The integral spliceosomal component CWC15 is required for development in Arabidopsis

Abstract

Efficient mRNA splicing is a prerequisite for protein biosynthesis and the eukaryotic splicing machinery is evolutionarily conserved among species of various phyla. At its catalytic core resides the activated splicing complex Bact consisting of the three small nuclear ribonucleoprotein complexes (snRNPs) U2, U5 and U6 and the so-called NineTeen complex (NTC) which is important for spliceosomal activation. CWC15 is an integral part of the NTC in humans and it is associated with the NTC in other species. Here we show the ubiquitous expression and developmental importance of the Arabidopsis ortholog of yeast CWC15. CWC15 associates with core components of the Arabidopsis NTC and its loss leads to inefficient splicing. Consistent with the central role of CWC15 in RNA splicing, cwc15 mutants are embryo lethal and additionally display strong defects in the female haploid phase. Interestingly, the haploid male gametophyte or pollen in Arabidopsis, on the other hand, can cope without functional CWC15, suggesting that developing pollen might be more tolerant to CWC15-mediated defects in splicing than either embryo or female gametophyte.

Figure 1

Expression pattern of genomic construct gCWC15-3xGFP during plant development. CWC15 expression pattern monitored with a genomic fusion to GFP is visible in all nuclei of gametophytes, embryo, and seedling. (A) Mature female gametophyte with nuclear GFP signal in all tissue types including central cell (asterisk), synergid (arrowhead), and egg cell (arrow). (B) Both vegetative and generative nuclei show CWC15-3xGFP signals in mature pollen grain. Inset: DAPI-stained nuclei in the mature pollen grain. (C) CWC15-3xGFP is present in all nuclei at globular embryo stage. (D) gCWC15-3xGFP expression in epidermal cells. The image is a maximum projection of z-stacks across abaxial cotyledon epidermal cells. Nuclear-localized CWC15-3xGFP is shown in green, cell outlines are stained with propidium iodide (magenta). (E) gCWC15-3xGFP expression in seedling root. Nuclear-localized CWC15-3xGFP is shown in green, cell outlines are stained with Renaissance SR2200 dye (grey). Transverse root section is shown as inset. (F) gCWC15-3xGFP expression in seedling shoot. Nuclear-localized CWC15-3xGFP in primary leaves of a 7-day-old seedling is shown in green, autofluorescence is shown in red. Scale bar: (A–D) 5 μm, (E,F) 100 μm.

Figure 2

Mis-regulation of CWC15 is causative for embryo and seedling phenotypes. (A,B) Normal wild-type (A) and cwc15-1 mutant seedling (B) with short primary root and three cotyledons. (C) Adult cwc15-1 mutant plants (marked by asterisks) grow smaller compared to wild type. (D) Range of cwc15-1 mutant seedling phenotypes with wild-type seedling on the left. Asterisk marks tricot seedling with normal root, arrows point to seedlings with asymmetrically positioned cotyledons, and arrowhead marks seedling with only one fully developed cotyledon. (E) The insertion site of the transgene is in the vicinity of 4 gene loci, among them CWC15 (At3g13200). Mutant alleles with corresponding insertion sites are depicted as cwc15-1 (promoter) and cwc15-2 (SALK insertion line, 3rd intron). Exons are shown as green arrows. Image of gene loci was exported from CLC Genomics Workbench software version 10.1.1 (https://digitalinsights.qiagen.com/products-overview/discovery-insights-portfolio/analysis-and-visualization/qiagen-clc-genomics-workbench/). (F) cwc15-1 rescue lines (at least 6 independent transgenic lines tested for each construct) with seedling phenotypes from left to right: (1) Wild type, (2) cwc15-1 mutant, (3) genomic rescue, (4) rescue with constitutive expression from ribosomal RPS5A promoter, (5) no rescue if expressed from putative promoter of gene At3g10100 which is only active during early embryogenesis. Scale bar: 2 mm.

Figure 3

Embryo phenotypes at early stages of development. (A–I) Corresponding embryos are shown for 2-cell (A–C), 16-cell (D–F), and globular stages (G–I). Wild-type embryos are depicted in (A,D,G) and mutant embryos in (B,C,E,F,H,I). Scale bar: 10 µm.

Figure 4

Down-regulation of CWC15 in cwc15-1 leads to global splicing defects and transcriptional differences. (A) Venn diagram showing number of genes differentially spliced in seedling and pollen tissue, between wild type and mutant. (B) Venn diagram comparing genes detected as differentially spliced and differentially expressed in pollen. (C) Venn diagram comparing genes detected as differentially spliced and differentially expressed in seedlings. Venn Diagram Plotter v1.5.5228 (https://omics.pnl.gov/software/venn-diagram-plotter) was used to generate Venn diagrams.

Figure 5

Loss of CWC15 function in cwc15-2 leads to defects in double fertilization. (A–D) Overlay images of wild-type control (A) or CWC15 deficient ovules (B–D) 20 h after pollination expressing pollen double marker visualizing histones of fertilized endosperm nuclei in red (A,C,D), sperm nuclei in green (B,C,D), and SR2200 counter-staining of pollen tube cell walls in white (A–D). Arrowheads mark endosperm nuclei (red), arrows point to sperm nuclei (green), and asterisks indicate pollen tubes (white). Scale bar: 20 µm.

Figure 6

Zygote and embryo-arrest phenotypes in cwc15-2^+/− mutant embryos. (A) Embryo arrested at zygote stage. (B, C) Embryos arrested at 1-cell stage. (D) Embryo arrested at 2-cell stage. Zygote and embryos are encircled by ellipses. Arrowheads indicate nuclei of fertilized endosperm. Scale bar: 10 µm.