Phenotypic evolution through variation in splicing of the noncoding RNA COOLAIR

Abstract

The extent to which natural polymorphisms in noncoding sequences have functional consequences is still unknown. A large proportion of the natural variation in flowering in Arabidopsis thaliana accessions is due to noncoding cis polymorphisms that define distinct haplotypes of FLOWERING LOCUS C (FLC). Here, we show that a single natural intronic polymorphism in one haplotype affects FLC expression and thus flowering by specifically changing splicing of the FLC antisense transcript COOLAIR. Altered antisense splicing increases FLC expression via a cotranscriptional mechanism involving capping of the FLC nascent transcript. Single noncoding polymorphisms can therefore be a major contributor to phenotypic evolution through modulation of noncoding transcripts.

Keywords: COOLAIR; FLOWERING LOCUS C; RNA capping/decapping; alternative splicing; flowering time.

Figure 1.

SNP259 in FLC influences COOLAIR splicing. (A,B) Schematic illustration of alternative COOLAIR splicing and polyadenylation in Col FRI^SF2 (FRI from SF-2 accession) and Var2-6. (Black boxes) FLC exons; (grey boxes) COOLAIR exons. The position of SNP259 (Col-0/Var2-6: G/T) is indicated; the nucleotides CT show the splice acceptor site for COOLAIR class II transcripts (“i” is marked). Arrows show PCR primers used to assay splicing; arrows plus a dashed line show primers that cover an exon–intron junction. The 54-base-pair (bp) shift of the COOLAIR acceptor site is highlighted with two vertical dashed lines, and the 64-bp additional exon is marked. (C) The COOLAIR class I splicing pattern is the same in a set of accessions representing the different FLC haplotypes. The RT–PCR primers F1 and R1 are as shown in B. For class I, the lower band is due to mispriming of the F1 oligo and represents unspliced class I COOLAIR. (D) COOLAIR class II is alternatively spliced in a set of accessions representing different FLC haplotypes. The RT–PCR primers F3 and R1 are as shown in B. The arrowheads indicate the characteristic COOLAIR splicing changes in accessions containing the SNP259T polymorphism.

Figure 2.

The Var2-6 FLC allele confers higher FLC expression and later flowering. (A) The Var2-6 FLC haplotype shows a distinct geographical distribution. Forty-six accessions carrying the Var2-6 haplotype are shown as red dots, and 101 accessions carrying other FLC haplotypes are shown as blue dots. The map was generated in R based on the latitude and longitude information of accessions (Supplemental Table S1). (B) Accessions carrying the Var2-6 FLC haplotype are later flowering than accessions carrying other FLC haplotypes. The flowering time of 120 d indicates that the plants did not flower when the experiment was finished. The Kolmogorov-Smirnov (KS) test indicated that the flowering time difference between Var2-6 haplotypes and the other accessions is significant (P < 0.001). (C) FLC expression of the Col allele in plants with and without active FRI. (D) FLC expression of the Var2-6 allele in plants with and without active FRI. Values in C and D are means ± SD from three biological repeats. The fold change in FLC expression between Col and Var2-6 FLC in fri is 28, and the fold change in FRI is 6. (E,F) SNP259T is associated with late flowering in an F2 population generated from a cross between Ka-0 and Ws-0 in fri (E) and FRI (F) homozygotes. The KS test indicated that the flowering time difference between plants containing the Ka-0 FLC allele and the Ws-0 allele is significant (P < 0.05).

Figure 3.

SNP259 specifically regulates COOLAIR alternative splicing. (A) Accessions with nucleotide SNP259T show reduced COOLAIR class II-i splicing compared with those containing SNP259G. Splicing efficiency was measured with quantitative PCR (qPCR) using the primers F2R2 and normalized to unspliced COOLAIR transcript levels at the same region. Next, the splicing efficiency was normalized to the value in ColFRI^SF2. The inset shows the position of primer F2R2 for qPCR analysis of COOLAIR variants in ColFRI^SF2 and Var2-6. Multiple accessions containing different FLC haplotypes are shown (Li et al. 2014). (B) Schematic illustration of the reciprocal SNP259 constructs in Col, Var2-6 FLC alleles, and Col FLC with RBCS terminator replacement at the COOLAIR promoter region. FLC and COOLAIR are illustrated at the top. RBCS in black bars shows the replacement of the COOLAIR promoter. The Col-0 and Var2-6 FLC alleles are indicated with white and grey boxes. (C) G259T mutation in Col FLC caused decreased COOLAIR class II-i splicing in multiple randomly selected transgenic lines. (D) T259G mutation in Var2-6 FLC caused increased COOLAIR class II-i splicing in multiple randomly selected transgenic lines. In C and D, each box plot shows the qPCR data of 10 randomly selected independent transgenic plants for each construct. (E) G259T heterogeneity did not influence FLC sense intron 1 splicing. The Student's t-tests indicated that the FLC sense intron 1 splicing between Col and Col-G259T or Var and Var-T259G are not significant ([NS]P > 0.05). (F) G259T heterogeneity did not influence FLC sense intron 6 splicing. Values in A, E, and F are means ± SD from three biological repeats. The Student's t-tests indicated that the FLC sense intron 6 splicing between Col and Col-G259T or between Var and Var-T259G is not significant ([NS] P > 0.05).

Figure 4.

G259T heterogeneity affects FLC expression in a COOLAIR-dependent mechanism. (A) The G259T mutagenesis in Col FLC increased FLC expression, as assayed using a pool of 48 independent transgenic lines (Student's t-test, [***] P < 0.001) and 32 randomly selected independent transgenic lines. (B) T259G mutagenesis in Var2-6 FLC decreased FLC expression, as assayed using a pool of 48 independent transgenic lines (Student's t-test, [*] P < 0.05) and 29 randomly selected independent transgenic lines. (C) G259T did not change FLC expression in a COOLAIR terminator exchange construct, as assayed using pools from 21 and 32 independent transgenic lines: Col-TEX and Col-TEX-G259T (Student's t-test, [NS] P = 0.216). (D) Flowering time comparison of Var/FRI flc-2 and Var-T259G/FRI flc-2 of plants given 4 wk of vernalization. Forty-five and 60 independent transgenic lines were scored, with eight individuals of each line included. The KS test indicated that the difference between transgenic lines is significant (P < 0.001). In A–C, values are means ± SD from three biological repeats. All of the transgenic plants shown in A–D were in a FRI flc-2 background.

Figure 5.

COOLAIR alternative splicing links cotranscriptionally to changed transcription and capping of the FLC nascent transcript. (A) FRI and the Var2-6 FLC allele both alter the proportion of transcripts with a 5′ cap. NIL(Var)fri is the near isogenic line (NIL) with Var2-6 FLC introgressed into a Col-0 fri background. (B) The single SNP259T polymorphism similarly alters the proportion of transcripts with a 5′ cap. The transgenic Col-G259T plants are in a Col flc-2 fri background. The amplified fragment from the uncapped transcript initiates at 5′-AATCAAGCGAATTGAGAACA-3′. (C) The 5′ capping analysis of chromatin-bound nascent FLC. The amplified fragment from the uncapped transcript initiates at 5′-AAAAAACAATTAATATACCG-3′. In A–C, TUBULIN was used as an internal control. (D) Comparison of chromatin-bound nascent FLC expression between Col-0fri, ColFRI^SF2, and NIL(Var)fri. (E) Chromatin immunoprecipitation (ChIP) analysis shows that total RNA polymerase II (Pol II) is more enriched over FLC in NIL(Var)fri and ColFRI^SF2 compared with Col-0fri. ACTIN was used as an internal control. The position of qPCR primers is relative to the FLC transcription start site (TSS). In D and E, values are means ± SD from three biological repeats (Student's t-test, [*] P < 0.05; [**] P < 0.01; [***] P < 0.001). (F) Model of how SNP259 polymorphisms cotranscriptionally regulate FLC expression (Hossain et al. 2013). (Solid purple circle) The 5′ cap at FLC transcripts; (open purple circle) an uncapped FLC transcript; (CBC) the cap-binding complex; (dashed lines) proposed effects of COOLAIR on capping/decapping.