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Developmental Cell

Article Long Noncoding RNA Modulates Alternative Splicing Regulators in Arabidopsis Florian Bardou,1 Federico Ariel,1 Craig G. Simpson,2 Natali Romero-Barrios,1 Philippe Laporte,1 Sandrine Balzergue,3 John W.S. Brown,2,4 and Martin Crespi1,* 1Institut

des Sciences du Ve´ge´tal, Centre National de la Recherche Scientifique, Saclay Plant Sciences, F-91198 Gif-sur-Yvette Cedex, France 2Cell and Molecular Sciences, The James Hutton Institute, Invergowrie, Dundee DD2 5DA, Scotland, UK 3Ge ´ nomique Fonctionnelle d’Arabidopsis, Unite´ de Recherche en Geńomique Ve´ge´tale (URGV), UMR INRA 1165, Universite´ d’Evry Val d’Essonne, ERL CNRS 8196, 91000 Evry, France 4Plant Sciences Division, College of Life Sciences, University of Dundee at the James Hutton Institute, Invergowrie, Dundee DD2 5DA, Scotland, UK *Correspondence: [email protected] http://dx.doi.org/10.1016/j.devcel.2014.06.017

SUMMARY

Alternative splicing (AS) of pre-mRNA represents a major mechanism underlying increased transcriptome and proteome complexity. Here, we show that the nuclear speckle RNA-binding protein (NSR) and the AS competitor long noncoding RNA (or ASCO-lncRNA) constitute an AS regulatory module. AtNSR-GFP translational fusions are expressed in primary and lateral root (LR) meristems. Double Atnsr mutants and ASCO overexpressors exhibit an altered ability to form LRs after auxin treatment. Interestingly, auxin induces a major change in AS patterns of many genes, a response largely dependent on NSRs. RNA immunoprecipitation assays demonstrate that AtNSRs interact not only with their alternatively spliced mRNA targets but also with the ASCO-RNA in vivo. The ASCO-RNA displaces an AS target from an NSR-containing complex in vitro. Expression of ASCO-RNA in Arabidopsis affects the splicing patterns of several NSR-regulated mRNA targets. Hence, lncRNA can hijack nuclear AS regulators to modulate AS patterns during development.

INTRODUCTION Most eukaryotic genes are alternatively spliced in a cell-typeand tissue-specific manner, and defects in alternative splicing (AS) can contribute to diseases in mammals (Blencowe, 2006; Matlin et al., 2005; Wang et al., 2008; Wang and Cooper, 2007). In plants, AS is involved in regulation of gene expression and developmental plasticity responses to environmental cues (James et al., 2012; Syed et al., 2012; Filichkin et al., 2010; Tanabe et al., 2007; Palusa et al., 2007). Non-protein-coding RNAs represent a significant part of the transcriptome (Zhu and Wang, 2012) that can elicit new mechanisms of gene regulation through, for example, their interaction with specific ribonucleoprotein complexes. Long noncoding RNAs (lncRNAs) are

predominantly involved in epigenetic patterning and chromatin remodeling or function as scaffolds that interfere or modulate the action of different RNA-related enzymatic complexes (Rinn and Chang, 2012). Interestingly, lncRNAs often exhibit tissueor cell-type-specific expression patterns (Djebali et al., 2012; Ben Amor et al., 2009; Liu et al., 2012), suggesting roles in certain cell types or developmental transitions. In the model legume Medicago truncatula, ENOD40 is a highly structured long RNA with poor protein coding potential and is involved in nodule organogenesis (Charon et al., 1999; Sousa et al., 2001). There is evolutionary conservation of this gene family at the nucleotide sequence level, with several plant species containing ENOD40 RNA-related sequences. As also shown for several lncRNAs using high throughput translational techniques (Ingolia et al., 2011), certain short open-reading-framederived peptides (12 amino acids) can be translated in vitro (Rohrig et al., 2002). However, the ENOD40 structured RNA region is required for biological activity (Sousa et al., 2001) and, by using a triple-hybrid approach with a conserved ENOD40 RNA region, we previously identified a Medicago truncatula RNA-binding protein (MtRBP1) that interacts with this RNA region in vivo (Campalans et al., 2004). This protein contains an RNA recognition motif in the C-terminal region, a nuclear localization signal, and localizes to nuclear speckles, so we renamed it MtNSR1 (for Medicago truncatula Nuclear Speckles RNA-binding protein 1). NSR1 was shown to be relocalized from the nucleus to the cytoplasm specifically in root tissues where ENOD40 is expressed, such as the nodule primordia and initiating lateral roots (LRs) (Campalans et al., 2004). Here, we have identified that nuclear speckle RNA-binding proteins (NSRs) are a family of RNA-binding proteins (RBPs) that act as nuclear AS regulators in Arabidopsis. These proteins interact with both their alternatively spliced mRNA targets and at least two structured lncRNAs, ENOD40 and lnc351 (Ben Amor et al., 2009), renamed Alternative Splicing Competitor RNA (ASCO-RNA). Expression of these lncRNAs affected AS of NSR-dependent mRNA targets and the ASCO-RNA can compete in vitro the binding of NSRs with an AS target. Our results suggest that lncRNAs can hijack AS regulators to modulate gene expression during developmental transitions, such as the formation of LRs in plants.

166 Developmental Cell 30, 166–176, July 28, 2014 ª2014 Elsevier Inc.

Developmental Cell lncRNA Modulates AS Regulators in Arabidopsis

RESULTS

Figure 1. AtNSRs Localize in Nuclear Speckles and Relocalize into Cytoplasmic Bodies When Coexpressed with the lncRNA ENOD40 (A) The NSRs from Medicago truncatula (MtRBP1/MtNSR1: Medtr6g034835.1) and Arabidopsis thaliana (AtNSRa and AtNSRb: At1g76940 and At1g21320, respectively) have a conserved gene structure (exons 1 to 5), an RNA recognition motif in the C-terminal part of the proteins (RRM), and a nuclear localization signal (NLS, represented by a red arrow in exon 1). The AtNSRb gene is expressed from two different transcription start sites, and the NSRb.2 mRNA isoform contains exon 1 but not exon 10 . The two transcripts have different translational start codons (indicated by blue arrows). (B and C) 35S-AtNSRa-GFP (B) and 35S-AtNSRb-GFP (C) colocalize with splicing-related SR protein AtSR34 (35S-SR34-RFP; AT1G02840) in nuclear ‘‘speckles’’ (colocalizations are in yellow). (D) 35S-AtNSRb-GFP fusion proteins localize in nuclear speckles (left panel), and coexpression of MtENOD40 RNA (from a 35S-ENOD40 construct) results

The Nuclear Speckle RBPs AtNSRa and AtNSRb Are Relocalized to the Cytoplasm on Expression of the ENOD40 RNA and Are Expressed in Root Meristems We first searched for MtNSR1-related sequences in Arabidopsis thaliana. We identified two different potential NSR1 orthologs, called NSRa and NSRb (Figure 1A). NSRa has been previously reported to be related to the serine/argininerich protein family (Schindler et al., 2008), a class of proteins involved in splicing regulation in eukaryotes (Barta et al., 2008). AtNSRa and AtNSRb differ due to the presence of a new 10 exon and two transcription start sites in the AtNSRb locus yielding two alternative mRNAs (Figure 1A, AtNSRb.1 and AtNSRb.2 mRNAs; Figure S1A available online). The NSRs belong to a plant-specific RRM-containing protein family (Figure S1B). Both AtNSRa and AtNSRb proteins localize in nuclear speckles, as demonstrated when translational green fluorescent protein (GFP) fusions are transiently expressed in tobacco leaves and the AtNSRs and MtNSRs colocalize in AtSR34-containing speckles (for serine/arginine-rich SplicingRelated [SR] Protein 34; Figures 1B, 1C, and S1C). Strikingly, when the AtNSRs are coexpressed with the ENOD40 RNA, the AtNSR-GFP fusion is relocalized to cytoplasmic bodies, indicating a conserved ENOD40 relocalization activity for both A. thaliana and M. truncatula NSRs (Figure 1D; Figures S1D and S1E). Hence, AtNSRs colocalize with splicing-related nuclear speckles and can be relocalized to the cytoplasm through the action of the ENOD40 RNA, indicating that they are true orthologs of MtNSR1. To determine the spatial expression pattern of AtNSRs in planta, the NSRa promoter and the two alternative NSRb promoters (corresponding to regions upstream of either exon 1 or exon 10 ) were fused to the reporter gene GUS (Figures 2A and 2B; Figures S2A–S2C). All promoters gave GUS expression in root meristems and in LRs. The three NSR-GUS plants also gave lower expression in root vascular tissues, while NSRb1GUS plants additionally stained cotyledon vascular tissues (Figures S2A–S2C). In addition, GFP-tagged NSRa and NSRb controlled by the NSRa endogenous promoter (NSRb promoters were too weak to produce detectable GFP signal) or the CaMV 35S promoter confirmed the localization of both NSRa and NSRb proteins in nuclear speckles in root meristematic cells, in vascular tissues, and throughout the LR primordia (Figure 2C and Figure S2D). Overexpression of NSR-GFP from the 35S promoter produced more abundant and clearly detectable nuclear particles but revealed a related localization in nuclear particles as the one observed with the endogenous promoter (Figure S2D). Interestingly, these easily detectable nuclear particles exhibited spontaneous cytoplasmic localization in trichoblast cells containing root hairs but not in the neighboring atrichoblast cells (Figures S2E–S2G), although nuclear localization was generally observed. We have previously suggested that AtNSRs are induced by auxin (Ben Amor et al., 2009), in relocalization from the nucleus to punctuate cytoplasmic regions (fluorescent dots) after transient expression in tobacco leaves (right panel). n, nucleus (circled by a red line); c, cytoplasmic dots. Scale bars, 8 mm. See also Figure S1.

Developmental Cell 30, 166–176, July 28, 2014 ª2014 Elsevier Inc. 167


and a similar regulation was detected in Arabidopsis databases (http://www.arabidopsis.org). Quantitative real-time PCR analysis revealed that NSRa is constitutively expressed, whereas NSRb is induced by auxin treatment in roots in a dose-responsive manner (Figures 3A and S3A). Therefore, AtNSR action is linked to primary and LR meristems, whose formation and patterning are known to be regulated by the key phytohormone auxin. AtNSRs Regulate Molecular and Growth Responses to Auxin To address the physiological role of NSRs, we isolated single nsra and nsrb Arabidopsis mutants and generated an nsra/nsrb double mutant (DM). Seven days after germination (in control conditions without naphthalene acetic acid [NAA]), Arabidopsis plants develop between one or two LRs (Figure S3B, mean number 1.5). The single mutants did not show any major phenotype, but the nsra/nsrb DM plants exhibited a significant reduction in the number and length of their LRs, despite their similar primary root length in relation to wild-type (WT) plants at this time point (Figures S3C and S3D). Plants germinated in the presence of 100 nM auxin have many more LRs (Figures 3B–3E; mean number, 12), and this phenotype is more drastic, suggesting that nsra/nsrb mutants are less sensitive to auxin (Pe´ret et al., 2009). In contrast, transgenic lines overexpressing NSRa or NSRb yielded dwarf plants, a phenotype that correlates with transgene expression levels (Figures S3E and S3F). The dwarf phenotype reflected the presence of smaller cells in these plants, as clearly observed in the leaf epidermis (Figures S3G and S3H). Moreover, these transgenic lines show reduced root growth in control conditions and under auxin treatment, although they have the same LR density (Figures S3I and S3J). To unravel the mechanism of action of NSRs, we analyzed the molecular response to auxin in the nsra/nsrb DMs. A transcriptomic approach revealed that over 2,200 genes were differentially regulated in comparison to WT plants after auxin treatment (24 hr, 1 mM NAA), and far fewer changes (535 genes) were observed without auxin treatment (Figure 3F). Interestingly, among the differentially expressed genes, we identified 11 lncRNAs that were deregulated in DM plants, representing approximately 15% of the lncRNAs that we had identified previously in Arabidopsis (Ben Amor et al., 2009; Figure S4A). The differential expression behavior for the auxin-inducible lnc34 and the lnc351 (that we have renamed ASCO-lncRNA for ASCO-lncRNA; discussed later) was confirmed using quantitative real-time PCR (Figures 3G and 3H). These results link NSRs to the regulation of gene expression of both mRNAs and lncRNAs in response to auxin. In addition, of the 49 genes most downregulated in nsra/nsrb compared to WT plants in response to auxin, 5 are auxin-responsive genes linked to LR initiation (dependent on SOLITARY ROOT-1; slr1), of which 3 are also controlled by the key regulators ARF7 and/or ARF19 involved in auxin signaling during LR formation (as asssessed with the Visual Lateral Root Transcriptome Compendium tool; Parizot et al., 2010; Figures S4B and S4C). On the other hand, among the 100 most upregulated genes in nsra/nsrb compared to WT plants in response to auxin, we found 28 genes that are normally repressed by auxin, of which 3 are also linked to LR initiation (slr1-dependent) and 6 are regulated by ARF7 and/or

ARF19 (Figures S4D and S4E). These results integrate the action of NSRs in LR initiation and known auxin signaling pathways. AtNSRs Regulate AS and Bind to Specific Target mRNAs As NSRs colocalized with spliceosome markers, we explored whether NSRs might modulate gene expression via splicing/AS. No major changes were detected in constitutive splicing patterns for several genes. We then used a high-resolution real-time PCR panel monitoring genome-wide AS events for 288 genes (Simpson et al., 2008, including one control gene). We analyzed AS patterns in RNA from root tissues of WT and nsra/nsrb mutant plants with or without auxin treatment (Table S1A, quantification of AS isoforms in different samples; Table S1B, significantly different values between samples >3% with a p value < 0.05; and Table S1C, number of genes and transcripts differentially affected in each class shown in Figure 4A). Auxin treatment induced significant changes in the relative amounts of AS isoforms in 85 genes of the 288 tested (Figure 4A). The vast majority (69 genes) of these auxin-induced AS events were dependent on NSRs (of these, 26 showed an additive effect of NSR dependency and auxin response; Figure 4A). AS events in 18 genes were exclusively affected under control conditions between WT and nsra/nsrb. These AS events included alternative 50 and 30 splice site usage, exon skipping, and intron retention (IR). Nonsense-mediated decay (NMD) can degrade specific AS transcripts; to exclude an NMD effect, we identified whether the transcripts affected in the DM and/or, by auxin treatment, whether they were NMD sensitive (Kalyna et al., 2012). Only a few of the known NMD-sensitive transcripts were altered, suggesting no general effect on NMD associated with NSRs or auxin (Table S1). Similarly, if there was a general reduction in splicing efficiency, an increase in IR events would be expected. We examined 30 transcript isoforms known to show detectable IR on the panel, but only a small number altered AS levels in the DM and auxin treatment, suggesting that there was no general splicing defect due to the treatments. We further confirmed the effects on AS using real-time PCR and a fluorescent gel assay for several genes. A relevant example of AS defects is the ATPase1 (At1g27770), which shows a single main isoform in both control and auxin-treated conditions but exhibits an IR event in the nsra/nsrb DM treated by auxin (Figure 4B; AS scheme in Figure S5E). Other examples are the F-box gene (At4g27050) transcripts and the auxin-related gene (At2g33830), which are normally spliced into two isoforms in control conditions but exhibit changes in AS in response to auxin (Figures S5A and S5B; AS schemes shown in Figures S5F and S5G, respectively). This auxin-induced splicing variation is not detected in the nsra/nsrb mutant background. Interestingly, among the 85 genes differentially spliced in nsra/nsrb compared to WT plants, 11 are linked to LR initiation (dependent on slr-1; Parizot et al., 2010; Figures S6A–S6C). These results suggest that NSRs may represent novel AS regulators in plants that modulate auxin effects on the transcriptome. To further demonstrate a direct molecular link between NSRs and alternatively spliced mRNAs, we generated transgenic lines where NSRa or NSRb proteins, fused to a hemagglutinin (HA) tail, were expressed under the control of the endogenous NSRa promoter in the nsra or nsrb mutant backgrounds, respectively. We then performed in vivo UV crosslinked RNA immunoprecipitation



Figure 2. AtNSRs Are Expressed during LR Formation (A and B) AtNSRa (pAtNSRs-GUS) (A) and the two AtNSRb promoters [pAtNSRb1-GUS and pAtNSRb2-GUS, schematized in (B)] fused to GUS are active in vascular tissues and during LR formation in dividing primordia until lateral root emergence. Scale bars, 100 mm. (C) Expression of the translational fusion pNSRaNSRa-GFP labels nuclear particles (higher magnification images of single nuclei are provided) during all steps of LR formation (I and II, IV, VII, and VIII according to Malamy and Benfey, 1997). Arrows indicate nuclei; yellow arrows show nuclei after migration during early steps of LR formation. See also Figure S2.

assays (RIP) and found that NSRa and NSRb specifically bind to specific AS mRNA targets in planta (Figure 5A). Hence, the NSR proteins regulate AS and bind to specific mRNA targets, notably during plant developmental processes regulated by auxin. The ASCO-RNA Modulates AS Patterns and Competes with the Binding of an NSR-Containing Complex to an AS Target In Vitro As mentioned in the Introduction, NSRs interact with lncRNAs (Campalans et al., 2004); hence, we explored whether Arabidopsis lncRNAs are recognized by AtNSRs. Among a collection of lncRNAs identified in Arabidopsis (Ben Amor et al., 2009), the ASCO-RNA (previously named lnc351) was the only one found to bind in vivo to AtNSRs in an RIP on total RNA or nuclear extracts (Figure 5B). In the nuclear fraction, the enrichment of ASCO-RNA in the NSRb-HA RIP was even more significantly increased, supporting the hypothesis that ASCO-RNA is involved in nuclear-related processes (Figure 5B). This correlated with the fact that this lncRNA is enriched in nuclear fractions (in comparison to total RNA; Figure 5C). However, in contrast to ENOD40, the ASCO-RNA was not able to induce cytoplasmic relocalization of AtNSRs in tobacco cells (Figure S1F). This lncRNA is also transcriptionally upregulated in the nsra/nsrb DM (Figure 3H). To further confirm the specificity of the NSR-HA interaction with AS mRNA targets and the ASCO-RNA, we showed that the levels of two control genes from the original panel (At5g13480 and At5g03240 coding for a Fy.1, WD-40 repeat family protein, and the polyubiquitin

UBQ3, respectively), whose splicing was not affected in the nsr mutants or by auxin treatment, were undetectable in the RIP products. As NSRs bind both their AS mRNA targets and the ASCO-RNA, we purified the NSR-containing complex and assayed its in vitro binding capacities to these molecules. By using an in vitro binding assay for an AS target (the auxinregulated protein gene At2g33830), we showed that incubation with nonlabeled At2g33830 mRNA or the ASCO-RNA significantly diminishes the amount of AS target immunoprecipitated (Figures 5D and S7). Hence, the ASCO-RNA modulates the in vitro binding of an AS target to an NSR-containing complex, likely through target displacement, indicating that the ASCO-RNA can act as an NSR-complex competitor for binding to their AS targets. To address this hypothesis in vivo, we generated Arabidopsis lines overexpressing the ASCO-RNA transcript and assayed the splicing of selected NSR targets. Two NSR mRNA targets (the previously in-vitro-assayed auxin-regulated protein gene At2g33830 and the ATPase1 gene At1g27770), exhibited AS changes in these plants (Figures 6A and 6B). Moreover, the latter one exhibited the same IR event as that observed in the nsra/nsrb DM treated by auxin (Figure 4B). Furthermore, the overexpression of ASCO-RNA additionally leads to decreased LR density when plants are grown on auxin, suggesting that ASCO-mediated modulation of NSR targets can affect auxin-induced LR formation (Figures 6C and 6D). This phenotype correlates with the fact that ASCO-RNA is upregulated in the nsra/nsrb mutant. Thus, the ASCO lncRNA can modulate AS events directed by interfering with AtNSRs in vivo. Since the ENOD40 RNA interacted with AtNSRs to induce their relocalization from the nucleus to the cytoplasm (Figure 1D), we also explored whether this lncRNA can change AS patterns of NSR targets. The auxin-related (At2g33830) genes and the CCA1 (At2g46830) exhibited visible changes in the ratio of AS isoforms in ENOD40-expressing plants (Figures S5C and S5D; schemes in Figures S5H and S5G, respectively). Therefore, expression of at least two lncRNAs led to significant alterations of AS in plants, suggesting that lncRNAs can



Figure 3. The nsra/nsrb DM Shows Reduced Number of Auxin-Induced LRs

a

(A) A kinetics of AtNSRb induction by auxin treatment (1 mM NAA) is shown (white bars), whereas AtNSRa is constitutively expressed (black bars). (B–D) The nsra/nsrb DMs show a reduced number (C) and length (D) of auxin-induced LRs (NAA, 100 nM). These phenotypes are more accentuated in response to auxin where LRs are formed all along the primary root, as shown in (B) corresponding to a WT and DM plant 7 days after germination in auxincontaining medium. (E) In contrast, no variation in primary root length was observed at this time point. n > 35; Error bars indicate confidence interval 5%; stars indicates Student’s t test a = 0.05. (F) Transcriptome analysis of the Atnsra/nsrb DM showed 535 genes differentially regulated compared to WT (NAA), whereas an auxin treatment revealed 2,214 genes with expression changes under the same criteria when compared to WT (+NAA; CATMA arrays V6). (G) The lncRNA lnc34 is strongly induced by auxin, an accumulation significantly lost in the DM. (H) The ASCO-RNA is upregulated in the nsra/nsrb DM in both control and auxin conditions. Error bars indicate SD. See also Figures S3 and S4.

modulate the action of nuclear splicing regulators during a developmental transition, the organogenesis of LRs. DISCUSSION Our results demonstrate that NSRs are plant RBPs that localize within nuclear speckles, interact with lncRNAs, and regulate AS. Regulation of gene expression during LR development partly depends on auxin-mediated AS involving NSRs. NSR-containing complexes bind to target pre-mRNAs to regulate their AS during LR formation in response to auxin. On the other hand, specific lncRNAs can interact with AtNSRs and, consequently, modulate NSR action and AS of NSR-dependent mRNA targets. We propose a model (Figure 7) where interactions of NSRs with the

ASCO-RNA compete the binding of NSRs to its AS targets and lead to specific changes in AS required to switch developmental fates such as those occurring during de novo organogenesis in plant roots. The NSRs fused to GFP localized in nuclear speckles, a nuclear compartment maintained by the specific association between diverse RNA and protein partners. Factors localized in nuclear speckles, such as SR proteins and small nuclear RNA ([snRNA] U1, U2, U4, U5), shuttle between the nucleus and the cytoplasm in HeLa cells. This translocation is important for their structural maturation and function (Patel and Bellini, 2008; Sapra et al., 2009), and localization patterns can change in response to the environment and/or in different tissues or developmental stages. The amount of RNA and protein components present can affect the nature of the speckles and, thus, their number and activity. In plants, many different speckles have been detected based on et al., 2008). the presence of particular proteins (Lorkovic The growing importance of AS in plants has been recently addressed, and at least 60% of intron-containing genes undergo AS (Marquez et al., 2012). The abundance and activity of splicing factors can have a major impact on gene expression by modifying the ratio between different AS isoforms having diverse functions (Syed et al., 2012). In plants, two protein isoforms of the SR-like splicing factor, SR45, differing only by eight amino acids including a putative phosphorylation site, can differentially complement petal or root developmental phenotypes in a sr45 mutant (Zhang and Mount, 2009). In addition, one AS isoform



family of splicing regulators and alters patterns of splicing (Yin et al., 2012). Another lncRNA, MALAT1, can modulate AS in HeLa cells by interfering with protein phosphorylation of spliceosomal proteins and the spatial integrity of the nuclear speckles (Tripathi et al., 2010). Here, we introduce a mechanism by which the interaction of lncRNAs with AS regulators, such as the NSR proteins, can modulate AS during specific developmental transitions, such as the formation of LRs by differentiated pericycle cells. This hypothesis is supported by the related molecular phenotypes of the nsra/nsrb mutant and the lines where the ASCO-RNA is overexpressed. Interestingly, this lncRNA is highly structured and binds NSR proteins as was previously shown for ENOD40 (Campalans et al., 2004; Ben Amor et al., 2009). This binding affects the interaction of NSRs with AS targets and may modulate the action of NSRs on their endogenous AS mRNA targets. Such lncRNAs may mimic endogenous AS transcripts to hijack regulators of the splicing machinery and induce rapid changes in AS patterns during development. EXPERIMENTAL PROCEDURES

Figure 4. Modulation of AS in the nsra/nsrb Mutant during Auxin Response (A) Relative proportion and number of genes showing a significant change in AS from the analysis using the high-resolution AS real-time PCR panel of the nsra/nsrb DM with and without 1 mM of auxin in comparison to WT (based on Table S1). An important class (43 genes) corresponds to auxin-dependent AS changes requiring AtNSRs (light gray). Genes with auxin-specific (green) and NSR-specific (blue) effects on AS are represented. The threshold used for the analysis was >3% change with a p value of < 0.05; detailed data and analysis are provided in Table S1. (B) The ATPase1 (At1G27770) is mainly transcribed as a single mRNA in control conditions or during auxin treatment (1 mM) whereas, in the Atnsra/nsrb mutant treated with auxin, a second isoform accumulates (an IR event, black bars; see Figure S5E). Arrows indicate the fully spliced (white) and AS (black) isoforms in PAGE gels, and their shading correspond to its quantification (relative to the amount of the same isoform in Col-0 during control conditions) in the accompanying bar graph panels. See also Figures S5 and S6 and Table S1.

of the ZIFL1 gene (ZIFL1.1) modulates indirectly cellular auxin efflux at the root tip, whereas a second isoform (ZIFL1.3) seems to mediate drought tolerance by regulating stomatal closure (Remy et al., 2013). Therefore, isoforms with very similar sequences can elicit substantially distinct morphological outcomes in different organs of the plant. Noncoding RNAs affect gene expression by modulating the action of RNP complexes, and certain lncRNAs were recently shown to affect AS by different mechanisms in mammalian systems. A class of sno-lncRNA (a class of nuclear-enriched intronderived lncRNAs processed on both ends by the small nucleolar RNA [snoRNA] machinery) is strongly associated with the Fox

Plant and Bacteria Material and Growth Conditions All mutants were in the Columbia-0 (Col-0) background. Atnsra (SALK_003214) and Atnsrb (Sail_717) were from the SALK and SAIL T-DNA collections, respectively. The plants were grown in long day (16 hr light/8 hr dark) or continuous light conditions at 23 C on soil or on solid half-strength Murashige and Skoog (½MS) medium. E. coli DH5a was used for subcloning. For the analysis of LR phenotypes, Arabidopsis seeds (WT; nsra/nsrb DMs; and lines overexpressing NSRa, NSRb, or ASCO-RNA) were grown in ½MS medium with or without 100 nM NAA after germination. Primary roots and LRs were measured 7 days after germination. For high-resolution real-time PCR panels for AS, 2week-old plantlets from WT and nsra/nsrb DMs were grown in ½MS medium and were transferred for 12 hr to 100 nM NAA; for this experiment, only roots were harvested. For the transcriptomic studies and for the real-time PCR, 2week-old plantlets were grown in ½MS medium and were treated or not with 1 mM NAA for 24 hr and harvested. Transient Expression in Tobacco Leaves or in Protoplasts All transient expression experiments were performed in Nicotiana benthamiana, infiltrated with 0.1 unit at optical density 600 nm Agrobacterium tumefaciens AGL-0 in infiltration medium containing ½MS medium, 0.01 M MgCl2, 0.01 M MES (pH 5.2), 0.001 M acetosyringone. All constructs (see Supplemental Experimental Procedures) were coexpressed with HC-Pro (Anandalakshmi et al., 1998). For the coexpression of AtNSRb-GFP and MtNSR1-DsRed, protoplasts transformation was used (Campalans et al., 2004). Briefly, Arabidopsis suspension cells were digested in Gamborg’s B5 medium supplemented with 0.17 M glucose, 0.17 M mannitol, 1% cellulase, and 0.2% macerozyme, and protoplasts were purified by flotation on 0.28 M sucrose. For transformation, 0.2 million cells were mixed with 5 mg of plasmid DNA in a solution containing 25% (w/v) polyethylene glycol (PEG) 6000, 0.45 M mannitol, 0.1 M Ca(NO3)2 (pH 9) and incubated in the dark for 20 min. Then, the PEG was washed twice with 0.275 M Ca(NO3)2, and protoplasts were resuspended in Gamborg’s B5 medium supplemented with 0.17 M glucose, 0.17 M mannitol and observed in the confocal microscope. Confocal Microscopy and Image Processing Fluorescent cells were imaged 24–48 hr after transient expression by confocal microscopy (Leica TCS SP2, Leica Microsystem) with excitation at 488 nm, and the fluorescence emission signal was recovered between 495 and 530 nm for GFP fusions, and excitation at 543 nm and emission signal recovered between 555 and 620 nm for DsRed or red fluorescent protein (RFP) fusions. The Leica confocal software was used for image acquisition and for the quantification of fluorescence profiles. Sequential scans were performed when necessary. Spectral profiles were calculated for five cells. Data processing was performed using ImageJ (http://rsbweb.nih.gov/ij/).



Figure 5. An NSR Complex Binds to Alternatively Spliced Targets and to ASCO-RNA In Vivo and In Vitro (A and B) RIP assays using HA-tagged NSRa or NSRb on total cell lysates (Total RNA) or nuclear extracts (Nuclear RNA) of 10-day-old seedlings treated with 10 mM NAA for 24 hr. Results of quantitative real-time PCR are expressed as the percentage of the respective input signal (INPUT: total signal before RIP). Genes analyzed in (A), with accession numbers, are as follows: housekeeping (HKeeping): At1G13320 (Czechowski et al., 2005); Fbox: At4G27050; PIWI factor: At2G29210; and auxin-regulated gene: At2G33830. In (B), the following lncRNAs were tested: lnc34, 43, 72, 78, 351 (ASCO-RNA), 375, and 536 (Ben Amor et al., 2009). The only significant enrichment was detected for ASCO-RNA (lnc351, At1G67105); the other RNAs gave values similar to those of housekeeping genes. (C) Quantitative real-time PCR showing the relative expression of U6 (a known nuclear RNA) and ASCO-RNA in total or nuclear RNA. Enrichment in the nuclear RNA fraction at higher values than U6 was observed for the ASCO-RNA. Errors bars indicate SD. (D) In vitro competition assay; the graph represents the average quantification of dot blots in three biological replicates (relative to the amount of the same dilution without competitor RNA [w/o]). These results indicate that the ASCO-RNA has the capacity to compete the binding of NSR to an AS target. The lncRNA72 (a lncRNA that does not bind to NSRs) was not able to compete NSR-AS target binding. Error bars indicate SD. See also Figure S7.

Statistical Analysis and Error Bars Error bars on real-time PCR and quantitative real-time PCR experiments were calculated using SDs, and Student’s t tests (a = 0.05) were calculated. For phenotypic analysis of root and epidermal cell measurements, errors bars were calculated using a confidence interval of 5%; stars indicate Student’s t test a = 0.05 (Figure 3 and Figure S3). For LR measurements (Figure 6), error bars were calculated using SDs, and a Kruskal-Wallis statistical test (a = 0.05) was conducted. For analysis of ASCO-RNA subcellular localization and in vitro NSRa-RNA binding assays (Figure 5), error bars were calculated using SDs. Real-Time PCR and Quantitative Real-Time PCR Total RNA was prepared from roots and plantlets at different developmental stages using the QIAGEN RNeasy plant mini kit. The DNase treatment was performed according to the manufacturer’s protocols. For reverse transcription with SuperScriptII (Invitrogen), 2.5 mg of total DNase-treated RNA was used. One microliter of the resulting complementary DNA (cDNA) solution was used for real-time PCR or quantitative real-time PCR analyses. The latter was conducted using standard protocols, and a complete list of quantitative real-time PCR primers is available (see Supplemental Experimental Procedures). Each cDNA sample was precisely calibrated and verified for two constitutive genes (AT1G13320 and AT4G26410; Czechowski et al., 2005). For real-time PCR, the amplification was performed as follows: one cycle of 4 min at 98 C, 26 cycles of 30 s at 98 C, 30 s at 59 C, and 1 min at 72 C. The products were separated on a 7.5% polyacrylamide gel stained with SyBr green (Invitrogen) and revealed by a Pharos imager (Biorad). Band pro-

files were quantified using ImageJ (http://rsbweb.nih.gov/ij/). Quantitative real-time PCR was performed using a Roche Light Cycler 480 using standard protocols (40 cycles, 60 C annealing). High-Resolution Real-Time PCR Panels for AS The original panel (Simpson et al., 2008) was expanded to 288 primer pairs by identifying AS events that were either published or annotated in The Arabidopsis Information Resource (TAIR8; http://www.arabidopsis.org/) or in the Alternative Splicing in Plants database (http://www.plantgdb.org/ASIP/). Primer pairs in which one primer was fluorescently labeled were designed as described elsewhere (Simpson et al., 2008; Patel and Bellini, 2008; Kalyna et al., 2012). Primer pairs used are listed in the Supplemental Experimental Procedures. Real-time PCR analysis was performed as described elsewhere (Simpson et al., 2008). In brief, reverse-transcriptase (RT) reactions were performed with total RNA using oligo-dT primers. Total RNA was extracted from the roots of WT and nsra/nsrb plants treated (or not treated) with 100 nM auxin for 12 hr. The first-strand cDNA was used for PCR using the gene/AS eventspecific primers for 24 cycles. Our high-resolution real-time PCR system is capable of detecting multiple different AS transcripts from a single gene, distinguishing AS events involving small size differences in transcripts (as few as 2–3 nucleotides) and identifying small but significant changes in the ratios of alternatively spliced variants. The AS variants for each of the genes were amplified simultaneously using the same primers within the same reaction. The different AS isoforms usually contain substantially conserved sequences, which reduce variation in amplification efficiency. In addition, if there were differences in amplification efficiency among particular AS isoforms, these



Figure 6. The ASCO-RNA Modulates AS of NSR-Dependent AS Targets In Vivo, and Its Overexpression Reduced the Number of AuxinInduced LRs (A) Plants overexpressing the ASCO-RNA showed changes in isoform distribution of the auxin-related protein (At2G33830) (an IR event; black bars). (B) The ATPase1 (At1G27770) is transcribed as a single mRNA in control conditions, whereas a second isoform (an IR event; black bars) is detectable in ASCORNA overexpressing plants, a pattern similar to the one observed in the nsra/nsrb DM treated with auxin (Figure 4B). Arrows indicate each AS isoform in PAGE gels, and their shading corresponds to its quantification (relative to the amount of the same isoform in Col-0 during control conditions) in the accompanying bar graph panels. (C and D) Two ASCO-RNA overexpressing lines (35S-ASCO.1 and 35S-ASCO.2) do not present any major variation in primary root length (C) but present a reduced LR density after and auxin treatment (100 nM NAA) 7 days after germination (D). For comparison, the nsra/nsrb phenotype is shown. n > 20; error bars indicate SD; asterisks indicate Kruskal-Wallis test a = 0.05. See also Figure S5. differences occurred in the PCR reactions with all samples. Electropherograms produced by the ABI 3730 genotyping software identified the exact size of the real-time PCR products for each primer pair. The peak areas for each real-time PCR product were extracted from three replicates; the ratios of the different peaks were calculated to generate a mean value and SE for each AS transcript as a percentage of the total transcript across the three replicates. Real-time PCR with the 287 primers to AS regions identify a total of around 945 transcripts, of which 128 showed no products in this study (Table S1). The mean ratio of the AS transcripts are tabulated for WT, the nsra/nsrb DM and auxin-treated WT and DM plants (WT + auxin and DM + auxin, respectively). Means were compared to WT (Col0) by one-way pairwise ANOVA after an angular transformation, comparing WT with WT + auxin; WT with DM; and WT with DM + auxin. Genes/transcripts showing variability above 3% and with a p value < 0.05 were considered significant (Table S1B). The number of genes

present in different classes (Table S1C) were grouped according to: (1) the comparison of WT with WT + auxin (auxin-regulated AS events); (2) the comparison of WT + auxin with DM + auxin (auxin-regulated events that do not require NSRs; the rest are NSR-dependent auxin-induced changes); (3) interaction effects (effects seen in DM + auxin that were not observed or were different from those in WT + auxin, so that both NSR action and auxin treatment is required to detect these events); and (4) the remaining detected AS events in control conditions (NSR dependent). Primers are indicated in the Supplemental Experimental Procedures. Transcriptomic Studies Microarray analysis was performed at the Unite´ de Recherche en Geńomique Ve´ge´tale using the CATMAv6.1 array, which is based on Roche-NimbleGen technology. A single high-density CATMAv6.1 microarray slide with 12



assumed to exhibit a standard normal distribution. To control the false discovery rate (FDR), adjusted p values were calculated using the optimized FDR approach (Storey and Tibshirani, 2003). The probes with an adjusted p value % 0.05 were considered to be differentially expressed. Statistical analysis was performed using the R software. The function SqueezeVar of the LIMMA library was used to smooth the specific variances by computing empirical Bayes posterior mean values. The kerfdr library was used to calculate the adjusted p values. Data Deposition Microarray data from this article were deposited at the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/; accession number GSE39659). Raw and normalized data coming from the Blanc-0801_2012_01_RNAPATHs_nsr project can be downloaded from a URGV FTP site at the following address (data_norm and gpr_cel folder): ftp://urgv.evry. inra.fr/CATdb/. Project can be visualized on the CATdb interface.

Figure 7. ASCO-RNA Interacts with the NSRs to Modulate AS Model for the action of NSRs. NSRs are splicing regulators that bind to premRNA targets and modulate the ratio between different isoforms. In addition, NSRs can bind lncRNAs such as the endogenous ASCO-RNA in the nucleus to alter AS. Our results support the hypothesis that lncRNAs can hijack NSRs to affect their binding to AS targets and modulate AS regulatory activity of the NSRs. These results indicate a link between alternative splicing regulation and the action of lncRNA.

chambers was used; each chamber contained 135,000 primers representing all of the Arabidopsis thaliana genes: 30,834 probes corresponding to TAIRv8 annotation (including 476 probes of mitochondrial and chloroplast genes) + 1,289 probes corresponding to EUGENE software predictions. Moreover, the array included 5,352 probes corresponding to repeat elements, 658 probes for microRNA, 342 probes for other RNAs (ribosomal RNA, transfer RNA, snRNA, and snoRNA) and 36 controls. Each long primer represents a triplicate in each chamber for robust analysis. Two independent biological replicates were produced. For each biological repetition and each point, RNA samples were obtained by pooling RNA from 10 plants. Plantlets were collected at 1.04 developmental growth stages (Boyes et al., 2001) and cultivated in ½MS conditions. Plantlets (2 weeks old) were grown in ½MS medium for 14 days and were treated for 24 hr with 1 mM NAA alongside untreated controls. Total RNA was extracted using the QIAGEN RNeasy kit according to the manufacturer’s protocol. For each comparison, one technical replicate with fluorochrome reversal was performed for each biological replicate (i.e., four hybridizations per comparison). The labeling of complementary RNAs with Cy3-dUTP or Cy5-dUTP (Perkin-Elmer-NEN Life Science Products) and hybridization to the slides were performed as described by Lurin et al. (2004). Two-micron scanning was performed using an InnoScan900 scanner (Innopsys), and the raw data were extracted using Mapix software (Innopsys). Statistical Analysis of Microarray Data Experiments were designed with the statistics group of the Unite´ de Recherche en Geńomique Ve´ge´tale. For each array, the raw data comprised the logarithm of median feature pixel intensity at wavelengths of 635 nm (red) and 532 nm (green). For each array, a global-intensity-dependent normalization was performed using the Loess procedure (Yang et al., 2002) to correct the dye bias. The differential analysis is based on the average log ratios over duplicate probes and over technical replicates. Hence, the number of sets of available data for each gene equals the number of biological replicates, and that number was used to calculate the moderated t test (Smyth, 2004). Under the null hypothesis, no evidence for specific variance between probes is highlighted by LIMMA, and, consequently, the moderated t statistic is

RNA Immunoprecipitation Atnsra and Atnsrb single mutant lines were complemented with the constructs pNSRa-NSRa-HA-HA and pNSRa-NSRb-HA-HA, respectively. Ten-day-old plants treated 24 hr with 1 mM NAA were irradiated three times with UV using a UV crosslinker CL-508 (Uvitec) at 0.400 J/cm2. Plants were ground in liquid nitrogen. Total cell proteins were extracted in RIP extraction buffer I (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 5 mM MgCl2; Triton 0.1%; 10% glycerol). The extracted suspension was filtered twice through Miracloth. After centrifugation for 15 min at 4 C, 4,000 rpm, the pellet was resuspended in 800 ml of nuclei lysis buffer I + SDS (0.1% SDS; 10 mM EDTA; 50 mM Tris-HCL, pH 7.4), as well as 20 ml of RNase inhibitor and 20 ml of proteinase inhibitor. After 1 hr incubation at 4 C in rotation, the sample was centrifuged at 1,500 rpm for 5 min at 4 C. One hundred microliters of supernatant was used to prepare RNA for the input sample. Independently, nuclear proteins were extracted in RIP extraction buffer II (20 mM Tris-HCl, pH 7.4; 25% glycerol; 20 mM KCl; 2 mM EDTA; 2.5 mM MgCl2; 250 mM sucrose; 20 ml protease inhibitor per sample). The extracted suspension was filtered twice through Miracloth. After centrifugation for 15 min at 4 C, 4,000 rpm, the pellet was resuspended in 200 ml/g of nuclei washing buffer II (20 mM Tris-HCl, pH 7.4; 25% glycerol; 2.5 mM MgCl2; 0.5% Triton; 20 ml protease inhibitor per sample). The sample was centrifugated again for 15 min at 4 C, 4,000 rpm, and the washing step was repeated. Then, the pellet was resuspended in 200 ml/g nuclei lysis buffer II (50 mM Tris-HCl, pH 7.4; 1% Triton; 100 mM NaCl; 1 mM MgCl2; 0.1 mM CaCl2; 20 ml protease inhibitor per sample; 20 U/ml RNAse inhibitor). RNasefree DNase (10 ml) was added and incubated at 37 C for 10 min. SDS was added to a final concentration of 0.1% (20% SDS, 2.5 ml/500 ml). The sample was incubated for 1 hr at 4 C and centrifuged at 13,000 rpm for 30 min at 4 C. The supernatant was transferred to a new tube, and 10% of the volume was conserved at 20 C as the input. To purify the NSR proteins, we used mMACs magnetic technology columns (Mitenyi). First, the columns were prepared following the manufacturer’s protocols. The extracts were incubated for 1 hr with the magnetic beads, which were coupled with HA antibodies and passed through the columns. Several washing steps were performed: twice with 200 ml of the Miltenyi nuclei lysis buffer (0.1% SDS), twice with 200 ml of the Miltenyi washing buffer 1, twice with 200 ml of the Miltenyi washing buffer 2 and, finally, once with 200 ml of sterile water. The beads were collected and subjected to a native elution as indicated by the manufacturer’s protocol. The resulting solutions were treated with proteinase K (RNase grade, Invitrogen) in 2 ml of RNase inhibitor at 55 C for 1 hr. Last, we passed the samples through new columns to exclude the beads. The total RNA was extracted from the template and from the input using Trizol reagent (Invitrogen) and then was resuspended in 60 ml of water. The templates and the inputs were treated with DNase, and random hexamers were used for subsequent RT. Quantitative real-time PCR reactions were performed using specific primers. In Vitro NSRa-RNA Binding Assay Arabidopsis seedling nuclei were isolated, and NSRa-HA protein was purified using the IPStar Robot (Diagenode), Invitrogen Protein A Dynabeads, and Sigma anti-HA antibodies, following a direct chromatin immunoprecipitation protocol without reverse-crosslinking. ARP, nc72, and ASCO-RNA RNA samples were synthesized in vitro using T7 promoter-included PCR product as a



template; T7 RNA polymerase (Roche) and ARP RNA samples were labeled using the DIG Labeling Mix (Roche). In vitro transcribed RNA samples were incubated for 10 min at 95 C and gradually cooled down to let them adopt structured conformations. The NSRa-RNA binding was performed in binding buffer 13 (10 mM HEPES, pH 7.0; 50 mM KCl; 10% glycerol; 1 mM EDTA; 1 mM dithiothreitol; 0.5% Triton X-100) 1 hr in ice, and then three washes were done with the same buffer in a magnetic field to attach the beads. 5fold nonlabeled RNA was used to compete NSRa-ARP binding. RNA elution was achieved by incubating the beads at 95 C for 15 min. Beads were removed in the magnetic field. Serial dilutions of the samples were applied on a Hybond N+ membrane (GE Healthcare) and crosslinked by UV radiation. Digoxigenin activity was detected using Roche regents, following the instructions of the manufacturer. The dot blot intensity was quantified on a dilution curve of the recovered RNA. The dilution allowing quantification (not saturated and detectable) was quantified using ImageJ (http://rsbweb.nih.gov/ij/).

, Z.J. (2008). Plant SR proteins and their Barta, A., Kalyna, M., and Lorkovic functions. Curr. Top. Microbiol. Immunol. 326, 83–102.

ASCO-RNA Cellular Compartmentalization To determine the nuclear enrichment of ASCO RNA, nuclei were purified as for RIP assays and RNA was extracted using the RNeasy Minikit (QIAGEN). Independently, total RNA was extracted using the same kit. cDNA was synthesized using Super Script II (Invitrogen) and random hexamers. For equal levels of two reference genes (AT1G13320 and AT4G26410; Czechowski et al., 2005), the ratio of the ASCO RNA between the nuclei extracts and total RNA was calculated. Nuclei-enriched U6 RNA levels were considered as a positive control.

Charon, C., Sousa, C., Crespi, M., and Kondorosi, A. (1999). Alteration of enod40 expression modifies medicago truncatula root nodule development induced by sinorhizobium meliloti. Plant Cell 11, 1953–1966.

ACCESSION NUMBERS Microarray data from this article were deposited at the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/; accession number GSE39659). SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, seven figures, and one table and can be found with this article online at http://dx.doi.org/10.1016/j.devcel.2014.06.017. ACKNOWLEDGMENTS This work was supported by the Agence Nationale de la Recherche (ANR-08BLAN-0082) and the Saclay Plant Sciences Labex (SPS, ANR-10-LABX-40, to M.C.); by the Biotechnology and Biological Sciences Research Council (BB/ G024979/1); the EU FP6 Programme—European Alternative Splicing Network of Excellence (LSHG-CT-2005-518238); The France-Brasilian COFECUB Exchange Program (to M.C.); and the Scottish Government Rural and Environment Science and Analytical Services Division (to J.W.S.B.). F.B. was supported by a grant of the French Ministry of Education, and F.A. was supported by an EMBO long-term fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This work has benefited from the facilities of the Imagif Cell Biology Unit of the Gif campus (https://www.imagif.cnrs.fr), which is supported by the Conseil Geńe´ral de l’Essonne. Scanning electron microscopy pictures were obtained with the help of Se´verine Domenichini (Institute de Biologie des Plantes). lncRNA34 expression was detected with the help of Olivier Vandeputte (Universite´ Libre de Bruxelles). We thank Dr. Jim McNicol of Biomathematics and Statistics Scotland for statistical support and Dr. Javier Paz Ares of the Centro Nacional de Biotecnologia for careful advice on the manuscript.

Ben Amor, B., Wirth, S., Merchan, F., Laporte, P., d’Aubenton-Carafa, Y., Hirsch, J., Maizel, A., Mallory, A., Lucas, A., Deragon, J.M., et al. (2009). Novel long non-protein coding RNAs involved in Arabidopsis differentiation and stress responses. Genome Res. 19, 57–69. Blencowe, B.J. (2006). Alternative splicing: new insights from global analyses. Cell 126, 37–47. Boyes, D.C., Zayed, A.M., Ascenzi, R., McCaskill, A.J., Hoffman, N.E., Davis, K.R., and Go¨rlach, J. (2001). Growth stage-based phenotypic analysis of Arabidopsis: a model for high throughput functional genomics in plants. Plant Cell 13, 1499–1510. Campalans, A., Kondorosi, A., and Crespi, M. (2004). Enod40, a short open reading frame-containing mRNA, induces cytoplasmic localization of a nuclear RNA binding protein in Medicago truncatula. Plant Cell 16, 1047–1059.

Czechowski, T., Stitt, M., Altmann, T., Udvardi, M.K., and Scheible, W.-R. (2005). Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol. 139, 5–17. Djebali, S., Davis, C.A., Merkel, A., Dobin, A., Lassmann, T., Mortazavi, A., Tanzer, A., Lagarde, J., Lin, W., Schlesinger, F., et al. (2012). Landscape of transcription in human cells. Nature 489, 101–108. Filichkin, S.A., Priest, H.D., Givan, S.A., Shen, R., Bryant, D.W., Fox, S.E., Wong, W.-K., and Mockler, T.C. (2010). Genome-wide mapping of alternative splicing in Arabidopsis thaliana. Genome Res. 20, 45–58. Ingolia, N.T., Lareau, L.F., and Weissman, J.S. (2011). Ribosome profiling of mouse embryonic stem cells reveals the complexity and dynamics of mammalian proteomes. Cell 147, 789–802. James, A.B., Syed, N.H., Bordage, S., Marshall, J., Nimmo, G.A., Jenkins, G.I., Herzyk, P., Brown, J.W.S., and Nimmo, H.G. (2012). Alternative splicing mediates responses of the Arabidopsis circadian clock to temperature changes. Plant Cell 24, 961–981. Kalyna, M., Simpson, C.G., Syed, N.H., Lewandowska, D., Marquez, Y., Kusenda, B., Marshall, J., Fuller, J., Cardle, L., McNicol, J., et al. (2012). Alternative splicing and nonsense-mediated decay modulate expression of important regulatory genes in Arabidopsis. Nucleic Acids Res. 40, 2454–2469. Liu, Y., Pan, S., Liu, L., Zhai, X., Liu, J., Wen, J., Zhang, Y., Chen, J., Shen, H., and Hu, Z. (2012). A genetic variant in long non-coding RNA HULC contributes to risk of HBV-related hepatocellular carcinoma in a Chinese population. PLoS ONE 7, e35145. , Z.J., Hilscher, J., and Barta, A. (2008). Co-localisation studies of Lorkovic Arabidopsis SR splicing factors reveal different types of speckles in plant cell nuclei. Exp. Cell Res. 314, 3175–3186. Lurin, C., Andre´s, C., Aubourg, S., Bellaoui, M., Bitton, F., Bruye`re, C., Caboche, M., Debast, C., Gualberto, J., Hoffmann, B., et al. (2004). Genome-wide analysis of Arabidopsis pentatricopeptide repeat proteins reveals their essential role in organelle biogenesis. Plant Cell 16, 2089–2103. Malamy, J.E., and Benfey, P.N. (1997). Organization and cell differentiation in lateral roots of Arabidopsis thaliana. Development 124, 33–44. Marquez, Y., Brown, J.W.S., Simpson, C., Barta, A., and Kalyna, M. (2012). Transcriptome survey reveals increased complexity of the alternative splicing landscape in Arabidopsis. Genome Res. 22, 1184–1195. Matlin, A.J., Clark, F., and Smith, C.W.J. (2005). Understanding alternative splicing: towards a cellular code. Nat. Rev. Mol. Cell Biol. 6, 386–398.

Received: November 27, 2013 Revised: March 24, 2014 Accepted: June 23, 2014 Published: July 28, 2014

Palusa, S.G., Ali, G.S., and Reddy, A.S.N. (2007). Alternative splicing of premRNAs of Arabidopsis serine/arginine-rich proteins: regulation by hormones and stresses. Plant J. 49, 1091–1107.

REFERENCES Anandalakshmi, R., Pruss, G.J., Ge, X., Marathe, R., Mallory, A.C., Smith, T.H., and Vance, V.B. (1998). A viral suppressor of gene silencing in plants. Proc. Natl. Acad. Sci. USA 95, 13079–13084.

Parizot, B., De Rybel, B., and Beeckman, T. (2010). VisuaLRTC: a new view on lateral root initiation by combining specific transcriptome data sets. Plant Physiol. 153, 34–40. Patel, S.B., and Bellini, M. (2008). The assembly of a spliceosomal small nuclear ribonucleoprotein particle. Nucleic Acids Res. 36, 6482–6493.



Pe´ret, B., De Rybel, B., Casimiro, I., Benkova´, E., Swarup, R., Laplaze, L., Beeckman, T., and Bennett, M.J. (2009). Arabidopsis lateral root development: an emerging story. Trends Plant Sci. 14, 399–408. Remy, E., Cabrito, T.R., Baster, P., Batista, R.A., Teixeira, M.C., Friml, J., SaĆorreia, I., and Duque, P. (2013). A major facilitator superfamily transporter plays a dual role in polar auxin transport and drought stress tolerance in Arabidopsis. Plant Cell 25, 901–926. Rinn, J.L., and Chang, H.Y. (2012). Genome regulation by long noncoding RNAs. Annu. Rev. Biochem. 81, 145–166. Rohrig, H., Schmidt, J., Miklashevichs, E., Schell, J., and John, M. (2002). Soybean ENOD40 encodes two peptides that bind to sucrose synthase. Proc. Natl. Acad. Sci. USA 99, 1915–1920. Sapra, A.K., Anko¨, M.-L., Grishina, I., Lorenz, M., Pabis, M., Poser, I., Rollins, J., Weiland, E.-M., and Neugebauer, K.M. (2009). SR protein family members display diverse activities in the formation of nascent and mature mRNPs in vivo. Mol. Cell 34, 179–190. Schindler, S., Szafranski, K., Hiller, M., Ali, G.S., Palusa, S.G., Backofen, R., Platzer, M., and Reddy, A.S.N. (2008). Alternative splicing at NAGNAG acceptors in Arabidopsis thaliana SR and SR-related protein-coding genes. BMC Genomics 9, 159. Simpson, C.G., Lewandowska, D., Fuller, J., Maronova, M., Kalyna, M., Davidson, D., McNicol, J., Raczynska, D., Jarmolowski, A., Barta, A., and Brown, J.W. (2008). Alternative splicing in plants. Biochem. Soc. Trans. 36, 508–510. Smyth, G.K. (2004). Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat. Appl. Genet. Mol. Biol. 3, Article3. Sousa, C., Johansson, C., Charon, C., Manyani, H., Sautter, C., Kondorosi, A., and Crespi, M. (2001). Translational and structural requirements of the early nodulin gene enod40, a short-open reading frame-containing RNA, for elicitation of a cell-specific growth response in the alfalfa root cortex. Mol. Cell. Biol. 21, 354–366.

Storey, J.D., and Tibshirani, R. (2003). Statistical significance for genomewide studies. Proc. Natl. Acad. Sci. USA 100, 9440–9445. Syed, N.H., Kalyna, M., Marquez, Y., Barta, A., and Brown, J.W.S. (2012). Alternative splicing in plants—coming of age. Trends Plant Sci. 17, 616–623. Tanabe, N., Yoshimura, K., Kimura, A., Yabuta, Y., and Shigeoka, S. (2007). Differential expression of alternatively spliced mRNAs of Arabidopsis SR protein homologs, atSR30 and atSR45a, in response to environmental stress. Plant Cell Physiol. 48, 1036–1049. Tripathi, V., Ellis, J.D., Shen, Z., Song, D.Y., Pan, Q., Watt, A.T., Freier, S.M., Bennett, C.F., Sharma, A., Bubulya, P.A., et al. (2010). The nuclear-retained noncoding RNA MALAT1 regulates alternative splicing by modulating SR splicing factor phosphorylation. Mol. Cell 39, 925–938. Wang, G.-S., and Cooper, T.A. (2007). Splicing in disease: disruption of the splicing code and the decoding machinery. Nat. Rev. Genet. 8, 749–761. Wang, J., Ou, S.-W., Wang, Y.-J., Zong, Z.-H., Lin, L., Kameyama, M., and Kameyama, A. (2008). New variants of Nav1.5/SCN5A encode Na+ channels in the brain. J. Neurogenet. 22, 57–75. Yang, Y.H., Dudoit, S., Luu, P., Lin, D.M., Peng, V., Ngai, J., and Speed, T.P. (2002). Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res. 30, e15. Yin, Q.-F., Yang, L., Zhang, Y., Xiang, J.-F., Wu, Y.-W., Carmichael, G.G., and Chen, L.-L. (2012). Long noncoding RNAs with snoRNA ends. Mol. Cell 48, 219–230. Zhang, X.-N., and Mount, S.M. (2009). Two alternatively spliced isoforms of the Arabidopsis SR45 protein have distinct roles during normal plant development. Plant Physiol. 150, 1450–1458. Zhu, Q.-H., and Wang, M.-B. (2012). Molecular Functions of Long Non-Coding RNAs in Plants. Genes (Basel) 3, 176–190.
