CYCLIN-DEPENDENT KINASE G1 is associated with the spliceosome to regulate CALLOSE SYNTHASE5 splicing and pollen wall formation in Arabidopsis

Abstract

Arabidopsis thaliana CYCLIN-DEPEDENT KINASE G1 (CDKG1) belongs to the family of cyclin-dependent protein kinases that were originally characterized as cell cycle regulators in eukaryotes. Here, we report that CDKG1 regulates pre-mRNA splicing of CALLOSE SYNTHASE5 (CalS5) and, therefore, pollen wall formation. The knockout mutant cdkg1 exhibits reduced male fertility with impaired callose synthesis and abnormal pollen wall formation. The sixth intron in CalS5 pre-mRNA, a rare type of intron with a GC 5' splice site, is abnormally spliced in cdkg1. RNA immunoprecipitation analysis suggests that CDKG1 is associated with this intron. CDKG1 contains N-terminal Ser/Arg (RS) motifs and interacts with splicing factor Arginine/Serine-Rich Zinc Knuckle-Containing Protein33 (RSZ33) through its RS region to regulate proper splicing. CDKG1 and RS-containing Zinc Finger Protein22 (SRZ22), a splicing factor interacting with RSZ33 and U1 small nuclear ribonucleoprotein particle (snRNP) component U1-70k, colocalize in nuclear speckles and reside in the same complex. We propose that CDKG1 is recruited to U1 snRNP through RSZ33 to facilitate the splicing of the sixth intron of CalS5.

Figure 1.

The cdkg1 Mutant Shows Reduced Male Fertility but Normal Vegetative Growth. (A) A DNA fragment of 800 bp in CDKG1 (At5g63370) is deleted in cdkg1. The triangle shows the annotated T-DNA of SALK_075762. The left and the right arrows show the primers used for the genotyping and for the genetic complementation assay. The corresponding protein structure of CDKG1 and the truncated CDKG1 in cdkg1 are also shown. Blue box, the CDS; gray lines, the untranslated regions; blue box with dotted line, deleted portion; white box/N, the N-terminal extension; black box, the Ser/Thr protein kinase catalytic domain; gray box, the C-terminal extension. (B) Genotype detection by PCR using four primer sets for cdkg1, wild type (WT; Col-0), and cdkg1 complementation transformants (Cmp). (C) Thirty-five-day-old plants of the wild type (Col-0), cdkg1, and one complementation plant are shown. (D) Open flowers (left panels) and stamens stained by Alexander’s stain (right panels) of the wild type, cdkg1, and a complementation line are shown. (E) to (P) Semithin cross-section analysis for anther development of Col-0 ([E] to [J]) and cdkg1 ([K] to [P]), from stage 7 to stage 12. DPG, degraded pollen grain; En, endothecium; Ep, epidermis; Msp, microspore; PG, pollen grain; T, tapetum; Tds, tetrads. Bars = 50 μm.

Figure 2.

Pollen Wall Development Is Defective in cdkg1. (A) to (B) Scanning electron microscopy analysis. (A) Typical pollen grain of the wild type with regular exine pattern. Bar = 5 μm. (B) Typical surviving pollen grains of cdkg1 with irregular shape and abnormal exine pattern. Bars = 5 (left and right panels) or 10 μm (middle panel). (C) to (H) TEM analysis. Pollen wall development of the wild type ([C] to [E]) and cdkg1 ([F] to [H]) at early stage 7 ([C] and [F]), late stage 7 ([D] and [G]), and stage 10 ([E] and [H]). Cls, callose; Msp, microspore; Pb, probacula; Pe, primexine; SP, sporopollenin particles or sporopollenin precursors. Bars = 1 μm.

Figure 3.

cdkg1 Shows Reduced Callose Synthesis and Abnormal pre-mRNA Splicing of CalS5. (A) Examples of callose fluorescence in tetrads of Col-0, cdkg1, and cals5-2 (left panel). Callose fluorescence quenching (right panel). Error bars represent sd of the mean of 30 biological replicates. IC, interstitial callose; PC, peripheral callose. Bars = 20 μm. (B) CalS5 gene structure. Black boxes, exons; black lines, introns; the name of the primer sets (e.g., Ex2-3) for gene expression analysis are shown. (C) Real-time RT-PCR expression analysis of CalS5 in Col-0 and cdkg1, using the primer sets EX2-3 and Ex39. Error bars represent sd of the mean of three biological replicates. (D) RT-PCR with different primer sets using the cDNAs of Col-0, cdkg1, and a complementation line (Cmp) TUB. (E) Model of partial splicing of the sixth intron of CalS5 pre-mRNA in cdkg1, according to RT-PCR and the sequence analysis of the additional PCR product.

Figure 4.

CDKG1 Is Associated with the Intron 6 Region of CalS5 pre-mRNA. (A) and (C) PCDKG1:CDKG1-Myc and PCDKG1:CDKG1-GFP constructs used for genetic complementation assays. (B) and (D) siliques and stained anthers of cdkg1 plants bearing PCDKG1:CDKG1-Myc (B) or PCDKG1:CDKG1-GFP (D). (E) and (F) RIP RT-PCR analysis of a PCDKG1:CDKG1-Myc line. Lanes 1, 3, and 5, the wild type; lanes 2, 4, and 6, PCDKG1:CDKG1-Myc line; lane 7, wild-type genomic DNA; lane 8, water. (G) and (H) RIP RT-PCR analysis of a PCDKG1:CDKG1-GFP line. Lanes 1, 3, and 5, the wild type; lanes 2, 4, and 6, PCDKG1:CDKG1-GFP line; lane 7, wild-type genomic DNA; lane 8, water. (I) RIP RT-PCR analysis of the RIP cDNA of the PCDKG1:CDKG1-Myc line using different primer pairs for CalS5. R, RIP cDNA; G, wild-type genomic DNA.

Figure 5.

CDKG1 Interacts with Splicing Factor RSZ33. (A) Domains of 14 CDKs in Arabidopsis. S_TKC domain, Ser/Thr, protein kinase catalytic domain; RS motif, Ser/Arg motif. (B) CDKG1 full length and an internal fragment both interact with RSZ33 in yeast cells; SR34+CDKG1 is a negative control, and pTAC12+pTAC14 is a positive control. BK, pGBK-T7; AD, pGADT7; 232-395, CDKG1 fragment amino acids 232 to 395. (C) BiFC assay between CDKG1 and RSZ33 in tobacco leaf cells. YC, YFP C terminus; YN, YFP N terminus. Bars = 10 μm. (D) mRFP-CDKG1 colocalizes with GFP-RSZ33. A root cell nucleus of a transgenic Arabidopsis plant expressing the two fusion proteins is shown. Bars = 10 μm.

Figure 6.

CDKG1 Is Targeted to Splicing Machinery in a Manner Dependent on the Region Including the Conserved RS Motif. (A) CDKG1-CFP colocalizes with SRZ22-YFP in tobacco leaf cells. Bars = 5 μm. (B) mRFP-CDKG1 is coimmunoprecipitated with SRZ22-YFP. Left panels, inputs. The inputs detected by anti-RFP antibody are nuclear extracts and the inputs detected by anti-β-actin antibody (down) are total soluble proteins. Right panel, nuclear extracts detected by anti-RFP antibody that were immunoprecipitated by anti-GFP magnetic beads or by magnetic beads alone. RG1, mRFP-CDKG1; SY, SRZ22-YFP; WT, the wild type. (C) Diagrammatic representation of CDKG1 and the fragments fused with GFP. Blue line, RS motifs; green line, nuclear localization signal; yellow line, PLTSLRE motif; cyan box, T-loop; white box, N terminus; black box, S_TKC domain; gray box, C terminus. (D) The cellular localization of CDKG1-GFP fragments in tobacco leaf cells. C, cytoplasm; N, nucleus. Bars = 5 μm.

Figure 7.

Model for CDKG1 in Regulation of pre-mRNA Splicing of CalS5 and Pollen Wall Development. For an intron with the GT 5′ SS, such as intron 5 of CalS5, U1 snRNP can recognize the splice site and initiate the removal of this intron efficiently without the help of CDKG1. For CalS5 intron 6 with the GC 5′ SS, U1 snRNP can’t recognize the intron efficiently because of the mismatch between the GC 5′ SS and U1 snRNA. The recruitment of CDKG1 through RSZ33 to the U1 snRNP stabilizes the binding of U1 snRNP to the region around intron 6 and facilitates the efficient splicing of intron 6. The matured CalS5 mRNA is transferred to the cytoplasm for translation of CalS5 protein, which is responsible for callose synthesis and pollen wall formation. 33, RSZ33; 22, SRZ22; 70K, U1-70k; U1, U1 snRNP. [See online article for color version of this figure.]