Download AtPTB-like 1 negatively regulates splicing inclusion of a plant

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Supporting Information Figs S1-S5.
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Supplementary Figure S1. AtPTB sequence comparisons.
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(a) Peptide sequence alignment of PTB genes from Arabidopsis. AtPTB1 (At3g01150,
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Q9MAC5); AtPTB2 (At5g53180, Q9FGL9); AtPTB3 (At1g43190, Q6ICX4) was performed
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using the Clustal W program (Thompson et al., 1994) at EMBL-EBI. The positions of the
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introns were identified from TAIR (http://www.arabidopsis.org/) and are indicated by
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triangular arrowheads. Closed arrow heads above the AtPTB1 peptide sequence indicates
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common intron positions between AtPTB1 and AtPTB2. Open arrow heads indicate a unique
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intron position in AtPTB1 and AtPTB2. Closed arrow heads below the AtPTB3 peptide
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sequence indicates the intron positions in AtPTB3. The positions of recognised RRM
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domains are shaded grey and were identified by SMART - simple modular architecture
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research tool http://smart.embl-heidelberg.de/. The horizontal open arrows indicate the
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position of the β5 strand extension.
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(b) Percentage peptide sequence identity between Arabidopsis PTBs, human PTB1, human
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hnRNPL and human hnRNPLL. Peptide comparisons to the human PTB-1 sequence (Acc:
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X62006) were determined using LALIGN a global alignment programme with an end gap
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penalty
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(http://www.ch.embnet.org/software/LALIGN_form.html). Comparison with 20 human
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hnRNP genes showed the three AtPTB peptide sequences had closest identity with human
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PTB (hsPTB) (see also supplementary Fig. 1c). AtPTB1 and AtPTB2 were highly related,
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showing 64.1% global peptide sequence identity and shared 7 introns in conserved positions.
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AtPTB3 showed ~25% global peptide sequence identity with the other two AtPTB genes and
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the positions of all except one intron were different. AtPTB3 showed the highest peptide
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sequence identity (32%) with hsPTB1 (see also Supplementary Fig. 1c). Human PTB1
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contains four RRMs which vary in their RNA-binding specificities and co-operate to loop out
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RNA (Wagner & Garcia-Blanco, 2001; Simpson et al., 2004; Oberstrass et al., 2005;
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Spellman & Smith, 2006). AtPTB3 also has four RRMs which may explain its greater
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similarity to hsPTB1 while AtPTB1 and AtPTB2 only contain three RRMs (Supplementary
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Fig. 1a). The regions of highest identity between the three plant genes were in RRM1 and
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RRM2 with the most highly conserved region between the three genes found at the β5 strand
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extension of RRM2 (M. Blatter personal communication; Conte et al., 2000; Oberstrass et al.,
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2005) (Supplementary Fig. 1a).
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(c) Evolutionary relationship of PTB orthologue peptide sequences from different plant
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species. The neighbor-joining phylogenetic tree (Saitou & Nei, 1987) was constructed using
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the CLC Workbench (CLC Bio). Comparison of the three PTB peptide sequences from
(Huang
&
Miller,
1991)
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on
the
Swiss
EMBnet
node
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Arabidopsis with other plant species show clustering of genes into three distinct groups and a
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further smaller fourth group found in some monocot plants (rice, sorghum and maize).
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AtPTB1 and AtPTB2 separated into two clades distinct from AtPTB3 and hsPTB. Human
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PTB1 and the related proteins hnRNPL and hnRNPLL, are included for comparison between
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plant and established regulators of splicing in human. The position in the tree of the three
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Arabidopsis PTB genes are indicated by *, human PTB1 by # and hnRNPL and hnRNPLL by
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◄. Numbers at each branch point represent the bootstrap values for percentages of 1,000
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replicate trees. The bar at the bottom indicates genetic distance. Accession numbers for tested
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genes are as follows:
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Arabidopsis: AtPTB1 (At3g01150), Q9MAC5; AtPTB2 (At5g53180), Q9FGL9; AtPTB3
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(At1g43190), Q6ICX4; Human: PTB1, P26599; hnRNPL, Q6NTA2; hnRNPLL, Q8WVV9;
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Rice: Os03g25980, Q8S7G0; Os01g43170; Os08g33830; Os01g64770; Os05g36120;
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Potato: StPTB1, Q38JI2; StPTB2, ABB55397; Chickpea: CaPTB1, Q84L59; Barley:
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HvPTB1, unigene 2403; HvPTB2 unigene 17519; HvPTB3 unigene 985; Castor Bean:
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RcPTB1, B9RIL3; RcPTB2, B9SE84; RcPTB3, B9T4F5; Pumpkin (Winter squash):
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CcPTB3 (RBP50), C0J4I8; Grape: VvPTB1, D7SXE2; VvPTB3, D7SRE5; VvPTB4,
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A5AQI7; VvPTB5, D7SWA3; Poplar: PtPTB1, A9PHS8; PtPTB2, B9IK00; PtPTB3,
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A9PHI7; PtPTB4, B9IDY5; PtPTB6, B9GT89; PtPTB7, B9H7U5; Sitka Spruce: PsPTB1,
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A9NUM8; PsPTB2, D5ABY8; PsPTB3, D5AA11; PsPTB4, D5A7V1; PsPTB5, D5A9E6;
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Sorghum: SbPTB1, C5WYL9; SbPTB2, C5YL85; SbPTB3, C5XR40; SbPTB4, C5YYP8;
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Maize: ZmPTB1, C0P8R8; ZmPTB5, C0PF88; Soybean: GmPTB1, C6TJN2; GmPTB3,
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C6TED2; Physcomitrella: PpPTB1, A9SIE8; PpPTB3, A9RYR1; PpPTB4, A9S4W5.
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References
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Conte MR, Grüne T, Ghuman J, Kelly G, Ladas A, Matthews S, Curry S. 2000.
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Structure of tandem RNA recognition motifs from polypyrimidine tract binding protein
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reveals novel features of the RRM fold. EMBO J. 19: 3132-3141.
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Huang X, Miller W. 1991. A time-efficient, linear-space local similarity algorithm. Adv.
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Appl. Math 12: 337-357.
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Oberstrass FC, Auwete, SD, Erat M, Hargous Y, Henning A, Wenter, P, Reymond L,
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Amir-Ahmady B, Pitsch S, Black, DL, Allain F.H-T. 2005. Structure of PTB bound to
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RNA: specific binding and implications for splicing regulation. Science 309: 2054-2057.
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Saitou N, Nei M. 1987. The neighbor-joining method: a new method for reconstructing
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phylogenetic trees. Mol Biol Evol, 4: 406-425.
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Simpson PJ, Monie TP, Szendröi A, Davydova N, Tyzack JK, Conte MR, Read CM,
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Cary PD, Svergun DI, Konarev PV et al. 2004. Structure and RNA interactions of the N-
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terminal RRM domains of PTB. Structure. 12: 1631-1643.
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Spellman R, Smith CWJ. 2006. Novel modes of splicing repression by PTB. Trends
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Biochem. Sci. 31: 73-76.
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Thompson JD, Higgins DG, Gibson TJ. 1994. CLUSTAL W: improving the sensitivity of
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progressive multiple sequence alignment through sequence weighting, position-specific gap
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penalties and weight matrix choice. Nucleic Acids Res. 22: 4673-4680.
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Wagner EJ, Garcia-Blanco MA. 2001. Polypyrimidine tract binding protein antagonizes
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exon definition. Mol Cell Biol. 21: 3281-3288.
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Supplementary Figure S2. Phenotypes of AtPTB cDNA over-expression lines. (a) Growth
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characteristics of over-expression lines of AtPTB1 and AtPTB2 in particular showing
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delayed growth and flowering in AtPTB2OE. (b) Western blot analysis of TAP tagged
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AtPTB over-expression lines showing expression of AtPTB1 and AtPTB2. Lane 1 AtPTB1
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OE, Lane 2 Untransformed Col-0, Lane 3, AtPTB2 OE. M is a size marker indicated in kDa.
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(c) “Spidery” phenotype in AtPTB2 OE lines. (d) Root length in 14 day old seedlings.
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Seedlings grown on graduated plates and root lengths measured using Rootimage software
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(Root Image Processing Laboratory (RIPL) at Michigan State University). Roots of both
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over-expression lines show non-vertical growth and those of AtPTB1OE were longer and
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AtPTB2OE were shorter compared to wild-type Col-0. Mean root length after 14 days growth
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was 52.4mm, 73.5mm and 23.4mm for wild type, AtPTB1OE and AtPTB2OE respectively.
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Supplementary Figure S3. Cross-regulation of alternative splicing of AtPTB1 and 2. (a)
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Schematic representations of AtPTB1 and AtPTB2 genes indicating AS events: alternative
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inclusion/exclusion of an alternative exon in intron 2 of both AtPTB1 and ATPTB2 and an
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alternative 5’ splice site in intron 8 of AtPTB1 (arrowheads). The positions of primer pairs
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195, 196 and 213 that amplify these AS events are shown as labelled arrows. Translation stop
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codons are indicated by stop signs. (b) Effect on AtPTB1 AS in AtPTB2OE and AtPTB2 in
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AtPTB1OE. Levels of the full-length RNA capable of producing functional protein expressed
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as percentage of total transcripts. Reductions in percentage of fully spliced transcript in
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primer pair 196 by AtPTB2 and 213 by AtPTB1 indicate an increase in the relative amounts
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of the transcripts containing the alternative exon while reduction in primer pair 195 indicates
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an increase in the relative amounts of transcripts spliced to the proximal 5’ splice site.
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Supplementary Figure S4. Splicing analysis of selected genes containing a mini-exon. (a)
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Schematic representation of the two ribulose-phosphate 3-epimerase genes containing a 5nt
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mini-exon indicated by an arrowhead. Coding sequences are shown as grey boxes and introns
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as lines between the boxes. UTR sequences are shown as black boxes. Primers pairs to
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amplify transcripts to detect splicing of the mini-exon are indicated as labelled arrows.
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Translation stop codons are shown as stop signs. The position of the CU- rich element in
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At3g01850 is indicated as an open circle with the sequence above. (b) Schematic
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representation of the Arabidopsis cell wall invertases containing a 9nt mini-exon indicated by
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an arrowhead. Coding, intron and UTR sequences are shown as above. Primer pairs are
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indicated as labelled arrows. Sequence alignments between the potato invertase sequence
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(PotInv) and the Arabidopsis invertases (At3g52600 or At2g36190) from the putative
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branchpoint (labelled with an arrowhead) to the mini-exon (boxed) are shown. The CU1
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element found in the potato invertase gene is shown boxed and is absent in the Arabidopsis
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invertases. (c) High-resolution RT-PCR analysis identified only transcripts that included the
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mini-exon (shown by arrowhead) in Arabidopsis invertases 2 and 4 in wild type Col-0,
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AtPTB1OE and AtPTB2OE lines. No evidence of exon skipping was found.
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Supplementary Figure S5. Examples of significant changes in alternative splicing in
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AtPTB over-expression lines. Examples of AS from the high resolution RT-PCR analysis in
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AtPTB1 and AtPTB2 over-expressing lines (Supplementary Table 2; Figure 5). Graphs show
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(a) No significant change in alternative splicing compared to the wild-type. (b) Significant
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changes in AS occur in both over-expressing lines compared to the wild-type and both affect
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alternative splicing by increasing or decreasing the ratio of alternatively spliced transcripts
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compared to the wild-type. (c) Significant changes in AS occur in both over-expressing lines
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compared to the wild-type, but affect AS in opposite directions compared to the wild-type.
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(d) Significant changes in AS specific to the AtPTB1 over-expressing line compared to the
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wild-type. (e) Significant changes in AS specific to the AtPTB2 over-expressing line
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compared to the wild-type. Graph bars indicate the relative percentage levels of the different
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transcripts and the product size in base pairs (bp) is indicated in the boxes to the right of the
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graph. Significance is indicated as * (p = <0.1); ** (p= <0.01); *** (p=<0.001) compared to
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the wild-type.
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