Article Figures & Data
Figures
- Figure 1.
Light exposure triggers opposite changes in levels of major SR30 splicing variants. A, Gene model of SR30 including splicing variants analyzed in this work. Primers binding within one exon are shown as arrowheads, whereas arrows with dotted lines indicate primer binding sites spanning splice junctions. The topmost pair are coamplification primers used in downstream analyses, while the primer pair directly above the first variant was used to measure total SR30 transcripts. Below each variant, the positions of primers used in RT-qPCR for detection of the corresponding splicing variants are indicated. Lines and boxes depict introns and exons, respectively; UTRs and cds are indicated by black and gray shading, respectively. Gray dashed line indicates binding site of artificial microRNA (amiR) spanning a specific splice junction of SR30.1. The triangle points at the T-DNA insertion site of sr30. Underneath the gene models, representative coverage plots are shown from a previous RNA-seq study (Hartmann et al., 2016) for dark (D) and 6-h white light (L). B to D, Splicing variants were quantified using RT-qPCR in seedlings exposed to white (B), blue (C), or red (D) light for indicated periods. Levels are relative to total SR30 transcripts and normalized to the 0 h sample. D, Dark; mean values + sd (n = 3–7 for white and n = 3 for blue and red light).
- Figure 2.
The SR30.2 variant is relatively stable and enriched in the nucleus. A, SR30 transcript levels determined via RT-qPCR in 10-d-old green wild-type, lba1, or upf3 seedlings, relative to total SR30 and the wild type. Mean values + sd; n = 3. B, Analysis of SR30.1 and SR30.2 RNA stability in 7-d-old Arabidopsis seedlings upon addition of cordycepin. Transcript levels were measured using RT-qPCR and normalized to a stable actin reference. Mean values ± sd are displayed on a log2 axis; n = 3. Half-lives based on exponential regression curves. C, Immunoblot analysis of total (T), cytosolic (C), and nuclear (N) fractions, with histone H3 and UGPase being detected as nuclear and cytosolic markers, respectively. Amidoblack staining shown beneath immunosignals; positions of relevant size marker bands are indicated. D, Coamplification of AS variants for SR30, SEF, and RS2Z33 from fractions described in C. Bands corresponding to fragments used for quantitation are marked with white and black dots next to nuclear samples. L, size ladder consisting of DNAs in 100-bp increments from 200 to 800 bp. E, Ratios of long to short AS variants in nuclear fractions relative to total fractions. Mean values + sd; n = 3. F and G, Levels of SR30 AS variants relative to total SR30 determined via RT-qPCR in indicated fractions from Arabidopsis (At) seedlings (F) and N. benthamiana (Nb) leaves expressing an SR30 reporter (G), using N6 (left) or dT20 (right) for cDNA generation. Mean values + sd; n = 3.
- Figure 3.
Splicing to SR30.2 results in diminished protein production. A, Immunoblot detection of Flag-tagged RPL18 in input (In) and immunoprecipitation (IP) fractions of 10-d-old green Arabidopsis seedlings from indicated genotypes. Twenty micrograms of total protein of In (∼0.1% of In) and 6% of IP sample was loaded. L, ladder containing proteins of indicated sizes. Upper and lower panels depict immune signal and amidoblack staining, respectively. B, RT-PCR coamplification of SR30.1/.2 from total RNA preparation (To; standard RNA extraction directly from freshly frozen material) and samples described in A. L, size ladder consisting of DNAs in 100-bp increments from 200 to 600 bp. C, Levels of SR30.1 and SR30.2 were determined via RT-qPCR in samples described before and are depicted relative to total SR30 transcripts and normalized to a total sample from the wild type. Reverse transcription of RNAs performed with dT20 (top) or N6 (bottom) primers. Mean values + sd; n = 3. Values for SR30.2 in relevant In and IP fractions are provided. D and E, Immunoblot detection of HA-tagged SR30.1 and SR30.2 in N. benthamiana upon transient expression of constructs based on the cds (D) or the cDNAs with 5′ and 3′ UTRs (E). Each sample pair came from corresponding leaf halves. WT, Noninfiltrated leaf. Fifteen micrograms of total protein each (D) or fresh weight equivalents (E) were loaded; lower panels show amidoblack staining as the loading control. F and G, Immunoblot analysis upon immunoprecipitation with α-SR30 from 10-d-old green wild-type or transgenic Arabidopsis seedlings, expressing indicated cDNA-HA3 constructs (F) or an amiR construct targeting SR30.1 (G). Fresh weight equivalents were loaded; cross-detection band (asterisk) serves as the loading control. Open arrowheads indicate endogenous SR30, and closed arrowheads in (F) mark tagged SR30.1.
- Figure 4.
Fluorescent protein fusions of SR30.1 and SR30.2 both localize to the nucleus in Arabidopsis protoplasts. A, A construct containing the cds of SR30.1 fused to YFP was transiently coexpressed with the nuclear marker NLS-DsRED in Arabidopsis protoplasts, followed by imaging using confocal microscopy. B and C, Colocalization of SR30.1 and SR30.2 fusions. D, Colocalization of SR30.2-YFP and SR30.1-CFP in the nucleoplasm and speckles. Bars = 10 µm in all panels.
- Figure 5.
Both SR30.1 and SR30.2 can alter splicing of the SR30 premRNA. A, Gene model of the reporter used for the splicing assay. Exons are shown as boxes and introns as lines. White, cds; hatched, 3′ UTR in SR30.2; gray, cds in SR30.1 and 3′ UTR in SR30.2; black, HA-tag. Arrowheads indicate binding positions of primers for coamplification of resulting splicing variants. With the exception of the HA-tag, model is drawn to scale. B, RT-PCR products upon coamplification of splicing variants SR30.1 and SR30.2 from the reporter coexpressed with a control protein (luciferase [LUC]) or the cds of SR30.1 and SR30.2. Shown is representative agarose gel including a no template control (−) and DNA size ladder (M) with 100-bp increments. C, Ratio quantification using a Bioanalyzer for splicing variants displayed in B and normalized to the control (LUC). Mean values + se; n = 14 to 15.
- Figure 6.
SR30.2 can be further spliced to SR30.3. A, Partial models of SR30 alternative splicing variants that were previously identified (Hartmann et al., 2016) to be significantly altered relative to SR30.1 upon light exposure of etiolated seedlings. Lines and boxes correspond to introns and exons, respectively. Gray and black fills indicate coding sequence and 3′ UTR, respectively; AS event identifiers from previous study (Hartmann et al., 2016) are provided. B, Levels of SR30 splicing variants relative to reference PP2A and ratio SR30.3/SR30.2 were determined using RT-qPCR from lba1 seedlings grown for 6 d in darkness and then retained in darkness or exposed for 6 h to white light in liquid control medium (MS) or medium containing 2% Suc. Data normalized to corresponding values from seedlings kept in dark and MS. Mean values + sd; n = 3 for sugar and light treatments and n = 2 for dark/MS controls. C, RT-PCR products upon coamplification of SR30.1 (circles) and SR30.2 (arrowheads) from Arabidopsis (A.t.) seedlings grown for 6 d in dark (D) or under long-day conditions (L) or N. benthamiana (N.b.) leaves transformed with SR30 constructs. Transformation results in constitutive expression of cDNA constructs (.1 and .2) based on the corresponding Arabidopsis SR30 variants or the genomic Arabidopsis SR30 sequence (Gen.). Black symbols indicate positions of products corresponding to endogenous splicing variants, and white symbols point at transgene-derived products that are size-shifted due to the presence of a tag. Size marker (M) consists of DNAs in 100-bp increments, with the lowest band representing 200 bp. D, RT-PCR products upon amplification of SR30.3 (indicated by diamonds), either from the endogenous locus and transgene (upper gel) or specifically from the transgene (lower gel). Samples as in C, with added nontransformed wild-type control for N. benthamiana. Size marker in 100-bp increments; top bands correspond to 200 and 300 bp in upper and lower gels, respectively.
- Figure 7.
Model of SR30 regulation via AS and downstream processes. Boxes and lines depict exons and introns, respectively, of SR30 transcripts, for which only the part from the exon upstream of the alternatively spliced region to the 3′ end of the mRNAs is displayed. Asterisks depict positions of first in-frame translational stop codons for each mRNA. RBP indicates a putative RNA-binding protein. Dark-gray shape represents nuclear envelope with pores for export (green rings). Active translation is indicated by light-green ribosome symbols. Magenta indicates impairment of the corresponding process.
Additional Files
Supplemental Data
Supplemental Figures and Tables
Files in this Data Supplement:
- Supplemental Data - Supplemental Figures 1-7 and Supplemental Tables 1 and 2
