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Supplementary Figure legends
Figure S1: Sequence analysis of Arabidopsis, Vitus vinifera and Oryza sativa RISP
Alignment of sequences from RISP homologues Arabidopsis (accession numbers NM_125513.1 and
NP_196406), Vitus vinifera (accession number CAO64487.1) and Oryza sativa (accession number
XP_479024) available in the database. Conserved amino acids are shown in black. Domains, where
computer analysis predicts coiled-coil are underlined (helixes H1, H2, H3 and H4).
Figure S2: RISP interactions with 60S
(A) Incubation of GST or GST-RISP bound to glutathione beads with 60S ribosomal subunits was
carried out for 30 min at 30°C and overnight incubation at 4°C in a 300 l reaction in buffer A
containing 50 mM Hepes, pH 7,5, 60 mM KCl, 3 mM MgCl2. The beads were washed, and the
unbound (U) and bound (B) fractions were analyzed by SDS PAGE followed by Coomassie blue
staining. Stars show 60S ribosomal proteins specifically co-precipitated with GST-RISP. (B) 60S
were washed twice, incubated with recombinant RISP, or TAV, or both at a 1:1.5 molar ratio and
then subjected to sucrose density gradient centrifugation followed by Western blot analysis with
antibodies against RISP and against TAV (western blot results of the gradient fractions are shown).
Gradients were fractionated with optical scanning at 254 nm. The sedimentation position of 60S
ribosomal subunits is shown.
Figure S3: In vitro interaction between L24, eIF3 and RISP
GST pull-down assays. GST-L24 attached to glutathione Sepharose 4B beads (lane 6) binding to
either eIF3 (lanes 7 and 8), or RISP and eIF3 (lanes 9-10); purified RISP and eIF3 are shown in
lanes 1 and 2, respectively. Unbound (U) and bound (B) fractions were analyzed by SDS-PAGE
followed by Coomassie blue staining.
Figure S4: Characterization of RISP-knockout plants.
(A) The T-DNA insertion in rispa.. Closed and open rectangles indicate the protein coding regions
and untranslated regions, respectively. A position of T-DNA inserted in the fourth exon in rispa is
shown by white triangle. (B) Col0 WT plants and 134C07 T-DNA-modified plants. (C) Transcript
accumulation in WT and 134C07 plants analyzed by RT-PCR.