1-8) were obtained more than once. The corresponding nucleotide changes are shown in Figure S2.

Figure 3

Dispersion of CBs in a coilin mutant. Owing to expression of the CB marker U2B″:GFP, CBs are visible as single, highly fluorescent spots, often close to the nucleolus (dark spherical area), in leaf nuclei of wild-type plants (top). By contrast, in the hgf1-7 coilin mutant, CBs are either uniformly absent or much smaller and less intensely fluorescent in all cells examined. The white bar indicates 10 µm.

Figure 4

Levels of GFP protein in coilin mutants. (A) Hyper-GFP fluorescence in coilin mutant seedlings. Appearance of ∼1–2-week-old seedlings of coilin mutants hgf1-1 through hgf1-8 as well as the wild-type T line and untransformed Col-0 growing on solid MS medium as visualized under a fluorescence stereo microscope. GFP fluorescence is high in the hypocotyls. Cotyledons appear red owing to auto-fluorescence of chlorophyll at the excitation wavelength of GFP. (B) Western blot analysis of GFP protein in coilin mutants. Separate lanes for the wild-type T line and nontransgenic Col-0 lanes are shown for samples that were run on separate gels. A tubulin loading control is visible at the top of each lane. The second smaller band migrating slightly below the GFP protein is likely a degradation product that is particularly noticeable when the GFP protein levels are high.

Figure 5

Abundance of GFP RNA isoforms in coilin mutants. (A) RT-PCR of GFP RNAs in coilin mutants. Semiquantitative RT-PCR was used to assess the accumulation of long, middle, and short GFP transcripts in two coilin mutants (hgf1-1 and hgf1-8), the wild-type T line, and nontransgenic Col-0. Actin is shown as a constitutively expressed control. –RT, no reverse transcriptase; gDNA, genomic DNA. (B) Percentages of the three major splice variants of GFP RNA were predicted based on RNA-seq data (Table S5, Table S8, and Table S9).

Figure 6

Examples of introns affected in splicing efficiency in coilin mutants. The number of reads for several introns that show either more-efficient splicing (MES; upper part) or increased intron retention (IR; lower part) in coilin mutants containing either the hgf1-8 or hgf1-1 allele compared with the wild-type T line are visualized by the Integrative Genomic Viewer. The target intron and the exons before and after the intron are shown by the blue bars and blue boxes, respectively. The Arabidopsis Genome Initiative (AGI) number for the target intron-containing gene and the range for counting the reads are shown at the top and bottom of each figure. The numbers on the left side of each figure indicate the number of technical replicates for each indicated plant line. The genes shown here are not among those that are differentially expressed genes in the T and coilin mutant lines.