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1 Research Arabidopsis PTB1 and PTB2 proteins negatively regulate splicing of a mini-exon splicing reporter and affect alternative splicing of endogenous genes differentially Craig G. Simpson 1, Dominika Lewandowska 1, Michele Liney 1, Diane Davidson 1, Sean Chapman 1, John Fuller 1, Jim McNicol 2, Paul Shaw 3 and John W. S. Brown 1,4 1 Cell and Molecular Sciences, The James Hutton Institute, Invergowrie, Dundee, DD2 5DA, UK; 2 Biomathematics and Statistics Scotland, Invergowrie, Dundee DD2 5DA, UK; 3 Information and Computational Sciences, The James Hutton Institute, Invergowrie, Dundee, DD2 5DA, UK; 4 Division of Plant Sciences, University of Dundee at JHI, Invergowrie, Dundee, DD2 5DA, UK Author for correspondence: Craig G. Simpson Tel: craig.simpson@hutton.ac.uk Received: 28 November 2013 Accepted: 14 March 2013 doi: /nph Key words: alternative splicing, mini-exons, splicing enhancers, splicing regulation, splicing repressors. Summary This paper examines the function of Arabidopsis thaliana AtPTB1 and AtPTB2 as plant splicing factors. The effect on splicing of overexpression of AtPTB1 and AtPTB2 was analysed in an in vivo protoplast transient expression system with a novel mini-exon splicing reporter. A range of mutations in pyrimidine-rich sequences were compared with and without AtPTB and NpU2AF 65 overexpression. Splicing analyses of constructs in protoplasts and RNA from overexpression lines used high-resolution reverse transcription polymerase chain reaction (RT-PCR). AtPTB1 and AtPTB2 reduced inclusion/splicing of the potato invertase mini-exon splicing reporter, indicating that these proteins can repress plant intron splicing. Mutation of the polypyrimidine tract and closely associated Cytosine and Uracil-rich (CU-rich) sequences, upstream of the mini-exon, altered repression by AtPTB1 and AtPTB2. Coexpression of a plant orthologue of U2AF 65 alleviated the splicing repression of AtPTB1. Mutation of a second CUrich upstream of the mini-exon 3 0 splice site led to a decline in mini-exon splicing, indicating the presence of a splicing enhancer sequence. Finally, RT-PCR of AtPTB overexpression lines with c. 90 known alternative splicing (AS) events showed that AtPTBs significantly altered AS of over half the events. AtPTB1 and AtPTB2 are splicing factors that influence alternative splicing. This occurs in the potato invertase mini-exon via the polypyrimidine tract and associated pyrimidine-rich sequence. Introduction Alternative splicing (AS) generates more than one mrna transcript from the precursor mrnas (pre-mrnas) of a gene through the selection of different splice sites (Black, 2003; Reddy, 2007; Nilsen & Graveley, 2010). Resultant alternatively spliced isoforms can be translated into functionally distinct proteins with different activities and localization, changes in their ability to interact with other proteins and substrates, or have altered sites for post-translational modification (Black, 2003; Stamm et al., 2005; Nilsen & Graveley, 2010; Carvalho et al., 2012). AS further regulates transcript abundance via degradation of particular AS isoforms by nonsense-mediated decay (NMD) (Ni et al., 2007; McGlincy & Smith, 2008; Kalyna et al., 2012; Schweingruber et al., 2013) or through the alternative inclusion of micro-rna (mirna) target sites (Yan et al., 2012). Regulation of splice site selection in AS depends on cis-splicing signals and their interactions with trans-acting factors. Besides the intronic splicing signals (5 0 and 3 0 splice sites, branchpoint and polypyrimidine tract), introns and exons contain sequences that enhance or suppress splicing. There are two main families of trans-acting RNA-binding splicing factors: serine-arginine-rich (SR) proteins and heterogeneous nuclear ribonucleoproteins (hnrnps). In general, SR proteins promote splice site selection by binding splicing enhancer sequences, while hnrnp proteins interact with suppressor sequences to inhibit or interfere with splice site selection (Long & Caceres, 2009; Shepard & Hertel, 2009; Xue et al., 2009; Han et al., 2010; Witten & Ule, 2011). The final outcome in terms of splicing phenotype depends on the combinatorial interactions of such factors with the pre-mrna and ultimately reflects the composition and activity of these proteins in different cells, developmental stages or conditions (Smith & Valcarcel, 2000; Chen & Manley, 2009). In animals, polypyrimidine tract binding protein (PTB) is an RNA-binding protein involved in splicing regulation (Spellman & Smith, 2006; Xue et al., 2009; Kafasla et al., 2012; Wachter et al., 2012) and other aspects of mrna production and function, such as 3 0 end processing, mrna stability, mrna localisation and internal ribosome entry site (IRES) translation (Pestova et al., 2001; Castelo-Branco et al., 2004; Knoch et al., 424

2 New Phytologist Research ; Sawicka et al., 2008; Babic et al., 2009). Although PTB is a negative regulator of AS of many alternative exons (Wagner & Garcia-Blanco, 2001; Spellman & Smith, 2006), a genome-wide analysis of PTB RNA interactions shows that PTB can also promote alternative exon inclusion by weakening constitutive splice sites (Xue et al., 2009). The altered splicing phenotype also reflected the position of binding of PTB on the transcript as also demonstrated for other hnrnp proteins (Witten & Ule, 2011). Recently, human PTB has been found to interact with 3 0 UTRs at key mirna binding sites, regulating access to mirnas that have the power to reprogram cellular gene expression (Engels et al., 2012; Xue et al., 2013). Human PTB contains four RNA recognition motifs (RRMs) and binds Cytosine and Uracil-rich (CU-rich) elements in polypyrimidine-rich sequences at multiple sites in a transcript, including the polypyrimidine tract (Singh et al., 1995; Perez et al., 1997; Xue et al., 2009). A number of models have been proposed for PTB suppression of splicing in different gene transcripts (Wagner & Garcia-Blanco, 2001; Spellman & Smith, 2006; Wachter et al., 2012). PTB can compete directly with U2AF 65 for binding to the polypyrimidine tract to block U2snRNP assembly or interfere with interactions between U2AF 65 and U1snRNP needed for intron or exon definition or spliceosome assembly (Lin & Patton, 1995; Singh et al., 1995; Izquierdo et al., 2005; Sharma et al., 2005, 2011; Sauliere et al., 2006; Matlin et al., 2007). PTB can bind cooperatively at multiple sites in the pre-mrna to either block access of splicing factors to splicing signals and regulatory sequences or to cause looping out of branchpoint or alternative exon sequences or form small local loops to allow assembly of protein complexes (Chou et al., 2000; Wagner & Garcia- Blanco, 2001; Amir-Ahmady et al., 2005; Oberstrass et al., 2005; Spellman & Smith, 2006; Cherny et al., 2010; Lamichhane et al., 2010). Finally, specific histone marks affected AS of specific genes through recruitment of PTB via a chromatin binding protein adaptor, strengthening a networking role for PTB in processes affecting gene expression (Luco et al., 2010). The importance of AS in plants has steadily risen with increasing numbers of plant genes that have been found to show AS (Xiao et al., 2005; Campbell et al., 2006; Wang & Brendel, 2006; Chen et al., 2007; Reddy, 2007; Barbazuk et al., 2008; Filichkin et al., 2010; Marquez et al., 2012; Syed et al., 2012). The most recent transcriptome survey, using a normalized library, demonstrated that 61% of intron-containing Arabidopsis genes undergo AS (Marquez et al., 2012). Although many examples of AS in plants have now been described (Jordan et al., 2002; Reddy, 2007; Sanchez et al., 2011; Carvalho et al., 2012; Syed et al., 2012; Staiger & Brown, 2013), relatively little is known about their regulation in terms of target binding sequences of splicing factors in plant introns or mrna targets of specific SR and hnrnp proteins. A small number of Arabidopsis enhancer sequences have been identified and shown to promote splice site selection, but their cognate RNA-binding proteins are unknown (Pertea et al., 2007; Thomas et al., 2102). Some SR proteins in Arabidopsis are relatively well characterized in terms of their protein protein interactions and subcellular localization, and overexpression lines display developmental or growth phenotypes, demonstrating the importance of SR protein abundance in the regulation of AS of many genes (Lopato et al., 1999; Kalyna et al., 2003; Ali et al., 2007; Duque, 2011). Similarly, hnrnp proteins and orthologues of spliceosomal proteins have been shown to be involved in AS. For example, cap-binding proteins (CBP20 and CBP80) often affect AS of alternative 5 0 splice sites in the first intron of plant genes (Raczynska et al., 2010); At- GRP7, AtGRP8 and PTB (see the following paragraph) affected a number of AS events (Stauffer et al., 2010; R uhl et al., 2012; Streitner et al., 2012); PRMT5, STIPL1 and SKIP are all involved in normal splicing of core circadian clock genes and mutants affect the period of the clock (Sanchez et al., 2010; Jones et al., 2012; Wang et al., 2012); and SUA is an AS factor that suppresses splicing of a cryptic ABSCISIC ACID INSENSITIVE3 (ABI3) intron regulating ABA during seed development (Sugliani et al., 2010). In addition, some SR and hnrnp proteins regulate AS of their own or other splicing factor transcripts (Kalyna et al., 2006; Sch oning et al., 2008; Simpson et al., 2008; Stauffer et al., 2010). That some of the AS events in SR protein genes are evolutionarily conserved highlights the importance of AS in plant growth and development (Iida & Go, 2006; Kalyna et al., 2006). Plants contain three genes with sequence similarity to human PTB (PTB1, PTB2 and PTB3) (Supporting Information, Fig. S1). PTB1 and PTB2 are closely related and are involved in pollen germination (Wang & Okamoto, 2009), while PTB3 was found in the phloem of pumpkin as a component of ribonucleoprotein complexes transported in the phloem sap (Ham et al., 2009). Potato PTB3 orthologues were also shown to interact with potato Nova1-like protein, an orthologue of a neuron-specific splicing factor in humans (Shah et al., 2013). The three Arabidopsis PTB genes are also subject to auto- and cross-regulation of AS of their pre-mrna transcripts (Wang & Brendel, 2006; Stauffer et al., 2010; R uhl et al., 2012). Their functions in AS have recently been demonstrated by transcriptome-wide RNA sequencing of PTB overexpression and knockout mutant lines. In contrast to AtPTB3, both AtPTB1 and ATPTB2 had major effects on AS. Reciprocal effects on AS of mutant and overexpression lines allowed the identification of c. 450 genes whose AS was putatively directly regulated by AtPTB1/2 (R uhl et al., 2012). AtPTB1 and AtPTB2 were able to both inhibit and stimulate intron splicing and affected cassette exon splicing, intron retention and alternative 5 0 splice site selection (R uhl et al., 2012). Here we exploited a mini-exon splicing reporter (Simpson et al., 2000, 2002) to investigate the ability of AtPTB1 and AtPTB2 to regulate splicing. AtPTB1 and AtPTB2 reduced splicing of the mini-exon, and using mutant constructs we defined pyrimidine-rich sequences between the branchpoint and 3 0 splice site of the intron upstream of the mini-exon as key signals controlling splicing and AtPTB1 activity. NpU2AF 65, an essential splicing factor which interacts with intron polypyrimidine tracts, competed with AtPTB1 and restored mini-exon splicing. Finally, using a high-resolution reverse transcription polymerase chain reaction (RT-PCR) system, we examined known AS events of endogenous genes in overexpression lines of AtPTB1 and AtPTB2, and they showed both common effects on AS patterns and effects specific to AtPTB1 and AtPTB2.

3 426 Research New Phytologist Materials and Methods Construction of invertase-green fluorescent protein (GFP) splicing reporter and invertase intron mutants The GF invertase gene from potato (accession no. AJ133765) consists of six exons and five introns (Maddison et al., 1999). Exon 2 is a 9 nt mini-exon that is spliced constitutively into invertase mrna (Simpson et al., 1996). A region of this invertase gene consisting of the last 9 nt of exon 1, the 219 nt intron 1, the 9 nt mini-exon 2, the 108 nt intron 2 and 9 nt of the 5 0 end of exon 3 was modified to include an initiation codon at the 5 0 end and an in-frame stop codon in mini-exon 2, and fused to the 5 0 end of the mgfp5 (GFP) gene (Siemering et al., 1996). Skipping of the mini-exon would produce a GFP protein with an N-terminal extension of seven amino acids, while inclusion of the miniexon would result in premature termination and possible expression of a five-amino-acid peptide (Fig. 1). This fragment was cloned between AscI and SacI sites of the binary vector pgrab. The plasmid pgrab contains the 35S promoter and terminator cassette from a derivative of prtl2 (Restrepo et al., 1990) with a modified multiple cloning site inserted between the Tobacco Etch Virus (TEV) leader sequence and the 35S terminator sequence. pgrab was produced by insertion of this modified 35S promoter and terminator cassette as a blunt-ended, PstI fragment between blunt-ended, KpnI and SacI sites of pgreenii 0229 (Hellens et al., 2000). To test the function of specific sequences in the invertase intron 1, the Inv1 splicing reporter was mutagenized. The Inv1 construct consists of 50 nt of exon 1, the 219 nt intron 1, the 9 nt mini-exon 2, the 108 nt intron 2 and 70 nt of exon 3 from the GF invertase gene inserted into the unique BamHI site found within the intronless zein gene of expression vector pdh515 (Simpson et al., 1996, 2000). Sequence changes made to the pyrimidine-rich region between the branchpoint and 3 0 splice site of intron 1 were made in Inv1, unless stated otherwise, using the Quickchange sitedirected mutagenesis system (Stratagene, La Jolla, CA, USA). The two CU-rich regions downstream of the polypyrimidine tract, CTCTCT (CU-1) and CCCCTT (CU-2), were mutated to give the constructs Inv46, 47, 69 and 70. In Inv46, CU-2 CCCCTT was mutated to CTCTCT (Fig. 2); in Inv47 the two CTCTCT in Inv46 were mutated to CACACA (Fig. 2). Inv69 and Inv 70 have CU-1 and CU-2 mutated to CACACA, respectively (Fig. 3). Polypyrimidine tract mutants where increasing numbers of Ts were exchanged for Cs in Inv39, Inv42, Inv59 and Inv61 have been described previously (Simpson et al., 2002) (Fig. 3). Cloning of RNA-binding protein genes The coding sequences of the Arabidopsis (At) and Nicotiana plumbaginifolia (Np) RNA-binding proteins, AtPTB1 (accession no. AF076924), AtPTB2 (accession no. BT015760), NpRBP45- HA (accession no. AJ292767) and NpU2AF65a (accession no. Y18351) (Domon et al., 1998; Marin & Boronat, 1998; Lorkovic et al., 2000), were cloned into the double 35S promoter containing vector, pjit60 (Guerineau & Mullineaux, 1993). (c) Fig. 1 Splicing reporter for negative regulation of mini-exon splicing. Schematic representation of potato invertase mini-exon splicing construct linked to green fluorescent protein (GFP; open box). Default splicing includes a 9 nt mini-exon (light grey box), which contains a translation stop codon. Skipping of the mini-exon bypasses the translation stop and produces an in-frame GFP coding sequence. Primers used for reverse transcription polymerase chain reaction (RT-PCR) analysis are shown as arrows. 786F is a 6-FAM-labelled oligonucleotide. Nicotiana benthamiana agroinfiltration-mediated expression of the splicing reporter construct with or without selected RNA-binding proteins. All inoculations shown used the splicing reporter construct coinoculated with: empty vector, no vector and vectors expressing GFP, NpRBP45, NpU2AF 65 and AtPTB1. (c) RT-PCR splicing analysis of the splicing reporter. Splicing to include the mini-exon gives a 221 bp product. Splicing that skips the miniexon (9 nt) gives a 212 bp RT-PCR product. Induction of skipping is found only as a result of expression of NtRBP45 and AtPTB1. Size bar indicates RT-PCR product size (bp). Influenza A virus Haemagglutinin (HA)-tagged NpRNP45 and NpU2AF65a were a kind gift from Dr Sergiy Lopato and Prof. Witek Filipowicz (Friedrich Miescher Institute, Basel). A cmyc epitope tag was cloned into the 3 0 end of the AtPTB1 and AtPTB2 sequences through two Quickchange site-directed mutagenesis reactions to produce patptb1-myc and patptb2-myc and used for tobacco protoplast cotransfections. Cloning of RNA-binding genes for Agrobacterium-mediated expression For Agrobacterium-mediated expression, the open reading frames for N. plumbaginifolia RBP45, U2AF65a and A. thaliana PTB1 were PCR-amplified and the products cloned in to pgem-t Easy (Invitrogen, Paisley, UK). The open reading frames were

4 New Phytologist Research 427 (c) Fig. 2 Mutations in CU-rich sequence elements affect mini-exon splicing and the effect of AtPTB1 and AtPTB2 expression. A 38 nt region between the branchpoint (bp) and U 11 (py) sequence and the 3 0 splice site of Inv1 shows two short CU-rich elements (CU1 and CU2). CU2 was mutated to make it more like a recognized human polypyrimidine tract binding protein (PTB) binding element (Inv46, underlined). The CU-rich elements were mutated individually (Inv69 and Inv70, underlined) and both elements were mutated (Inv47, underlined) to replace Us with As. The position of primers O8 and O9 are indicated. Reverse transcription polymerase chain reaction (RT-PCR) analysis of mini-exon splicing of intron constructs (Inv1, Inv46, Inv69, Inv70 and Inv47) coexpressed with AtPTB1 and AtPTB2 in tobacco (Nicotiana xanthi) protoplasts. The ratio of mini-exon inclusion and exclusion was calculated from the peak areas of RT-PCR products with primers O8 and O9 and the percentage of transcripts that demonstrate mini-exon inclusion is shown graphically. Assays were performed in the absence of AtPTB1 or AtPTB2 (black bars) or in the presence of AtPTB1 (white bars) or AtPTB2 (grey bars). Standard errors are calculated from three or more independent experiments with the exception of Inv70 with AtPTB2, which was calculated from two independent experiments. One-way ANOVA is shown as: *, P 0.1; **, P 0.05; ***, P 0.001; compared with experiments lacking the overexpressed protein. released from the pgem-t Easy clones by EcoRI digestion, blunt-ended by T4 DNA polymerase and the released bluntended fragments cloned in sense orientation between bluntended XhoI and FspI sites of pich18000 (Marillonnet et al., 2005) to replace the GFP gene in this Agrobacterium-delivered, Tobacco mosaic virus-based vector. The resulting binaries were transformed into Agrobacterium strain LBA4404. For AtPTB1 and AtPTB2 whole plant transformation, coding sequence was inserted into the CTAPi binary vector (Rohila et al., 2004) and transformed into Agrobacterium GV3101 by the freeze thaw method (H ofgen & Willmitzer, 1988). Agrobacterium-mediated expression Agrobacterium-mediated infiltrations were adapted from Voinnet et al. (2003). Agrobacterium strains were grown to stationary Fig. 3 Increased mini-exon splicing repression by AtPTB1 and AtPTB2 in polypyrimidine tract mutants with increased C content. Polypyrimidine tract mutations with increasing numbers of C nucleotides (Inv39, Inv42, Inv59, Inv61). RT-PCR analysis of mini-exon splicing of intron constructs (Inv1, Inv39, Inv42, Inv59 and Inv61) co-expressed with AtPTB1 in tobacco (Nicotiana xanthi) protoplasts. The ratio of mini-exon inclusion and exclusion was calculated from the peak areas of reverse transcription polymerase chain reaction (RT-PCR) products with primers O8 and O9 and the percentage of transcripts that demonstrate mini-exon inclusion is shown graphically. Assays were performed in the absence of AtPTBs (black bars) or in the presence of AtPTB1 (white bars) or AtPTB2 (grey bars). Standard errors are calculated from three or more independent experiments, with the exception of Inv42 with AtPTB2, which was determined from a single experiment. One-way ANOVA is shown as: *, P 0.1; **, P 0.05; ***, P 0.001; compared with experiments lacking the overexpressed protein. (c) Increasing mini-exon skipping by AtPTB1 and AtPTB2 in mutants with increasing C content is shown as the difference in percentage of mini-exon splicing between samples with or without AtPTB1 and AtPTB2 for the four pyrimidine tract mutants (Inv39, Inv42 (not AtPTB2), Inv59 and Inv61) which have increasing C content and decreasing U content. AtPTB1, solid line; AtPTB2, dashed line. phase, pelleted and suspended in 10 mm MgCl 2, 150 lm acetosyringone, 10 mm MES, ph 5.6mKOH at an OD 600 of between 0.1 and For coinfiltration experiments, bacterial strains containing constructs for the splicing reporter and the RNAbinding protein were mixed before performing the Nicotiana benthamiana leaf infiltration. Infiltrated regions of the leaf were marked and visualized by illumination with a long-wave (365 nm) ultraviolet lamp and the results photographed. For RT- PCR, infiltrated sections of leaves were excised and RNA extracted as previously described (Simpson et al., 1996). Oligo

5 428 Research New Phytologist 786F 5 0 -GCGCCAAAAATGGGATCAATG, corresponding to invertase exon 1 sequence, and oligo GTGACAAGTGTT GGCCATGG, complementary to the GFP sequence downstream of the mini-exon system, were used for RT-PCR (Fig. 1a). Protoplast expression and splicing analysis Myc-tagged patptb1 or patptb2 was mixed with an equal molar amount of the plasmids containing the invertase mini-exon and mutant reporter constructs. Protoplasts of Nicotiana tabacum var. Xanthi were transfected with the plasmid mixtures and RNA isolated as described previously (Simpson et al., 1996). RT-PCR analysis of the different mutated splicing reporters used the 5 0 fluorescent phosphoramidite 6-FAM labelled O8, 5 0 -CCC AATTGTTCAACCCTAC and O9, 5 0 -GGTAAGATGCCTGT TGCGATTGC primers. O8 and O9 correspond to the zein gene sequences that border the site of intron construct insertion in pdh515 (Simpson et al., 1996, 2000). RT-PCR fragments were separated on a 3730 DNA Analyzer (Applied Biosystems, Paisley, UK) and data were collected and analysed using Genemapper software (Applied Biosystems). Quantification of RT-PCR products was by measurement of the fluorescent peak areas of the detected fragments after 24 cycles (Simpson et al., 2000). Percentage inclusion or exclusion of the mini-exon was calculated from the peak areas for each processed transcript. Each construct was analysed in at least three independent transfection experiments and standard errors were determined. To confirm expression of the tagged proteins, protein was extracted from protoplasts, separated on 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gels and blotted onto Immobilon transfer membranes (Millipore). Immunoblots were performed using rabbit anti-ha (Millipore Antibodies) or mouse anti-myc (Abcam, Cambridge, UK) depending on the protein tag. Proteins were detected using Amersham Enhanced Chemiluminescence Western blotting detection reagents using horseradish peroxidase-linked secondary antibodies following the manufacturer s instructions (GE Healthcare, Little Chalfont, UK). Whole plant transformation Whole-plant transformation was by the floral dip method (Clough & Bent, 1998). Arabidopsis plants were grown in pots to the stage where inflorescences were developed and buds were beginning to flower. The above-ground part of the plants was dipped in Agrobacterium cultures for 10 min. Plants were laid on their side in the dark overnight, transferred to a containment glasshouse and allowed to set seed. Transformed seeds were grown on soil and selected by glufosinate ammonium (0.25 g l 1 ). Segregating T2 plants were sprayed with glufosinate ammonium (0.25 g l 1 ) and seed from homozygous lines was collected. Transgene expression was confirmed by western dot blot on Immobilon P filters (Millipore) and probed using whole rabbit IgG antibody (Upstate Biotechnology, Lake Placid, NY, USA) (Stott, 1989). Endogenous gene splicing analysis Total RNA was extracted from the leaves of 5-wk-old seedlings or flowers of homozygous lines of AtPTB1 and AtPTB2 using the RNeasy Plant Mini Kit (Qiagen, Venlo, the Netherlands) and AS analysed by fluorescent RT-PCR as described previously (Raczynska et al., 2010). Mean AS efficiencies with standard errors were calculated for three separate biological replicates. Means were compared by ANOVA after an angular transformation, between wildtype plants and the different AtPTB overexpressing lines. Results Expression of AtPTB1 induces exon skipping of a mini-exon To address the functional characteristics of plant PTB-like proteins, we investigated whether AtPTB1 was able to affect plant splicing. We constructed a splicing reporter gene in a binary vector that consisted of exon 1, exon 2 (mini-exon) and part of exon 3 with authentic introns from a potato invertase gene (Simpson et al., 2000, 2002) fused to the GFP coding sequence (Fig. 1a). The mini-exon is 9 nt long and was modified to include a translation termination codon. Constitutive splicing includes the miniexon, and the presence of the stop codon in the mini-exon blocks production of GFP. Inhibition of splicing of the mini-exon (exon skipping) generates an open reading frame and production of the GFP fusion protein. The full-length coding sequences of AtPTB1, N. plumbaginifolia (Np) U2AF 65 and RBP45 were used to replace the GFP gene in pich18000, a binary-delivered, virus-based expression vector (Marillonnet et al., 2005), to assess their effect on the splicing reporter. U2AF 65 is a splicing factor that normally interacts with the polypyrimidine tract early in spliceosome assembly (Zamore et al., 1992; Valcarcel et al., 1996) and RBP45 was included as a control RNA-binding protein of unknown function with affinity for binding U-rich sequences (Lorkovic et al., 2000). To initially assess whether AtPTB1 affects splicing, the miniexon GFP splicing reporter construct was agroinfiltrated into regions of N. benthamiana leaves along with vectors for AtPTB1, NpU2AF 65 and NpRBP45 expression (Figs 1b, 4 6). Infiltrated leaves were visually assayed for GFP expression before excision of the infiltrated region from which total RNA was extracted and RT-PCR performed with primers to exon 1 and GFP (Fig. 1b,c). Control leaf regions were infiltrated with the splicing reporter construct alone or with a vector expressing GFP or an empty vector (Fig. 1b). The splicing reporter construct alone and the splicing reporter in combination with an empty vector did not produce GFP fluorescence (Fig. 1b), and RNA transcribed from the reporter only showed mini-exon inclusion (Fig. 1c). GFP expressed from the vector gave strong GFP fluorescence, but only inclusion of the mini-exon in the GFP reporter mrna was detected, showing that the vector did not alter splicing of the reporter (Fig. 1b,c). Coexpression of AtPTB1 with the splicing reporter generated a GFP signal, indicating increased mini-exon

6 New Phytologist Research 429 GFP fluorescence at a level considerably lower than AtPTB1 and some exon skipping was observed by RT-PCR (Fig. 1b). Thus the agroinoculation/viral vector system provided an initial indication that AtPTB1 induced skipping of the mini-exon and negatively regulated potato invertase mini-exon splicing. Fig. 4 Coexpression of U2AF 65 compensates for splicing repression by AtPTB1. Polypyrimidine tract mutant constructs of Inv1 where three Us have been replaced with three As (Inv41) and five Us with five Cs (Inv61). Reverse transcription polymerase chain reaction (RT-PCR) analysis of mini-exon splicing of intron constructs (Inv1, Inv41, Inv61) coexpressed with AtPTB1 and AtPTB2 in tobacco (Nicotiana tabacum var. Xanthi) protoplasts. The ratio of mini-exon inclusion to exclusion was calculated from the peak areas of RT-PCR products with primers O8 and O9 and the percentage of transcripts that demonstrate mini-exon inclusion is shown graphically. Assays were performed on Inv1 (black bars), Inv41 (white grey bars) and Inv61 (grey bars) in the absence of AtPTB1 or U2AF65 (no protein) in the presence of AtPTB1 (+AtPTB1) or U2AF 65 (+U2AF 65 )orin the presence of both AtPTB1 and U2AF 65. Standard errors are calculated from three independent experiments and one-way ANOVA is shown as: **, P 0.05; ***, P 0.001; compared with experiments lacking the overexpressed protein. skipping and production of GFP fusion protein (Figs 1b, 6), which was confirmed by detection of RT-PCR products lacking the mini-exon (Fig. 1c). Coexpression of NpU2AF 65 did not produce a GFP signal and exon skipping was not detected (Fig. 1b, c). Finally, the control RNA-binding protein, NpRBP45, gave CU-rich regions downstream of the polypyrimidine tract are required for efficient splicing and function with AtPTB1 and 2 Having shown that AtPTB1 can reduce splicing of the mini-exon in the N. benthamiana agroinoculation system, we used an N. tabacum L. cv Xanthi protoplast transient expression system to investigate the activity of AtPTB1 and AtPTB2 with a number of mutants of the mini-exon reporter (Fig. 2a). The key signals for efficient inclusion of the mini-exon are contained in the intron upstream of the mini-exon (intron 1) and were shown previously to be the branchpoint sequence, its extended distance from the 3 0 splice site (53 nt) and the adjacent polypyrimidine tract (Simpson et al., 2000, 2002) (Fig. 2a). Human PTB binds CU-rich sequences within polypyrimidine regions, with CUCUCU representing the optimal binding site (Perez et al., 1997; Simpson et al., 2004; Oberstrass et al., 2005; Xue et al., 2009). Examination of intron 1 of the mini-exon reporter identified two CU regions of 8 (CU1) and 7 nt (CU2) long, 5 nt downstream of the polypyrimidine tract and separated by 4 nt (Fig. 2a). The upstream region, CU1, contained an optimal CUCUCU sequence for hsptb, and CU1 and CU2 potentially represented PTB binding sites. To test whether these elements were required for efficient splicing of the mini-exon, a series of mutants were made. First, the CU2 region was mutated to contain a more optimal CUCUCU sequence (Inv46 CU2opt) and this had little effect on splicing of the mini-exon (Fig. 2a,b). Secondly, the Us in either CU1 (Inv69) or CU2 (inv70) or both CU elements (inv47) were mutated to As (Fig. 2a). Mutation of the upstream CU sequence (CU1mut Inv69) had no effect on splicing, while mutation of CU2 (CU2mut Inv70) reduced mini-exon inclusion by over 50% (Fig. 2b). This suggests that CU2 is critical for Fig. 5 Overexpression of AtPTB1 and AtPTB2 alters alternative splicing (AS) profiles of endogenous genes. Venn diagram showing the number of AS events that were significantly affected by overexpression of AtPTB1 (dark grey) and AtPTB2 (light grey). Graph showing the 26 AS events that were significantly affected by both AtPTB1 and ATPTB2. Bars on the graph show the difference in the ratio (expressed as a percentage) of the alternatively spliced transcript to total transcripts for overexpression lines (OE) and wildtype (WT) plants. Differences were calculated by subtracting the WT ratio from the OE line and expressing as a percentage. Black bars show difference between WT and AtPTB1. White bars show difference between WT and AtPTB2. *Indicates results that show opposite effects between AtPTB1 and AtPTB2.

7 430 Research New Phytologist (c) Fig. 6 Different alternative splicing (AS) profiles of Arabidopsis thaliana endogenous genes in overexpression lines of AtPTB1 and AtPTB2. Examples of AS events that show: opposite effects between AtPTB1 and AtPTB2 on an alternative 3 0 splice site; specific effect by AtPTB1 on an alternative 5 0 splice site; and (c) a specific effect by AtPTB2 on an alternative 5 0 splice site. Schematics show exon-intron structure: coding sequence (white boxes) 5 0 and 3 0 UTRs (black boxes). Constitutive and alternative splicing is indicated by the diagonal lines and the alternative splice sites are indicated by an arrowhead. Premature and authentic translation stop codons are indicated by stop signs and the primers used for reverse transcription polymerase chain reaction (RT-PCR) analysis by short arrows. Standard errors are calculated from three independent experiments and one-way ANOVA is shown as: **, P 0.05; ***, P 0.001; compared with experiments lacking the overexpressed protein. efficient splicing of the mini-exon and acts as a splicing enhancer sequence, presumably binding a trans-acting factor that promotes splicing. Coexpression of AtPTB1/2 with the same series of CU sequence mutations showed significant reductions in splicing in all mutants, with the exception of CU1mut (Inv69) (Fig. 2b). In the wildtype sequence (Inv1), mini-exon inclusion was reduced from 97% to 62 and 71% upon overexpression of AtPTB1 and AtPTB2, respectively. The CU2opt (Inv46) construct containing the optimal CUCUCU sequence showed a similar reduction in splicing as the wildtype intron (Inv1), suggesting that the effect of the CU2 region was unchanged by altering the CU-rich sequence. By contrast, the CU1mut (Inv69) showed no significant reduction in splicing of the mini-exon, suggesting that the CU1 sequence is required for AtPTB1/2 repression. The CU2 sequence acts as an enhancer of mini-exon splicing, and mutation to a CArich sequence (Inv70) reduces splicing significantly without At- PTB overexpression, suggesting that the CU2 region, unlike CU1, is not primarily involved in the AtPTB effect. With overexpression of AtPTB1, there was a further reduction in splicing consistent with the CU1 sequence mediating the AtPTB effect. The CU1-CU2 double mutant (Inv47) also had greatly reduced splicing compared with Inv1 (97 to 37%) and was further reduced with coexpression of AtPTB1/2. These results suggest that the negative effect of AtPTBs are mediated via the CU1 sequence, suggesting that it is at least part of a binding site for AtPTB1. This contrasts with the positive influence of CU2 in the highly efficient splicing of the wildtype mini-exon reporter such that, in terms of the effect of AtPTB, the CU-rich regions had different roles. AtPTB1 and AtPTB2 act at the polypyrimidine tract We have previously shown that mutation of the mini-exon reporter polypyrimidine tract to replace increasing numbers of Us with Cs progressively reduced splicing efficiency of the miniexon to 90 and 63% when four and five Cs were present, respectively (Simpson et al., 2002; and Fig. 3b). The polypyrimidine tract is 5 nt upstream of the AtPTB-sensitive CU1 sequence element (Fig. 3a). To examine whether AtPTB1/2 may also act at the polypyrimidine tract, we expressed the py mutant series with and without AtPTB1/2 and analysed the efficiency of splicing of the mini-exon. Coexpression of AtPTB1 and AtPTB2 with the wildtype construct (Inv1) reduced splicing to 62 and 71%, respectively. Coexpression of AtPTB1/2 with the C mutant series (Inv39, Inv42, Inv59 and Inv61) reduced splicing of the miniexon further, particularly when four or five Cs were present in the polypyrimidine tract and the downstream CU1 and CU2 elements remained unchanged (Inv59 and Inv61) (Fig. 3b). The data were visualized by plotting the difference in percentage mini-exon inclusion between cells expressing the mutant construct alone and with the two AtPTBs (Fig. 3c). Thus, increasing U to C mutations in the polypyrimidine tract increased repression of mini-exon splicing when AtPTB1/2 was coexpressed. This suggests that both AtPTBs potentially function at the py tract and that this effect is stronger with UC-rich sequences (although it is possible that the sequence changes affect interactions with

8 New Phytologist Research 431 other proteins in this region). Thus, AtPTB1 and AtPTB2 may function at both the polypyrimidine tract and the closely associated CU-rich CU1 sequences to repress mini-exon splicing. NpU2AF 65 promotes mini-exon splicing and compensates for the repression by AtPTB1 Human U2AF 65 interacts with the polypyrimidine tract early in spliceosome assembly, is recruited by SR proteins to promote splicing and interacts with U1snRNP in exon definition (Zamore et al., 1992; Valcarcel et al., 1996). NpU2AF 65 has a role in plant splicing and shows affinity primarily for poly(u) sequences (Domon et al., 1998). To examine whether NpU2AF 65 could promote mini-exon splicing and counteract the repressive effect of AtPTB1, polypyrimidine tract mutants (Inv41 and Inv61) were selected because they showed approximately equal inclusion/skipping of the mini-exon (Fig. 4a,b no protein). Coexpression of AtPTB1 (AtPTB2 was not used in these experiments, as the two PTBs gave similar results with the mini-exon system) reduced mini-exon splicing substantially from 57 and 55% to 16 and 13% for Inv41 and Inv61, respectively. Coexpression of NpU2AF 65 with the wildtype splicing reporter (Inv1) had no significant effect on splicing, as in this construct all transcripts include the mini-exon. However, coexpression of NpU2AF 65 with the weakened Inv41 and Inv61 promoted mini-exon splicing to 92 and 87%, respectively (Fig. 4b). Coexpression of both NpU2AF 65 and AtPTB1 restored splicing efficiency to a value intermediate between those when AtPTB1 and NpU2AF 65 were expressed alone. Thus, NpU2AF 65 promotes inclusion of the mini-exon in constructs with weakened polypyrimidine tracts, and expression of AtPTB1 and NpU2AF 65 have opposite effects. However, upon coexpression, they compensate for one another, suggesting that these proteins compete for binding, most likely at the polypyrimidine tract. Overexpression of AtPTB1 and AtPTB2 affects alternative splicing of endogenous Arabidopsis genes Having shown that AtPTB1/2 and AtPTB2 repress splicing of the mini-exon in reporter constructs, we examined the ability of these proteins to affect the splicing of endogenous Arabidopsis genes using an established high-resolution AS RT-PCR system (Simpson et al., 2008; Raczynska et al., 2010; Sanchez et al., 2010; Jones et al., 2012; Kalyna et al., 2012; Streitner et al., 2012). Homozygous T3 lines of Tandem Affinity Purification (TAP)-tagged AtPTB1 and AtPTB2 overexpression lines (At- PTB1OE and AtPTB2OE) were produced by transformation of Arabidopsis Col-0 and confirmed by western analysis (Fig. S2b). The principal phenotypes observed in these lines were rapid germination and growth in AtPTB1OE, delayed germination and growth in AtPTB2OE, and angled root growth compared with the wildtype (Fig. S2a,d). AtPTB2OE lines further showed a narrowing of the leaves to give a spidery phenotype and shorter root growth (Fig. S2c). High-resolution RT-PCR analysis of the At- PTB1OE and AtPTB2OE lines confirmed expression of the cognate AtPTB cdna transcripts and cross-regulation of alternative splicing. Overexpression of AtPTB1 promoted inclusion of the regulated alternative exon in AtPTB2 from 32% in the wildtype to 51% (primer pair 213), while overexpression of AtPTB2 increased inclusion of regulated alternative exon of AtPTB1 from 13% in the wildtype to 18% (primer pair 196). However, overexpression of AtPTB2 had a much greater effect on the alternative 5 0 splice site selection found in exon 8. This alternative 5 0 splice site removes 47 nt from exon 8, thereby truncating the C-terminal sequence of AtPTB1 towards the end of RRM3 (from 399 to 314 amino acids). Overexpression of AtPTB2 reduces use of this site from 93% in the wildtype to 78% in the AtPTB2OE line (primer pair 195) (Fig. S3; Table S1). In vivo transient overexpression of AtPTB1/2 disrupted the splicing of the potato invertase mini-exon. We therefore used the high-resolution RT-PCR approach to screen a set of 20 endogenous genes that contain short, 5 25 nt, mini-exons, to determine whether overexpression of AtPTB1/2 generally affects the splicing of mini-exons (Table S2). The majority of these mini-exon-containing genes include the mini-exon constitutively into the final mrna transcript in wildtype plants and splicing of the miniexon was unaffected by overexpression of AtPTB1/2 (Table S2). The single exception was the ribulose-phosphate 3-epimerase gene (At3g01850), where overexpression of AtPTB1 and AtPTB2 increased and decreased inclusion of the mini-exon from 94% in wildtype to 100 and 76%, respectively (Table S2). Sequence comparison with a second ribulose-phosphate 3-epimerase (At1g63290), which was not affected in the overexpression lines, showed that At3g01850 contained a distinct CUCUCU element positioned between the 3 0 splice site and the predicted branchpoint (Fig. S4a). Similarly, Arabidopsis has two invertase genes that, like the potato invertase, have a conserved 9 nt miniexon. We specifically looked at the splicing of the Arabidopsis invertase mini-exons in flower tissue and found that both genes were unaffected by overexpression of the two AtPTB cdnas and lacked CU-rich regions found in the potato invertase gene (Fig. S4b,c). AtPTB1 and AtPTB2 share 64% identity at the amino acid level (Fig. S1b). In a number of plant species, orthologues of the two genes separate into distinct clades, suggesting that they have diverged since ancestral gene duplication and may therefore have evolved differential functions. To examine the comparative effects of AtPTB1 and AtPTB2 on a range of different AS events, we selected 89 defined events that cover different types of AS (Table S1) and used the high-resolution RT-PCR system to measure changes in AS. There were varying effects on AS as a result of overexpression of the two PTB proteins: no effect; similar or opposite effects; and AS events affected by either AtPTB1 or ATPTB2 (Table S1; Fig. S5). Overall, 35 and 51% of the AS events analysed showed a significant change (P 0.05; 3% or greater change from wildtype) in the AtPTB1OE and AtPTB2OE lines, respectively, compared with the wildtype plant. Twenty-six of the primer pairs used to analyse AS events were significantly affected by both AtPTB proteins (Fig. 5a). Of these 26, around a third increased or decreased alternative splice site selection in the opposite direction depending on the overexpression line, indicating quite different responses to the two

9 432 Research New Phytologist AtPTB proteins for some genes (Fig. 5b). For example, AS of the sigma 4 factor gene (At5g13730) involved alternative 3 0 splice site selection in intron 2, which generated a fully spliced transcript or a transcript that included 14 additional nucleotides and introduced a premature termination codon (PTC) (Fig. 6a). Overexpression of AtPTB1 increased the relative abundance of the shorter functional transcript by 7%, while overexpression of AtPTB2 decreased the relative abundance of this transcript by 27%, thereby reducing the functional transcript of this gene (Fig. 6a). In addition, a number of AS events were only significantly altered by overexpression of either AtPTB1 or AtPTB2 (Fig. 5a). For example, AtPTB1OE uniquely affected the alternative 5 0 splice site in the 5 0 UTR of the ribonucleoprotein SRP14. Use of the distal 5 0 splice site increased from 18 to 25% in the At- PTB1OE line, but was unaffected in the AtPTB2OE line (Fig. 6b). Similarly, the AtPTB2OE line uniquely affected the alternative 5 0 splice site in a transcribed gene with unknown function (At2g04790). Overexpression of AtPTB2 significantly changed selection of the distal 5 0 splice site from 69% of the total transcripts in the wildtype to 49% in the AtPTB2OE line, while AtPTB1OE had very little effect at this splice site (Fig. 6c). In summary, AtPTB overexpression lines had a limited effect on the constitutive splicing of a selection of mini-exons but had widespread effects on alternatively spliced transcripts. Overexpression of the two different PTB proteins showed similar effects for some target genes but often affected AS differently, indicating that, despite their peptide similarity, they have differences in functional targets and binding activities. plant PTB has a preference for CU-rich sequences reflecting the preferred binding site for human PTB1, which is a CU-rich element located in pyrimidine-rich sequence (Singh et al., 1995; Perez et al., 1997), but we currently cannot rule out the changes in sequence affecting the function of other factors at this site. Using mutants capable of detecting both enhanced and repressed mini-exon splicing, we also showed that overexpression of a plant U2AF 65 orthologue, NpU2AF 65 (Domon et al., 1998), competed with AtPTB1 to promote splicing and that these proteins operate antagonistically to each other. The CU1 element is only 6 nt downstream of the poly U polypyrimidine tract (Simpson et al., 2002). The CU1 sequence appears to be the main binding region for AtPTBs, as mutation of CU1 (Inv69) abolishes the repressive effect of the AtPTBs. However, mutations in the polypyrimidine tract also showed an increased ability of AtPTB1/2 to repress mini-exon splicing, suggesting that they may also interact with the polypyrimidine tract (Fig. 7). This is reminiscent of the cooperative binding of human PTB to a high-affinity site and secondary weaker site in the regulation of splicing of the smooth muscle (SM) alternative exon of a-actinin. The intron upstream of the SM exon contains a distinct branchpoint and UCUU motifs at the 3 0 end of the polypyrimidine tract that promote binding of PTB to repress SM exon splicing. The UCUU sequences were crucial for PTB binding (high-affinity sites) but not enough for splicing repression, which needed the cooperative binding of PTB to the polypyrimidine tract to compete with U2AF 65 (Matlin et al., 2007). Similarly, PTB repression of N1 in c-src has high-affinity binding to one site Discussion Polypyrimidine tract binding proteins have a firmly established role in vertebrates as regulators of splicing. They compete or combine with other factors to control alternative splice site choice (Black, 2003; Matlin et al., 2005; Spellman et al., 2005, 2007; Sawicka et al., 2008; Xue et al., 2009). Plant PTB-like sequences have also been shown to function in splicing and AS through auto- or cross-regulation of splicing of alternative exons in PTB genes and, from a genome-wide analysis, to affect AS of over 400 different genes (Stauffer et al., 2010; R uhl et al., 2012). Here we have exploited a well-characterized and sensitive plant invertase mini-exon splicing system (Simpson et al., 2000, 2002) to characterize the function of AtPTB1 and AtPTB2. First, we developed a GFP-based splicing reporter to detect mini-exon skipping induced by transient expression of splicing factors such as PTBs. Aligning this visual system with high-resolution RT-PCR of RNA from agroinfiltrated leaf regions confirmed the AS phenotype at the RNA level. Secondly, we generated new mutants and exploited existing polypyrimidine tract mutants to analyse the sequence elements with which AtPTBs act to regulate splicing of the invertase mini-exon. Transient overexpression of AtPTBs significantly reduced mini-exon splicing in vivo and repression of the mini-exon reporter was mostly via the CU1 sequence (UCUCUCUU) and the polypyrimidine tract. Repression by At- PTBs also increased in mutants where the poly U polypyrimidine tract contained increased C nucleotide content. This suggests that Fig. 7 Model for role of py tract and CU-rich sequences in mini-exon splicing and in repression by AtPTB1. Efficient splicing of the mini-exon depends on the py tract and CU2 sequence and these are presumably bound by splicing factors that promote the formation of the complex involving U1snRNP at the 5 0 splice site of the downstream intron to promote its splicing (see Simpson et al., 2002). When CU2 is mutated, CU1 can act as a weak enhancer sequence, and when both CU-rich sequences are mutated, the remaining splicing may be a result of the less efficient complex formation (indicated by long curved arrow). AtPTB1 can bind to the CU1 sequence and the polypyrimidine tract. The CU1 sequence appears critical, such that it may be the initial site of interaction followed by binding to the py tract. Whether this reflects binding by different RNA recognition motifs (RRMs) in the same AtPTB1 molecule or by different AtPTB1 molecules is unknown.

10 New Phytologist Research 433 followed by binding to a second lower-affinity site which correlates with splicing repression (Amir-Ahmady et al., 2005). Although not systematically analysed in the same way as the potato invertase gene, the presence or absence of a CU-rich sequence downstream of the polypyrimidine tract also correlated with the effect of AtPTBs on mini-exon repression of two ribulose-phosphate 3-epimerase genes (Fig. S4a). While examining the effect of mutation of two CU-rich sequences downstream of the polypyrimidine tract in the invertase splicing system, the CU2 sequence was required for normal splicing of the mini-exon and therefore acted as an intronic splicing enhancer. By contrast, the upstream CU1 sequence was needed for the repressive effect of AtPTB and therefore acted as an intronic splicing suppressor. To date, very few splicing enhancer or suppressor sequences have been identified in plant pre-mrnas (Pertea et al., 2007; Thomas et al., 2102). Here we demonstrate the presence of two adjacent CU-rich sequence elements with different enhancing (CU2) and repressive (CU1) effects and show that the latter may also be important in cooperative binding of PTB to the polypyrimidine tract, suggesting that this is an important region for binding of different plant splicing factors to modulate AS of the mini-exon. Nearly 50% of human genes have PTB binding sites and nearly a quarter of human AS involves PTB (Xue et al., 2009). Using our high-resolution system, we found that over half of the AS events tested were significantly altered by overexpression of AtPTB1 and/or AtPTB2 (Fig. 5). We also found that AtPTB1 and AtPTB2 often showed distinct effects on different AS events. AtPTB1 and AtPTB2 are clearly related in sequence, exon-intron structure and regulation by AS and NMD. However, phylogenetic analysis separates the two genes and their orthologues into distinct clades, suggesting that these genes have arisen through duplication and diverged in function. We also observed different phenotypes in the overexpression lines, further supporting differential functions. A recent RNA-seq screen of misexpression lines of AtPTB1 and AtPTB2 showed that these genes regulated c. 450 events and that, as also shown here, both AtPTBs had specific and redundant effects (R uhl et al., 2012). Only eight alternative splicing events were common between the c. 90 AS events on the high-resolution RT-PCR splicing panel studied here and the significant events discovered by RNA-seq. The low amount of overlap between the two systems may result from the different resolution of the analysis on different overexpression lines. Nevertheless, AtPTB1 and AtPTB2 have both common and differential effects on different pre-mrnas. Although only a relatively small number of genes were analysed here, different AS profiles were observed in genes encoding RNA-binding proteins, including SR and hnrnp splicing factors, kinases and transcription factors that may underlie the discrete germination and growth phenotypes shown between the two overexpressing lines. For example, overexpression of AtPTB1 causes exclusive production of the PSEUDO RESPONSE REGULATOR 9 (PRR9) functional mrna transcript, while overexpression of AtPTB2 causes over half of the PRR9 transcripts to use an alternative 5 0 splice site in intron 2, which adds 8 nt, introducing PTCs and targeting the transcript for NMD (Fig. S5c.) (James et al., 2012). Mutants of the arginine dimethyltransferase, PRMT5, have reduced functional PRR9 as a result of increased nonfunctional transcripts (as described earlier) and prmt5 mutants show a delay in the period of the clock and late flowering (Deng et al., 2010; Sanchez et al., 2010). Considering the effect these genes have on AS, it is likely that maintaining the correct balance of these hnrnps will be important in plant development, reflecting their auto- and cross-regulation (Stauffer et al., 2010). Detailed AtPTB1 and AtPTB22 protein-rna sequence binding analysis and targeted protein mutation analysis will help to dissect further the functional specificity of these genes as distinctive plant hnrnp regulatory splicing factors. Acknowledgements We acknowledge funding for this research from the Scottish Government Rural and Environment Science and Analytical Services division (RESAS) and the European Alternative Splicing Network of Excellence (EURASNET), LSHG-CT We thank Dr Sergiy Lopato and Prof. Witek Filipowicz (FMI, Basel) for clones of NpU2AF65 and NpRBP45; Prof. Albert Baronet (University of Barcelona) for PTB1; Dr Viktor Kimyuk (Icon Genetics) for plasmid pich18000; Dr Andreas Wachter (Universit at T ubingen, Germany) for sharing PTB data and Dr Markus Blatter (ETH, Z urich) for his bioinformatic RRM domain screen of plant PTB and advice on PTB structure. References Ali GS, Palusa SG, Golovkin M, Prasad J, Manley JL, Reddy AS Regulation of plant developmental processes by a novel splicing factor. PLoS ONE 2: e471. Amir-Ahmady B, Boutz PL, Markovtsov V, Phillips ML, Black DL Exon repression by polypyrimidine tract binding protein. 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Nature Reviews Molecular Cell Biology 10: Chen WH, Lv G, Lv C, Zeng C, Hu S Systematic analysis of alternative first exons in plant genomes. BMC Plant Biology 7: 55. Cherny D, Gooding C, Eperon GE, Coelho MB, Bagshaw CR, Smith CW, Eperon IC Stoichiometry of a regulatory splicing complex revealed by single-molecule analyses. EMBO Journal 29: Chou M-Y, Underwood JG, Nikolic J, Luu MHT, Black DL Multisite RNA-binding and release of polypyrimidine tract binding protein during the regulation of c-src neural-specific splicing. Molecular Cell 5: