Supplementary Materials for

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1 Supplementary Materials for n mirn-mediated therapy for S6 blocks IRES-driven translation of the N second cistron Yu Miyazaki, Xiaofei Du, Shin-ichi Muramatsu, hristopher M. omez he PDF file includes: orresponding author. cgomez@neurology.bsd.uchicago.edu Published 3 July 6, Sci. ransl. Med. 8, 347ra94 (6) DOI:.6/scitranslmed.aaf566 Fig. S. Schematic representation of V9 vectors and V9 transduction efficiency into Purkinje cells. Fig. S. Histopathological examination of the V9-injected mouse cerebral cortex and hippocampus. Fig. S3. he long-term follow-up of V9-injected mouse behavioral phenotypes and N IRES driven expression in mice. Fig. S4. Stem-loop structure of N IRES and the predicted binding sites of mir-7, mir-39-5p, and mir p. Fig. S5. Effects of mir-39-5p on mutated N IRES templates. Fig. S6. Effects of eif4ii and eif4ii on mutated N IRES templates in the presence or absence of mir-39-5p. Fig. S7. otransduction of V9- -Q33 with either V9 mir-mock or V9 mir-39-5p. Fig. S8. he long-term follow-up of therapeutic effect of mir-39-5p on mouse behavioral phenotypes and N IRES driven S6 expression in mice. Fig. S9. Potential mouse mrns targeted by human mir-39-5p. Fig. S. Histopathological examination of the brain, cerebellum, heart, lung, liver, and kidney of wild-type mice injected with V mir-39-5p. Legend for table S Legends for movies S to S5 Other Supplementary Material for this manuscript includes the following: (available at

2 able S (Microsoft Excel format). Source data. Movie S (.mov format). Digiait video of an 8-week-old V9-FP mouse on treadmill. Movie S (.mov format). Digiait video of an 8-week-old V9- -Q mouse on treadmill. Movie S3 (.mov format). Digiait video of an 8-week-old V9- -Q33 mouse on treadmill. Movie S4 (.mov format). Digiait video of an 8-week-old V9 Q33 mirmock mouse on treadmill. Movie S5 (.mov format). Digiait video of an 8-week-old V9 Q33 mir- 39-5p mouse on treadmill.

3 Supplementary figure Supplementary figure. Schematic representation of V9 vectors and V9 transduction efficiency into Purkinje cells. () Schematic representation of truncated transgenes of N second cistron that lack the sequence of 5 upstream from N IRES (IRES-α). he sequence of nt 496 to 7757 of full-length N cdn (NM_7.) corresponds to that of N IRES and α open reading frame. he sequence of nt 53 to 55 is modified from to of stop codon. (B) Schematic representations of the V9 vectors expressing either N IRES-driven α-q or N IRES-driven α-q33 tagged with a -terminal FL epitope. IR: inverted terminal repeats; WPRE: woodchuck hepatitis virus posttranscriptional regulatory element; SV4: simian virus 4; poly: polyadenylation signal sequence. () Representative immunofluorescence images of V9-FP-injected mouse cerebellum from 4-week-old mice co-stained with calbindin-specific and FP-specific antibodies and fluorescent secondary antibody (scale bars: μm). Phosphate saline buffer (PBS)-injected mice were used as controls. (D) Purkinje cell count of V9-injected mouse cerebellum relative to PBS-injected mouse cerebellum (n = 6) ± 8.9 % of calbindin positive cells were positive for FP staining in the cerebellum of V9-FP mice. albindin(+): calbindin positive cells. FP(+): FP positive cells.

4 Supplementary figure V9-FP V9-α-Q V9-α-Q33 B V9-FP V9-α-Q V9-α-Q33 Supplementary figure. Histopathological examination of the V9-injected mouse cerebral cortex and hippocampus. (, B) Representative images of () cerebral cortex and (B) hippocampus of V9-FP mice, V9-α-Qmice, and V9-α-Q33 mice stained with Hematoxylin and eosin. Scale bars: 5 μm in () and (B).

5 F P Q Q33 V- F P V-Q V- Q 33 V- FP V-Q V-Q 33 V- FP V-Q V-Q 33 V- FP V- Q V-Q 33 FP Q Q33 FP Q Q 33 FP Q Q33 FP Q Q 33 FP Q Q 33 FP Q Q33 Supplementary figure 3 B weeks weeks Latency to fall (s) D Stride length (cm) t t t33 trial FP Q Q33 otal distance traveled (m) 4 3 E weeks weeks 6 Stride frequency (steps/sec) Left forelimb Right forelimb Left hindlimb Right hindlimb Left forelimb Right forelimb Left hindlimb Right hindlimb E F 3 weeks 3 weeks 3 weeks Latency to fall (s) 5 5 t t t33 trial FP Q Q33 otal distance traveled (m) 4 3 Supplementary figure 3. he long-term follow-up of V9-injected mouse behavioral phenotypes and N IRES driven α expression in mice. ( to F) linical features of V9-injected mice. () Rotarod test and (B) open field assay, () stride length and (D) stride frequencies assessed by Digigait analysis at weeks of age. (E) Rotarod test and (F) open field assay at 3 weeks of age (n = : 6 male and 6 female mice per each group). () Western blot and densitometric analyses showing α-fl expression in the cerebellum of V9-injected mice at 3 weeks of age (n = 4). FP: V9-FP mice; Q: V9-α- Q mice; Q33: V9-α-Q33 mice. ll data represent mean ± SEM. P<.5, P<.. wo-way NOV in () and (E). One-way NOV in (B) to (D), and (F). Student s t- test in (). α -FL PDH α-fl / PDH.5

6 Supplementary figure 4 Supplementary figure 4. Stem-loop structure of N IRES and the predicted binding sites of mir-7, mir-39-5p, and mir p. () Secondary structure of stem-loop of N IRES calculated by the entroidfold. (B to D) he nucleotide sequence of binding sites of (B) mir-7, () mir-39-5p, and (D) mir p within N IRES predicted by the mirn_argets program.

7 N 39 g g c N 39 Supplementary figure 5 N IRES B N IRESmut g c g c c c g c g c g g c g cc c.... mir-39-5p acuu - -UUUUuc N IRES gg N IRESmut c c c c c c g g c c D E Normalized IRESmut luciferase activity.5 (kda) α -FL α -FL PDH α-fl / PDH.5 N 39 α-fl / PDH.5 IRESmut IRESmut Supplementary figure 5. Effects of mir-39-5p on mutated N IRES templates. (, B) Schematic representations of () the original N IRES and (B) mutated N IRES (N IRESmut) templates. > and > substitutions within the predicted mir-39-5p binding site are shown in lowercase. We also performed > and > substitutions within the complementary binding site of the sequence targeted by mir-39-5p, also shown in lowercase, to maintain the stem-loop structure of N IRESmut as same as that of the original. () Nucleotide sequence of the mir-39-5p binding site within N

8 IRES and N IRESmut templates. (D) Relative dual luciferase N IRESmut reporter activities in HEK93 cells treated with mir-39-5p or a mirn negative control (N) (n = 6). he ratio of firefly luciferase to Renilla luciferase activities of a bicistronic N IRESmut reporter vector was normalized to that of a bicistronic control reporter vector. (E) Western blot and densitometric analyses showing α-q33-fl and α-q33-fl expression from N IRESmut vectors in HEK93 cells treated with mir-39-5p or N (n = 6). ll data represent mean ± SEM. : not significant. Student s t-test in (D) and (E).

9 N I, N I, 39 N I, N I, 39 N I, N I, 39 N I, N I, 39 N I, N I, 39 Supplementary figure 6 Normalized IRESmut luciferase activity IRESmut B Normalized IRESmut luciferase activity N I, N I, 39 IRESmut (kda) 5- α -FL α-fl / PDH D (kda) 5- α -FL α-fl / PDH 37- IRESmut α -FL PDH α-fl / PDH IRESmut α -FL PDH α-fl / PDH 3 E N IRES F N IRES eif4ii eif4ii eif4ii N mrn eif4ii N mrn go4 mir-39-5p Initiation of IRES-driven α translation Supplementary figure 6. Effects of eif4ii and eif4ii on mutated N IRES templates in the presence or absence of mir-39-5p. (, B) Relative dual luciferase N IRESmut reporter activities in HEK93 cells treated with over-expression of either () eif4ii (II), (B) eif4ii (II), or a control vector () in the presence of mir-39-5p or a mirn negative control (N) (n = 6). he ratio of firefly luciferase to Renilla luciferase activities of a bicistronic N IRESmut reporter vector was normalized to that of a

10 bicistronic control reporter vector. (, D) Western blot and densitometric analyses showing α- Q33-FL and α-q33-fl expression from N IRESmut vectors in HEK93 cells treated with over-expression of either () II, (D) II, or in the presence of mir-39-5p or N (n = 6). (E) Schematic diagram of the effect of eif4ii and eif4ii on N IRES-driven α translation. he complex of eif4ii and eif4ii directly acts on N IRES to enhance the initiation of IRES-driven α translation. (F) Schematic diagram of the inhibitory effect of mir-39-5p bound to go4. mir-39-5p, in collaboration with go4, binds to N IRES to inhibit the interaction between N IRES and the complex of eif4ii and eif4ii. ll data represent mean ± SEM. P<., P<.. : not significant. Student s t-test in () to (D).

11 moc k 39 Supplementary figure 7 V9-miR-mock V3 IR MV promoter FP mir-mock WPRE SV4 poly V3 IR V9-miR-39-5p V3 IR MV promoter FP mir-39-5p WPRE SV4 poly V3 IR B V9-FP V9-Q33-miR-mock V9-Q33-miR-39-5p FP.5 Merged α-fl Ratio of both FL and FP positive cells / FL positive cells Supplementary figure 7. otransduction of V9-α-Q33 with either V9 mirmock or V9 mir-39-5p. () Schematic representations of the V9 vectors expressing FP with either mir-39-5p or a scrambled mir (mir-mock). IR: inverted terminal repeats; WPRE: woodchuck hepatitis virus posttranscriptional regulatory element; SV4: simian virus 4; poly: polyadenylation signal sequence. (B) Representative immunofluorescence images of V9-injected mouse cerebellum from 4-week-old mice stained with FP- and FL-specific antibodies and fluorescent secondary antibody (scale bars: µm). () he ratio of Purkinje cells stained with both FL- and FP-specific antibodies (both FL and FP positive cells) to those with FL-specific antibody (FL positive cells). FL positive cells were transduced with V9-α-Q33. FP positive cells were transduced with either V9 mir-mock or V9 mir-39-5p (n = 6). V9-FP: V9-FP mice; V9 Q33-miRmock: V9 Q33 mir-mock mice; V9 Q33 mir-39-5p: V9 Q33 mir-39-5p mice. ll data represent mean ± SEM. : not significant. Student s t-test in ().

12 FP mo ck 39 F P mock 39 F P moc k 39 F P mock 39 FP mock 39 FP mo ck 39 FP mo ck 39 FP mo ck 39 F P mo ck 39 F P mo ck 39 F P mock 39 Supplementary figure 8 Latency to fall (s) 5 5 weeks B weeks FP 39 mock t t t33 trial otal distance traveled (m) D weeks weeks 7 Stride length (cm) Stride frequency (steps/sec) Left forelimb Right forelimb Left hindlimb Right hindlimb Left forelimb Right forelimb Left hindlimb Right hindlimb E Latency to fall (s) 5 5 F 3 weeks 3 weeks FP 39 mock otal distance traveled (m) weeks α -FL PDH α-fl / PDH.5 t t t33 trial Supplementary figure 8. he long-term follow-up of therapeutic effect of mir-39-5p on mouse behavioral phenotypes and N IRES driven α S6 expression in mice. ( to F) linical features of V9-injected mice. () Rotarod test and (B) open field assay, () stride length and (D) stride frequencies assessed by Digigait analysis at weeks of age. (E) Rotarod test and (F) open field assay at 3 weeks of age (n = : 6 male and 6 female mice per each group). () Western blot and densitometric analyses showing α-q33-fl expression in the cerebellum of V9-injected mice at 3 weeks of age (n = 4). FP: V9-

13 FP mice; mock: V9 Q33 mir-mock mice; 39: V9 Q33 mir-39-5p mice. ll data represent mean ± SEM. P<.5, P<.. wo-way NOV in () and (E). Student s t-test in (B) to (D), (F), and ().

14 Supplementary figure 9 Supplementary figure 9. Potential mouse mrns targeted by human mir-39-5p. () he list of 3 human genes of which 3 UR has binding sites targeted by hsa-mir-39-5p predicted by the argetscanhuman 7. ( mong these, 7 genes (PRX, ZNF78, orf46, ZNF3, ZNF86, ERBB4, PBP) have hsa-mir-39-5p binding sites within 3 UR of their conserved mouse orthologs. (B to H) Relative expression of the mouse (B) Prx, () Zfp78, (D) 49347IRik, (E) Zfp6, (F) Zfp86, () Erbb4, and (H) Ptbp mrns in the cerebellum of V9-injected mice (n = 4). N/: not assigned; W, mock: wildtype mice injected with V9 mir-mock; W, 39: wild-type mice injected with V9 mir-39-5p. ll data represent mean ± SEM. P <.5. : not significant. Student s t-test in (B) to (H).

15 Supplementary figure Supplementary figure. Histopathological examination of the brain, cerebellum, heart, lung, liver, and kidney of wild-type mice injected with V mir-39-5p. ( to F) Histopathological examination of the () brain, (B) cerebellum, () heart, (D) lung, (E) liver, and (F) kidney of the -week-old wild-type mice injected with V mir- 39-5p (W-miR-39-5p) compared to age-matched wild-type mice (W). ll were stained with Hematoxylin and eosin. Scale bars: (), 5 μm; and (B) to (F), μm.

16 able S. Source data. Movie S. Digiait video of an 8-week-old V9-FP mouse on treadmill. Movie S. Digiait video of an 8-week-old V9-α-Q mouse on treadmill. Movie S3. Digiait video of an 8-week-old V9-α-Q33 mouse on treadmill. Movie S4. Digiait video of an 8-week-old V9 Q33 mir-mock mouse on treadmill. Movie S5. Digiait video of an 8-week-old V9 Q33 mir-39-5p mouse on treadmill.