BRR2a Affects Flowering Time via <i>FLC</i> Splicing

<div><p>Several pathways control time to flowering in <i>Arabidopsis thaliana</i> through transcriptional and posttranscriptional gene regulation. In recent years, mRNA processing has gained interest as a critical regulator of flowering time control in plants. However, the molecular mechanisms linking RNA splicing to flowering time are not well understood. In a screen for Arabidopsis early flowering mutants we identified an allele of <i>BRR2a</i>. BRR2 proteins are components of the spliceosome and highly conserved in eukaryotes. Arabidopsis BRR2a is ubiquitously expressed in all analyzed tissues and involved in the processing of flowering time gene transcripts, most notably <i>FLC</i>. A missense mutation of threonine 895 in BRR2a caused defects in <i>FLC</i> splicing and greatly reduced <i>FLC</i> transcript levels. Reduced <i>FLC</i> expression increased transcription of <i>FT</i> and <i>SOC1</i> leading to early flowering in both short and long days. Genome-wide experiments established that only a small set of introns was not correctly spliced in the <i>brr2a</i> mutant. Compared to control introns, retained introns were often shorter and GC-poor, had low H3K4me1 and CG methylation levels, and were often derived from genes with a high-H3K27me3-low-H3K36me3 signature. We propose that BRR2a is specifically needed for efficient splicing of a subset of introns characterized by a combination of factors including intron size, sequence and chromatin, and that <i>FLC</i> is most sensitive to splicing defects.</p></div

Intron retention in <i>brr2a</i>-2.

<p>(A) Number of alternative splicing events (AS) based on three biological replicates each of Col and <i>brr2a</i>-2. ES, exon skipping; AA, alternative acceptor site; AD, alternative donor site; IR, intron retention; C, complex, a combination of one or several of ES, AA, AD or IR. The increased number of AS events in <i>brr2-a</i> is mainly due to increased intron retention. (B) Number of complex alternative splicing events. 2IR, two intron retention events in the same transcript; IRIR, either one of two different introns is retained; 3IR, three intron retention events in the same transcript; 2IRIR, either a pair of introns or a more 3' located single intron is retained; IR2IR, either a single intron or a more 3' located pair of introns is retained; 4IR, four intron retention events in the same transcript. For (A) and (B), AS events were quantified from RNA-seq data using ASTALAVISTA [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005924#pgen.1005924.ref049" target="_blank">49</a>]. The complex AS events observed in <i>brr2a</i>-2 are mainly combinations of IR events. (C) Schematic representation of basic AS events. (D) Total intron number per transcript with detected IR (left) and number of retained introns per transcript with detected IR (right). Whereas transcripts with detected IR have on average 6 transcripts, only one or two of those are retained, suggesting that IR is mostly an intron and not a transcript property. (E) Length of introns with increased retention in <i>brr2a</i>-2 (“retained”), with decreased retention (“released”) and with no change in retention (“equal”). Introns with increased retention are often shorter whereas introns with decreased retention are often longer than unaffected introns. (F) GC content of introns with increased retention in <i>brr2a</i>-2 (“retained”), with decreased retention (“released”) and with no change in retention (“equal”). Introns with increased retention have often lower GC content whereas introns with decreased retention have often higher GC content than unaffected introns. P-values are from one-sided t-tests.</p

<i>FLC</i> splicing efficiency is reduced in <i>brr2a</i>-2.

<p>Intron retention was calculated as the ratio of unspliced to total (spliced + unspliced) transcripts for three representative <i>FLC</i> introns (A) and for intron 1 in <i>MAF1</i> and <i>SEP3</i>, and intron 2 in <i>AG</i> (B) in both Col and <i>brr2a</i>-2. For <i>FLC</i> and <i>MAF1</i>, RNA was extracted from 15 day-old seedlings grown under SD conditions at ZT = 7. For <i>SEP3</i> and <i>AG</i>, RNA was extracted from inflorescences of LD-grown plants. Results were normalized to <i>PP2a</i>; shown are mean ± SE (n = 3). Note the different scales used for each gene showing that that majority of <i>MAF1</i>, <i>SEP3</i> and <i>AG</i> transcripts are correctly spliced even in the <i>brr2a</i>-2 mutant.</p

Intron retention in <i>brr2a</i>-2 is associated with specific chromatin properties.

<p>Box plots show averaged genome-wide bisulfite sequencing and ChIP signals for exons (blue) and introns (green). DRI, differentially retained introns in <i>brr2a</i>-2. DRI gene, genes containing at least one differentially retained intron. Thus, the boxes in each plot represent, from left to right, (i) exons from genes that have no DRIs, (ii) exons from genes that have at least one DRI, (iii) normally spliced introns in genes that have at least one DRI, (iv) DRIs, and (v) introns from genes that have no DRIs. Control genes without DRIs were selected to have the same median expression as the DRI genes. Significant differences are indicated; numbers are–log₁₀ of p-values from Wilcoxon signed-rank tests.</p

Developmental alterations in <i>cäö</i>.

<p>(A) Silhouette of the sixth rosette leaf from wild type (Col, left) and <i>cäö</i> (right) showing the serrated margin of the <i>cäö</i> leaf. Plants were grown for 4 weeks under LD conditions. (B) Rosette morphology of Col and <i>cäö</i> plants at time of bolting in LD; scale bar: 5 cm. (C) Leaf morphology of Col and <i>cäö</i> in 20 days old plants; scale bar: 1 cm. (D) Reduced silique length in <i>cäö</i> mutants. Scale bar: 1 mm (E) Cleared wild type (left panel) and <i>cäö</i> ovules with arrested (middle) or absent female gametophytes (right panel). Scale bar: 25 μm. Cells of the egg apparatus are indicated: CC, central cell; EC, egg cell; SY, synergids.</p

<i>cäö</i> is an early flowering mutant in Arabidopsis.

<p>Flowering time of wild type (Col), <i>msi1-tap1</i>, <i>cäö</i> and <i>cäö msi1-tap1</i> in number of total rosette leaves at bolting under LD (left) and SD (right). Shown are mean ± SE (n ≥ 14). Numbers in the graph represent p-values of two-sided t-tests.</p

<i>CÄÖ</i> encodes the ATP-dependent RNA helicase protein BRR2a.

<p>(A) SNP annotations in the identified region with reduced recombination on left arm of chromosome 1. (B) Schematic representation of the protein domain structure of BRR2a. A detailed description of the protein domains can be found in the main text. (C) Threonine 895 is conserved among eukaryotic BRR2 proteins. Sequence alignment of the end of helicase domain 1 in BRR2A proteins from yeast, animals and plants. The asterisk highlights threonine 895, which is altered to an isoleucine in <i>brr2a</i>-2. Conserved amino acid residues are highlighted in black. Residues not identical but similar are highlighted in gray.</p