Nature Chemical Biology Supplementary Information

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1 Nature Chemical Biology Supplementary Information Synthetic Translational Regulation by an L7Ae-Kink-turn RNP Switch Hirohide Saito *, Tetsuhiro Kobayashi, Tomoaki Hara, Yoshihiko Fujita, Karin Hayashi, Rie Furushima & Tan Inoue * 1

2 Supplementary Methods DNA and RNA preparation for the in vitro assays All DNA templates and primers used in this study were purchased from Hokkaido System Science. Either the synthetic DNA templates were amplified by PCR with the appropriate 5'- and 3'- primers or single stranded templates were annealed with the T7 annealing primer (see below). PCR amplifications were carried out using Ex Taq DNA polymerase (TAKARA BIO INC.) or KOD-PLUS-DNA polymerase (TOYOBO). The corresponding synthetic mrnas for use with the PURE system were prepared by two-step PCR according to the manufacturer s instructions (PURESYSTEM classic II 96, POST GENOME INSTITUTE CO., LTD). The second step PCR was carried out using the universal primer (see below) and the corresponding reverse primer. PCR amplifications were carried out for ~20 cycles (94 C, 15 sec; 50 C, 30 sec; C, 60 sec). The pegfp-n1 or DsRed Express vector (Clontech) was used as an initial template. The minimal box C/D motif was prepared based on the sequence from Archaeoglobus fulgidus, and the GAAA tetraloop was added to the upper stem region 2

3 (box C/D mini ) 34. Non-Watson-Crick base pairing between G7 and A20 is essential for the kink-turn structure; these two bases were therefore mutated to C (box C/D mut ) to disrupt the kink-turn, or the sequences that form the kink-turn were replaced with a GAAA tetraloop (box C/D ). After purification, the PCR products were transcribed using T7 RNA polymerase for ~3 h at 37 C. The DNA templates were then degraded using TURBO DNase (MEGAshortscript TM, Ambion) for 15 min at 37 C. The resulting RNA products were purified by denaturing PAGE and precipitated using ethanol. For the gel-mobility shift assay (EMSA), we prepared radio-labeled RNAs in the presence of [ 32 P-α]GTP (Perkin Elmer). For the translational activation systems, we transcribed the regulator RNAs from template DNAs composed of synthetic single-stranded DNAs and the T7 annealing primer. The synthetic mrnas were transcribed using MEGAscript T M (Ambion) and purified using the RNeasy MinElute TM Cleanup Kit (QIAGEN), according to the manufacturer s instructions. The DNA templates and primers used in this study were prepared as follows: (the italicized bases correspond to the T7 promoter sequence). 3

4 T7 annealing primer: 5'-GCTAATACGACTCACTATA-3' box C/D mini template: 5'-GGGTCAGCTTTCGCATCACGCCCTATAGTGAGTCGTATTAGC -3' box C/D mut template: 5'-GGGGCAGCTTTCGCATGACGCCCTATAGTGAGTCGTATTAGC-3' box C/D template: 5'-GGGTTTCCCCTATAGTGAGTCGTATTAGC-3' universal primer: 5'-GAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAA ATAATTTT GTTTAACTTTAAGAAGGAGATATACCA-3' 4

5 Primers for mrnas: box C/D mini _ EGFP mrna box C/D mini _EGFP Fwd-primer: 5'-AAGGAGATATACCAATGGGGCGTGATGCGAAAGCTGACCCTGTGAGCA AGGGCGAGGAG-3' EGFP Rev-primer: 5'-TATTCATTACCCGGCGGCGGTCACGAA-3' box C/D mut _ EGFP mrna box C/D mut -EGFP Fwd-primer: 5'-AAGGAGATATACCAATGGGGCGTCATGCGAAAGCTGCCCCTGTGAGCA AGGGCGAGGAG-3' EGFP Rev-primer: 5'-TATTCATTACCCGGCGGCGGTCACGAA-3' 5

6 box C/D _ EGFP mrna box C/D -EGFP Fwd-primer: 5'-AAGGAGATATACCAATGAGGGGAAACCCAGTGAGCAAGGGCGAGGAG-3' EGFP Rev-primer: 5'-TATTCATTACCCGGCGGCGGTCACGAA-3' box C/D mini -DsRed -mrna box C/D mini -DsRedEx Fwd-primer: 5'-AAGGAGATATACCAATGGGGCGTGATGCGAAAGCTGACCCT GCCTCCTCCGAGGACGTC-3' DsRedEx Rev-primer: 5'-TATTCATTACTACAGGAACAGGTGGTGGC-3' 6

7 box C/D mut -DsRed mrna box C/D mut -DsRedEx Fwd-primer: 5'-AAGGAGATATACCAATGGGGCGTCATGCGAAAGCTGCCCCT GCCTCCTCCGAGGACGTC-3' DsRedEx Rev-primer: 5'-TATTCATTACTACAGGAACAGGTGGTGGC-3' box C/D _DsRed -mrna box C/D -DsRed Ex Fwd-primer: 5'-AAGGAGATATACCAATGAGGGGAAACCCAGCCTCCTCCGAGGACGTC-3' DsRedEx Rev-primer: 5'-TATTCATTACTACAGGAACAGGTGGTGGC-3' box C/D anti _ EGFP mrna (ON switch mrna) 7

8 box C/D-anti_ EGFP mrna Fwd-primer: 5'-AAGGAGATATACCAATG-CAGCTTTCGCATCAC-GTGAGCAAGGGCGAG-3' EGFP Rev-primer: 5'-TATTCATTACCCGGCGGCGGTCACGAA-3' Regulator RNAs: box C/D_25 RNA box C/D_25 RNA-T7-annealing primer 5'-GGGGTCAGCTTTCGCATCACGCCCCTATAGTGAGTCGTATTAGC-3' box C/D mut _25 RNA box C/D mut _25 RNA-T7-annealing primer 5'-GGGGGCAGCTTTCGCATCACGCCCCTATAGTGAGTCGTATTAGC-3' box C/D_29 RNA 8

9 box C/D_29 RNA-T7-annealing primer 5'- GGTGGGTCAGCTTTCGCATCACGCCCACCTATAGTGAGTCGTATTAGC -3' box C/D mut _29 RNA box C/D mut _29 RNA-T7-annealing primer 5'- GGTGGGGCAGCTTTCGCATCACGCCCACCTATAGTGAGTCGTATTAGC -3' Preparation of the recombinant L7Ae protein from A. fulgidus The pet 28-b+ vector (Novagen) was selected for the cloning and expression of the recombinant protein L7Ae from A. fulgidus. We followed the protein expression protocol that was reported previously 32 with a slight modification. Briefly, the plasmid was transformed into E. coli. BL-21 (DE3) (plyss) cells. Protein expression was induced using 1 mm IPTG and the culture was incubated overnight at 30 C. The cells were harvested by centrifugation at 6000 rpm for 20 min at 4 C and resuspended in sonication buffer (50 mm phosphate buffer, ph 8.0, 300 mm NaCl) at 4 C. The suspension was then sonicated. The lysate was then incubated for 15 min at 80 C to 9

10 denature endogenous protein, which was removed by centrifugation at 6000 rpm for 20 min at 4 C. The supernatant contained the recombinant hexahistidine-tagged L7Ae protein. L7Ae was purified from the supernatant using Ni-NTA Agarose following the manufacturer s directions (QIAGEN). The purity of the protein was confirmed using SDS-PAGE. The eluted protein was concentrated using a YM-3 microcon (Millipore), and the protein was dialyzed against buffer containing 20 mm HEPES-KOH (ph 7.5), 150 mm KCl, 1.5 mm MgCl 2, and 5 % glycerol. The concentration of the purified L7Ae protein was determined using the Bradford protein assay (BIO-RAD). The purified L7Ae protein was stored in storage buffer (20 mm Hepes-KOH (ph 7.4), 150 mm KCl, 1.5 mm MgCl 2 containing 40 % glycerol) at -20 C. To construct an L7Ae derivative (L7AeK37AK79A), QuikChange II Site-Directed Mutagenesis Kit was purchased from Stratagene. The mutations (K37 to A and K79 to A) were introduced into the plasmid that coded L7Ae (The pet 28-b+-L7Ae) by using the following primers. The constructed plasmid were confirmed by sequencing. K79A Fw-primer: 5'- CATTTACGTTAAAAGCGCGAACGACCTTGGAAGG -3' K79A Rev-primer: 10

11 5'- CCTTCCAAGGTCGTTCGCGCTTTTAACGTAAATG -3' K37A Fw-primer: 5'- GTACCAACGAGACGACAGCGGCTGTGGAGAGGGGAC -3' K37A Rev-primer: 5'-GTCCCCTCTCCACAGCCGCTGTCGTCTCGTTGGTAC-3' Electrophoretic Mobility-Shift Assays (EMSAs) EMSA assays were carried out in 20 µl of RNP-binding buffer (1 x buffer: 20 mm Hepes-KOH, 150 mm KCl, 1.5 mm MgCl 2, 2 mm DTT, U/ml trna, 3% glycerol, ph 7.5). The RNAs that contain the box C/D mini or box C/D mut motifs were body-labeled using [ 32 P-α]GTP. Initially, the labeled RNAs were denatured by heating at 80 C for 5 min, and then 5-fold RNP binding buffer concentrate was added to the solution. The resulting solution was pre-incubated at 37 C for 5 min. After RNA refolding, various concentrations of recombinant L7Ae (0-500 nm) were added to the solutions. The L7Ae-RNA complexes were then incubated at 4 C for 60 min. The RNA-protein complex was separated from free RNA by running 8 % native 11

12 polyacrylamide gels at 4 C for ~4 hours, which were analyzed using a BAS 2500 (Fuji Film). SPR assay RNP binding experiments using SPR were performed at 25 C on a Biacore 3000 instrument (GE Healthcare) according to the manufacturer s instructions. The biotinylated DNA oligonucleotide (5'-Biotin-CCGGGGATC CTCTAGAGTC-3', 1 µm) was dissolved in 500 mm KCl and immobilized on the surface of a streptavidin-coated sensor chip SA (GE HealthcareBIAcore AB) at a flow rate of 10 µl min -1 for 8 min. After the DNA was immobilized on the SA chip, the RNA was diluted to a final concentration of 1 µm in HBS-EP buffer (10 mm HEPES at ph 7.4, 150 mm NaCl, 3 mm EDTA, % surfactant P20), heated at 80 C for 5 min, and then refolded at room temperature for 5 min. The sample was then diluted 10-fold in 1 M KCl and injected over the sensor chip surface at 5 µl min -1 for 3 min. In this way, Box C/D mini RNA was conjugated to DNA-hybridizable RNA sequences and immobilized on SA sensor chips. L7Ae was serially diluted in running buffer (10 mm Tris-HCl at ph 8.0, 12

13 150 mm NaCl, 5 % glycerol, 125 µg ml -1 yeast trna [Roche], 62.5 µg ml -1 BSA, 1 mm DTT, 0.05 % Tween 20) and injected at 25 C at a flow rate of 50 µl min -1 for 1 min. Any complex that remained bound after a 5 min dissociation by the same running buffer was disrupted using several 30 sec injections of 8 M Urea at 20 µl min -1. Binding experiments were performed in triplicate at each protein concentration, and all of the experiments were repeated at least four times to confirm the binding kinetics. In order to subtract any background noise from each dataset, all of the samples were also run over an unmodified sensor chip (containing no RNA). In addition, running buffer without any protein was randomly injected throughout every experiment (double referencing). We also analyzed the interaction between L7Ae and box C/D, and the sensorgrams that we obtained were similar to the sensorgrams that were obtained using the unmodified sensor chips without RNA. The data were analyzed by BIA evaluation software (GE Healthcare) and a simple 1:1 Langmuir interaction model. The kinetic parameters were determined through global fitting analysis. Monitoring the time course of the cell-free translation 13

14 To monitor the expression of the cell-free protein in real time (Supplementary Figure 5a), we mixed 5 µl of solution A with 1 µl of 3.75 µm mrna (final concentration, 375 nm) in the presence or absence of 1 µl of 10 x L7Ae solutions. The mixtures were then diluted to 8 µl in water. The mrna-l7ae complex was incubated at 4 C for 30 min, and then 2 µl of solution B was added to the solution to initiate translation. The solutions were then transferred to 384-well microplates, and the expression of EGFP was monitored every 5 min at 37 C using microplate readers (Infinite F500, TECAN, excitation filter 485/20 nm, emission filter 535/25 nm). Western Blotting for the in vitro translation assays After the cell-free translation assay, 10 µl of the solution was diluted to 200 µl with water, and 5 µl of the diluted solution was used for western blotting. The membranes were incubated with mouse monoclonal anti-gfp antibody (1:200, Santa Cruz Biotechnology, sc-9996), followed by goat anti-mouse IgG (H + L)-HRP conjugate (1:2000, BIO-RAD). The chemiluminescence signals from the protein bands were detected using ECL-Plus Western blotting detection reagents (GE Healthcare). The 14

15 images were analyzed using an LAS-3000 (Fuji-film). See also Supplementary Figure 5b. Construction of a tetracycline-inducible cell line (T-REx TM HeLa- DsRed M -L7Ae) The tetracycline (Tet) -inducible cell line (T-REx TM HeLa-DsRed M -L7Ae) was established as follows. T-REx TM HeLa cells (Invitrogen), cultured in fresh medium supplemented with 10 % CBS (#2001H, MP-Biomedicals), were transfected with pcdna4/to/dsred-l7 containing the tetracycline-regulatable promoter and the DsRed M -L7Ae gene. The medium was changed to fresh medium supplemented with 10 % CBS after a 4 hour incubation at 37 C. After 24 hours, the medium was again replaced with fresh medium supplemented with 10 % CBS, 5 µg/ml of blasticidin (Invivogen), and µg/ml of zeocin (Invitrogen) for selection. The cells were cultured in selective medium every 3-4 days until foci could be identified. The selected cells were cloned using a cell sorter (FACS Aria, BD Biosciences). The clone exhibiting the highest expression level of DsRed M -L7Ae in the presence of Tet was confirmed by western blotting and named H5. The H5 cells were cultured in D-MEM/F-12 15

16 supplemented with 10 % CBS, 5 µg/ml of blasticidin, and 150 µg/ml of zeocin. Plasmid construction for in vivo translation assays The L7Ae gene was amplified by PCR from pet28b-l7ae using the following primers: L7-Fwd (5'-AAGGATCCATCATATGCGGCCGCTTATGTACGTGAGATTTGAGG-3') and L7-Rev (5'-CACTCGAGTTGAATTCTCTTCTGAAGGCCTTTAATC-3'). The gene was then cloned into pcdna3.1-mychisa (pcdna, Invitrogen) between the BamHI and XhoI sites to construct the pl7ae plasmid. The DsRed-Monomer (DsRed M ) gene fragment was amplified by PCR from pdsred-monomer (Clontech) using the following primers: DsRed M -Fwd (5'-CCGGATATCCCTGGGAGCCGGAGTGGC-3') and DsRed M -Rev (5'-CATGAATTCTTGCCACCATGGACAACACCGAGGACG-3') before cloning into pcdna3.1-mychisa at the EcoRI and EcoRV sites to construct pdsred M. The sequences of each of the clones were confirmed. To generate a chimeric protein tagged with L7Ae, each fragment from pl7ae and pdsred M was ligated to construct the fusion proteins, pdsred M -L7Ae or pl7ae-dsred M, using NotI and XhoI, or BamHI and EcoRI, respectively. To generate a Tet-controllable expression system, 16

17 the pcdna4/to/dsred M -L7Ae plasmid was constructed by ligating NotI- and XhoI-digested fragments of pcdna4/to/mychisa and pcdna3.1-dsred M -L7Ae. The pbox C/D-EGFP construct, which contains the boxc/d mini motif placed after the AUG codon, was amplified by whole-plasmid PCR of pegfp-n1 (Clontech) and the following primers: (5'-GGGCGTGATGCGAAAGCTGACCCTGTGAGCAAGGGC GAGGAGCTG-3') and (5'-CATGGTGGCGACCGGTGGATC-3'). The template plasmid was then digested using DpnI. The pboxc/d -EGFP construct, which contains a GAAA loop instead of boxc/d mini, was amplified by the same methods using the following primers: (5'-AGGGGAAACCCAGTGAGCAAGGGCGAGGAGCTG-3') and (5'-CATGGTGGCGACCGGTGGATC-3'). The amplified PCR products were self-ligated. The cloned plasmids were confirmed by sequencing. The p1-box C/D-EGFP construct was constructed as follows: the EGFP gene with the boxc/d mini in the 5'-UTR was amplified from pegfp-n1 by PCR using the following primers: (5'-CCTAGATCTGGGGCGTGATCCGAAAGGTGACCCGGATCCATGGTGAGCAA GGGCGAGGAGCTGTTC-3') and (5'-GAGTCGCGGCCGCTTTACTTGTAC-3'). The EGFP in pegfp-n1 was replaced with the PCR product (EGFP with the boxc/d mini in 17

18 the 5'-UTR) between the BglII and NotI sites and then sequenced. [Note: After constructing the above plasmid, a Kozak sequence was introduced into the plasmid by replacing the 5'-UTR region (between the BamHI and NdeI sites) of the plasmid with that of pegfp-n1.] Plasmid construction for shrna expression The pentr/h1/to vector (Invitrogen) was used to construct two shrna expression plasmids, p481 and psk-7n. The p481 plasmid was designed to express shrna encoding residues of the EGFP-coding sequence. The psk-7n plasmid was designed to express shrna that encodes a stop codon in all three reading frames to prevent nonspecific gene knockdown. These plasmids were produced by ligating the annealed oligonucleotides with the linearized pentr/h1/to vector. The oligonucleotide pair (top strand and bottom strand) used was as follows: p481 top strand: 5'-CACCGGCATCAAGGTGAACTTCAAGATCCAGCATAGGGATCTTGAAGTTC ACCTTGATGCC 3' 18

19 p481 bottom strand : 5'-AAAAGGCATCAAGGTGAACTTCAAGATCCCTATGCTGGATCTTGAAGTTCA CCTTGATGCC 3' psk-7n top strand : 5'-CACCGCACTAGCGTATGAATGAAAGATCCAGCATAGGGATCTTTCATTCATA CGCTAGTGC 3' psk-7n bottom strand : 5'-AAAAGCACTAGCGTATGAATGAAAGATCCCTATGCTGGATCTTTCATTCATA CGCTAGTGC 3' A target sequence of the EGFP gene (25 bp :5'-GGCAUCAAGGUGAACUUCAAGAU CC-3') was provided by Katoh et al. 44. Western blotting for L7Ae expression in the cells 19

20 After transfecting the resultant plasmids into HeLa cells (2.5 x 10 5 cells /well) in 6-well plates, the cells were lysed for 30 min on ice in 0.3 ml of RIPA buffer (1 x PBS, 1 % NP 40, 0.5 % sodium deoxycholate, 0.1 % SDS, 0.3 mg/ml PMSF, and 30 µl/ml aprotinin). The lysed cells were then centrifuged at 15,000 x g for 20 min at 4 C, and the supernatant was used as the total cell lysate. The concentration of proteins in each sample was normalized using the DC-protein assay kit (BioRad). The membranes were incubated with the mouse monoclonal anti-c-myc antibody (1:500, Anti-c-Myc (Ab-1) Mouse mab (9E10), Calbiochem). Cell proliferation assay (WST-1 assay) Initially, HeLa cells were plated in 96-well plates (1 x 10 4 cells/well), pre-incubated for 24 hours at 37 C, and then transfected with the corresponding plasmids (pcdna or pl7ae; µg). The metabolic activity of the cells was assayed after 24 hours by measuring the mitochondrial dehydrogenase activity using the tetrazolium salt WST-1 according to the manufacturer s instructions (Roche Diagnostics). 20

21 Quantitative Reverse Transcription-PCR (qrt-pcr). qrt-pcr was carried out according to the manufacturer s protocol (Roche Diagnostics). Briefly, total RNA was prepared from HeLa cell cultures using an RNAqueous-4PCR Kit (Ambion). RNA (0.5 µg) was reverse transcribed using a High-Capacity cdna Reverse Transcription Kit (Applied Biosystems). The forward ( ; 5'-ATGGCCGACAAGCAGAAG-3') and reverse ( ; 5'-GTAGTGGTCGGCGAGCTG-3') primers used to detect EGFP mrna were purchased from Hokkaido System Science. Aliquots of cdna were subjected to real-time PCR using a Lightcycler480 SYBR green I Master (Roche Diagnostics) according to the manufacturer s protocols. For each reaction, no amplification was observed when reverse transcription was omitted. The relative expression levels of EGFP mrna were normalized using the expression levels of GAPDH mrna as a reference. 21

22 Supplementary Results Supplementary Figure Legends & Figures Supplementary Figure 1 The specific and tight interaction between L7Ae and the box C/D K-turn. (a) EMSA, showing the formation of the box C/D mini -L7Ae complex. Radiolabeled box C/D mini or box C/D mut (25 nm) was assayed in the presence of increasing amounts of unlabeled L7Ae. The RNP complex was incubated at 4 C for 60 min. The concentration of L7Ae is indicated above each lane. (b) SPR analysis. Sensorgrams showing kinetic analyses of the interaction between box C/D mini and L7Ae. Box C/D mini RNA was conjugated to DNA-hybridizable RNA sequences and immobilized on an SA sensor chip. The concentrations of L7Ae that were injected are indicated to the right of each sensorgram. The A 1 min association phase was followed by a 5 min dissociation phase in which the surface was washed with a protein-free running buffer (as indicated by the black arrows). The experiments were repeated at least 4 times, and representative data are 22

23 shown. The kinetic parameters (i.e., the dissociation rate constant, kd, and the equilibrium dissociation constant, K D ) that were obtained from the global fitting analysis are described in the text. Supplementary Figure 2 Schematic description of the in vitro translational repression assay using the interaction between L7Ae and the box C/D K-turn. The PURE translation system was used to analyze the translation efficiency of the mrna containing the box C/D mini motif near the initiation site. Supplementary Figure 3 Synthetic mrnas used in the in vitro ORF-OFF systems. The box C/D mini motif was inserted immediately after the AUG start codon of the targeted mrna (either box C/D mini EGFP- or box C/D mini DsRed- mrnas). Non-Watson-Crick base pairing between G7 and A20 (indicated by bold italic letters), which are essential for the K-turn structure, were mutated to C (box C/D mut - EGFP (DsRed) mrnas), or the sequences 23

24 that form the K-turn structure were replaced with the GAAA tetraloop (box C/D - EGFP (DsRed) mrnas). Supplementary Figure 4 Specific interaction between L7Ae and the box C/D mini -EGFP mrna. (a) Synthetic RNAs used for EMSA. The partial sequences of the box C/D mini -EGFP mrnas (a total length of 110 nt or 200 nt of RNA from the 5'-terminus) used in this study. The minimal box C/D motif (box C/D mini ) and its derivatives (box C/D mut ) were inserted into the RNAs. Two mutated bases are indicated by bold italic letters. (b) EMSA showing the formation of the box C/D mini -containing RNA and L7Ae complex. Radiolabeled RNA (either box C/D mini, 110nt-EGFP, 200nt-EGFP, or their mutants; all at 10 nm) was assayed in the presence of increasing amounts of unlabeled L7Ae (from left; 0, 2, 5, 10, 20, 50, 100, 200, and 500 nm). The RNA-L7Ae complex was incubated at 4 C for 60 min. [Note: 10 nm of the box C/D mini or 110nt-EGFP was interacted efficiently with 20 nm of L7Ae to form the RNP complex (RNA:protein = 1:2). The RNP complex clearly formed between 200nt-EGFP and L7Ae in the presence of 50 nm or more of L7Ae 24

25 (RNA:protein = 1:5)]. Supplementary Figure 5 In vitro Translational repression assays. (a) Time course of translational repression by L7Ae. Before the translational assays, the RNP complex was incubated at 4 C for 30 min. The translation efficiency of box C/D mini EGFP mrna is indicated by circles. The addition of L7Ae effectively repressed the translation of the mrna (as indicated by the squares). The bars shown at the circles depict the means + SD of three independent experiments. (b) Western blot analysis of the translation repression. Different concentrations of L7Ae (0, 0.625, 1.25, 2.5, or 5 µm) were used to monitor the expression levels of EGFP. The EGFP expressed from box C/D mini (box C/D ) EGFP mrna was quantified to analyze the translational repression efficiency (right). (c) Translational repression of box C/D mini EGFP- mrna by L7Ae or L7AeK37K79A. Two amino acids (K37 and K79) of L7Ae were mutated to alanine to weaken the binding affinity with the box C/D K-turn motif. The efficiency of the translational repression of L7Ae was monitored as a control (white bars). Translational repression by 25

26 L7AeK37K79A was less efficient than that by L7Ae (black bars). The bars shown depict the mean + SD of three independent experiments. Supplementary Figure 6 Synthetic mrnas used in the in vitro UTR-OFF systems. The box C/D mini motif was inserted into the 5'-UTR region. For the UTR-2 mrna, 2 bases were used as a linker (indicated by green letters with a line) between the box C/D mini motif and the SD sequence (indicated by purple letters) of the targeted EGFP-coding mrna (UTR-2 mrna). For the UTR-2mut mrna, the two mutations (G7 to C and A20 to C, shown by bold italic letters) were introduced into the box C/D region. The box C/D motif was also inserted into the same region of the mrna as a control (UTR-2 mrna). The UTR-5, UTR-9, and UTR-13 mrnas were prepared by inserting different lengths of linkers (shown by green letters) between the box C/D mini motif and the SD sequence. Supplementary Figure 7 26

27 Translational repression assay using the UTR-OFF system. (a) Translational repression of UTR-2 mrna and its derivatives by L7Ae. To normalize the translational repression efficiency, we used a solution without L7Ae as a control (relative fluorescence = 1.0) for each mrna. Translation of UTR-2 mrna was effectively repressed by L7Ae in a concentration-dependent manner (white bars). The translational efficiencies of the UTR-2 or UTR-2mut mrnas are also shown. The bars depict the mean + SD of three independent experiments. (b) Competition assay for the translation repression of UTR-2 mrna. Competitor RNA containing the box C/D motif (see also Methods) was used to modulate the translational repression by L7Ae. The experimental conditions were the same as in Fig. 2d. Supplementary Figure 8 Construction of the plasmids for use in vivo. (a) Illustrations of the plasmid constructs produced by ApE ( All of the plasmids have a CMV promoter for transcription of EGFP with or without box C/D mini. (b) Illustrations of the secondary structure of the mrna used for the in vivo study. The pbox C/D-EGFP construct contains a box C/D mini after the start codon (ORF-OFF). The 27

28 pbox C/D -EGFP construct contains a GAAA loop instead of the box C/D mini. The p1-box C/D-EGFP construct contains a box C/D mini in the 5'-UTR of the EGFP gene (UTR-OFF). For the p1-box C/D-EGFP construct, a WC base pair (G-C) was changed to (C-G) (as shown by the red squares) to remove the start codons located in the box C/D mini. The G and A bases marked by asterisks were mutated to C to disrupt the K-turn (box C/D mut ), as described in the text. The bases shown in green indicate the ORF region of EGFP. (c) Primary sequences of the plasmids at the translation initiation site. The annotations were described above. Supplementary Figure 9 Expression of L7Ae in the cells does not interfere with cell proliferation and morphology. (a) Western blotting of L7Ae expressed in the cells. HeLa cells were plated in 6-well plates (2.5 x 10 5 cells/well), pre-incubated for 24 hours at 37 C, and transfected with the corresponding plasmids (pcdna or pl7ae). Twenty-four hours after transfection, we analyzed the expression of L7Ae in the cells using anti-myc antibody. (b) Cell proliferation analysis in the presence of L7Ae. HeLa cells were plated 28

29 in 96-well plates (1.0 x 10 4 cells /well), pre-incubated for 24 hours at 37 C, and transfected with DNA ( µg) encoding the L7Ae protein (pl7ae) or an empty vector (pcdna). Twenty-four hours after transfection, we evaluated the cell viability using WST-1 assays (see Supplementary Methods). The bars depict means ± SD of three independent experiments. (c) Microscopic analysis of the L7Ae-transfected cells. HeLa cells were plated in 6-well plates (2.5 x 10 5 cells/well), pre-incubated for 24 hours at 37 C, and transfected with DNA (1 µg) encoding the L7Ae protein (pl7ae) or an empty vector (pcdna). Twenty-four hours after transfection, cell morphology was analyzed by phase contrast microscopy (objective lenses; 4x or 10x). Note that L7Ae- or pcdna-transfected cells exhibited a similar morphology under the same conditions. Supplementary Figure 10 Flow cytometry analysis of the translational repression by L7Ae-K-turn. Twenty-four hours after transfection of the corresponding plasmids, the highly GFP-positive cells (>10 4, FITC-A) were counted and divided by the total number of cells (10,000 cells for each analysis). The same data as used in Fig. 4b were used for the analysis. The 29

30 transfection of pl7ae effectively decreased the expression of EGFP from pbox C/D-EGFP in a concentration-dependent manner (the red square), but it had little effect on the repression of box C/D -EGFP-mRNA (the purple cross). Supplementary Figure 11 Western blotting and quantitative RT-PCR analyzes. (a) A western blot analysis of the L7Ae expressed in the cells. pl7ae (0, 0.5, 1.0, 1.5, or 2.0 µg) and pbox C/D- or pbox C/D -EGFP (1.0 µg) were co-transfected in HeLa cells (2.5 x 10 5 cells /well). After 24 hours of transfection, the expression of EGFP was monitored using the anti-myc antibody. (b) Quantitative RT-PCR to analyze the amount of two mrnas (pbox C/D-EGFP and pbox C/D -EGFP) in the presence or absence of pl7ae. GAPDH mrna was used to normalize the relative mrna level. Supplementary Figure 12 Fluorescence microscopic images of the cells. EGFP expression was observed 24 hours after the co-transfection of pbox C/D-EGFP (or p1-box C/D-EGFP) and pl7ae (or 30

31 p481) into 70-90% confluent HeLa cells ( /well) in 24-well plates. The box C/D-defective mutant indicates the box C/D mut and box C/D for p1-box C/D-EGFP and pbox C/D-EGFP, respectively. pl7ae minus (-) indicates that the cells were co-transfected with pcdna instead of pl7ae. The expression of EGFP was repressed only when both L7Ae and the intact box C/D-EGFP were present. For RNAi induced by shrna produced from p481, EGFP both with and without the intact box C/D was repressed. The shrna produced from psk-7 was used as a negative control because it is incapable of carrying out RNAi. The experimental conditions were the same as in Fig. 4d, and the same images as used in Fig. 4d were also shown in this figure. Supplementary Figure 13 Schematic illustration of the translational repression of EGFP by the expression of chromosomally integrated DsRed M tagged with L7Ae. The DsRed M -L7Ae protein is under the control of a Tet-inducible promoter. The DsRed M -L7Ae that is produced binds to the box C/D mini motif of the target mrna encoding EGFP, resulting in the repression of its translation. 31

32 Sup. Fig. 1 Saito et al. a L7Ae (nm) L7Ae RNP free RNA box C/Dmini box C/Dmut b Response Units (RU) 150 nm 120 nm 40 nm 30 nm 20 nm Time (sec)

33 Sup. Fig. 2 Saito et al.

34 Sup. Fig. 3 Saito et al. synthetic mrnas used in this study (ORF-OFF system) A C A A C C G A A G U UG G UGA U G CGA A G C box C/D mini UGA C G C G CUCUAGAAAUAAUUUUGUUUAACUUUAAGAAGGAGAUAUACCAAUG GGG C CC 5' UGUGAGCAAGGGC... 3' 5'-UTR SD sequence... GCCTCCTCCGAG... 3' MGRDAKADP... box C/Dmini (mut)-egfp-mrna box C/Dmini (mut) -DsRed-mRNA A C A A C C G A A G U UG G A A A box C/D Δ G C G C G CUCUAGAAAUAAUUUUGUUUAACUUUAAGAAGGAGAUAUACCAAUG GG C 5' A AGUGAGCAAGGGC... 3' box C/DΔ-EGFP-mRNA 5'-UTR SD sequence... GCCTCCTCCGAG... 3' box C/DΔ-DsRed-mRNA MRGNP...

35 Sup. Fig. 4 Saito et al. a box C/D mini A C A A C C G A A G U UG G UGA U G CGA A G C UGA C G C G CUCUAGAAAUAAUUUUGUUUAACUUUAAGAAGGAGAUAUACCAAUG GGG C CC 5' U... 5'-UTR SD sequence nt (EGFP or DsRed) nt (EGFP or DsRed) 3' b L7Ae L7Ae L7Ae L7Ae L7Ae L7Ae RNP RNA box C/Dmini box C/Dmut 110nt-EGFP 110nt-EGFPmut 200nt-EGFP 200nt-EGFPmut

36 Sup. Fig. 5 Saito et al. a b L7Ae 1.2 box C/Dmini box C/DΔ fluorescence (A.U.) control L7Ae 5 µm Anti-GFP box C/Dmini-EGFP box C/DΔ-EGFP EGFP expression levels (A.U.) time (min) box C/Dmut-EGFP L7Ae (µm) c 1 L7Ae L7AeK37K79A Relative fluorescence (A.U.) protein (µm)

37 synthetic mrnas used in this study (UTR-OFF system) Sup. Fig. 6 Saito et al. A C A A C C G A A G U UG G UGA U G CGA A G C box C/Dmini UGA CG C G C UTR-2 (mut) mrna G C 5' G C UC AGAAGGAGAUAUACCAAUGGUGAGCAAGGGC... 3' 5'-UTR SD sequence ORF (EGFP) A C A A C C G A A G U UG G UGA U G CGA A G C box C/Dmini UGA CG C G C UTR-9 mrna G C 5' G C UC AACUUUAAGAAGGAGAUAUACCAAUGGUGAGCAAGGGC... 3' 5'-UTR A C A A C C G A A G U UG G A A A box C/DΔ G C G C UTR-2Δ mrna G C 5' G C UC AGAAGGAGAUAUACCAAUGGUGAGCAAGGGC... 3' 5'-UTR A C A A C C G A A G U UG G UGA U G CGA A G C box C/Dmini UGA CG C G C UTR-13 mrna G C 3' 5' G C UC GUUUAACUUUAAGAAGGAGAUAUACCAAUG GUGAGCAAGGGC... 5'-UTR A C A A C C G A A G U UG G UGA U G CGA A G C UGA box C/Dmini CG C G C UTR-5 mrna G C 5' G C UC UUAAGAAGGAGAUAUACCAAUGGUGAGCAAGGGC... 3' 5'-UTR

38 Sup. Fig. 7 Saito et al. a UTR-2 UTR-2Δ UTR-2mut b competition assay Relative fluorescence (A.U.) Relative fluorescence (A.U.) L7Ae (5 µm) L7Ae (µm) Competitor (µm)

39 Sup. Fig.8 Saito et al. a b BoxCD-EGFP F1 ori SV40 early promoter pbox C/D-EGFP BoxCD mini CMV_promoter pbox C/D-EGFP 4757 bp Kan/neoR ColE1 origin EGFP F1 ori SV40 early promoter pbox C/DΔ-EGFP CMV_promoter pbox C/DΔ-EGFP 4745 bp Kan/neoR ColE1 origin ColE1 origin CMV_promoter BoxCD mini G/C p1box C/D-EGFP 4712 bp EGFP p1box C/D-EGFP Kan/neoR SV40 early promoter F1 ori c pbox C/D-EGFP pbox C/DΔ-EGFP p1box C/D-EGFP p1box C/D mut-egfp CGCCACCATGGGGCGTGATGCGAAAGCTGACCCTGTGAGCAAGGGCGAGGA box C/D mini CGCCACCATGAGGGGAAACCCAGTGAGCAAGGGCGAGGA box C/D Δ CCTAGATCTGGGGCGTGATCCGAAAGGTGACCCGGATCCACCGGTCGCCACCATGGTGAGCAAGGG box C/D mini * * CCTAGATCTGGGGCGTCATCCGAAAGGTGCCCCGGATCCACCGGTCGCCACCATGGTGAGCAAGGG box C/D mut * *

40 Sup. Fig. 9 Saito et al. a pl7ae (0.5 µg) pl7ae (1.0 µg) Anti-Myc pcdna (2.0 µg) 150 cell viability (%) b 50 pl7ae x 10 x4 control (pcdna) pcdna (control) pl7ae c WST-1 assay (1.0x104cells/well) DNA (µg)

41 Sup. Fig. 10 Saito et al. 4 EGFP positive cells (>10 intensity / total cells)(%) pbox C/D-EGFP + pcdna pbox C/D-EGFP + pl7ae pbox C/DΔ-EGFP + pcdna pbox C/DΔ-EGFP+ pl7ae Transfected DNA (pcdna or pl7ae) (µg)

42 Sup. Fig. 11 Saito et al. a pboxc/d-egfp 1.0μg pboxc/d -EGFP 1.0μg pl7ae (μg) Anti-Myc L7Ae b Relative mrna level (A.U.) L7Ae pboxc/d-egfp pboxc/d -EGFP control 1.0μg +pl7ae 2.0μg +pl7ae

43 Sup. Fig. 12 Saito et al. p1-box C/D-EGFP pbox C/D-EGFP psk-7 defective box C/D- box C/D pl7ae pegfp-n1 untreated p481

44 Sup. Fig. 13 Saito et al. tetracycline DsRed L7Ae boxc/d EGFP transcription mrna boxc/d-egfp