Fig 1: Knockdown (KD) of RNA processing machinery components increases SMN protein levelsSMA patient-derived fibroblasts were transfected with siRNA for 48 h.(A) Gene expression validates KD of WDR33, CPSF1, and THOC3. Data were analyzed using an unpaired t test (WDR33 mRNA: p < 0.0001; CPSF1 mRNA: p < 0.0001; THOC3 mRNA: p < 0.0001). n = 5.(B) Western blot and quantification of SMN protein levels 48 h after siRNA transfection. SMN protein levels are increased with KD of each gene. Cells transfected with siRNA targeting either U2AF2 or SRSF3 were run as a control. Data were analyzed using unpaired t tests (control versus siWDR33: p = 0.0064; control versus siCPSF1: p = 0.0043; control versus siTHOC3: p = 0.0205). n = 4–5.(C) SMA patient-derived fibroblasts were simultaneously transfected with siRNAs targeting WDR33, CPSF1, and THOC3 (triple KD) for 48 h. Gene expression validates KD of WDR33, CPSF1, and THOC3. Data were analyzed using unpaired t tests (WDR33 mRNA: p < 0.0001; CPSF1 mRNA: p < 0.0001; THOC3 mRNA: p < 0.0001). n = 5.(D) Western blot and quantification of SMN protein levels 48 h after transfection with all three siRNAs. SMN protein levels are increased in fibroblasts with all three genes knocked down. Data were analyzed using an unpaired t test (p = 0.0418). n = 5.All data are represented as mean ± SEM. *p < 0.05, **p < 0.01, ****p < 0.0001. See also Figure S2.
Fig 2: KD of RNA processing machinery components increases cytoplasmic SMN mRNASMA patient-derived fibroblasts were transfected with siRNA targeting WDR33, CPSF1, or THOC3 for 48 h.(A and B) SMN2 pre-mRNA (A) and total SMN2 mRNA (B) were examined using qRT-PCR. Total SMN mRNA was unchanged with KD of each gene. SMN pre-mRNA data were analyzed using one-way ANOVA (F(3,16) = 4.233, p = 0.0221) followed by post hoc Dunnett’s multiple comparisons test (control versus siWDR33: p = 0.0131; control versus siCPSF1: p = 0.5059; control versus siTHOC3: p = 0.9635). Total SMN2 mRNA data were analyzed using one-way ANOVA (F(3,16) = 0.7025, p = 0.5643). n = 5.(C) SMA patient-derived fibroblasts were transfected with siRNA for 48 h and were then treated with actinomycin D to inhibit transcription. SMN2 mRNA expression was examined for 8 h after the addition of actinomycin D. SMN2 mRNA stability was unchanged with reduction of each gene. n = 3–4.(D) Splicing of the SMN2 transcript and quantification of the ratio of full-length SMN to SMNΔ7. SMN2 splicing was unchanged with reduction of each gene. SMN2 splicing data were analyzed using one-way ANOVA (F(3,12) = 4.071, p = 0.0329) followed by post hoc Dunnett’s multiple comparisons test (control versus siWDR33: p = 0.8984; control versus siCPSF1: p = 0.0537; control versus siTHOC3: p = 0.9990). n = 5.(E) Cytoplasmic and nuclear SMN2 mRNA levels were examined using qRT-PCR. We found the predominantly nuclear MALAT1 mRNA in the nuclear and not the cytoplasmic RNA fractions, confirming the purity of fractionation. Importantly, cytoplasmic SMN mRNA expression was increased with reduction of WDR33, CPSF1, and THOC3. Data were analyzed using one-way ANOVA (F(3,16) = 19.45, p < 0.0001) followed by post hoc Dunnett’s multiple comparisons test (control versus siWDR33: p < 0.0001; control versus siCPSF1: p < 0.0001; control versus siTHOC3: p = 0.0001). n = 5. Nuclear SMN mRNA expression was decreased with KD of WDR33, CPSF1, and THOC3. Data were analyzed using one-way ANOVA (F (3,16) = 8.846, p = 0.0011) followed by post hoc Dunnett’s multiple comparisons test (control versus siWDR33: p = 0.0040; control versus siCPSF1: p = 0.0014; control versus siTHOC3: p = 0.0014). n = 5.All data are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Figure S3.
Fig 3: Motor function assessments in SMA and healthy infants in the first 6 months of life. (A) Motor function testing paradigm. All infants were tested using the TIMPSI. After the TIMPSI, a mandatory rest period of 20 minutes was followed by either the CHOP‐INTEND or AIMS assessment. Infants who scored less than 41 on the TIMPSI were tested using the CHOP‐INTEND, otherwise the infant was tested using the AIMS test. (B) Results of infant motor function tests for all infants as a function of the age at the time of enrollment visit. For the SMA cohort, the SMN2 copy number for each infant is indicated by the color as indicated in the key by each graph. For the healthy cohort the SMN1 copy number for each infant is indicated by the color as indicated in the key by each graph.
Fig 4: Peripheral blood SMN mRNA and protein levels in SMA and healthy control infants. (A) Full‐length SMN mRNA levels from whole blood measured using ddPCR expressed as a ratio of SMN to HPRT. (B) SMN protein levels detected in PBMCs measured by SMN‐ECL ELISA expressed as pg/107 cells. For the SMA cohort, the SMN2 copy number for each infant is indicated by the color as indicated in the key by each graph. For the healthy cohort, the SMN1 copy number for each infant is indicated by the color as indicated in the key by each graph.
Fig 5: Ulnar compound muscle action potential is significantly reduced in SMA infants compared to healthy infants. Ulnar CMAP peak amplitude (mV) in SMA and healthy control infants as a function of the age at the time of enrollment visit. For the SMA cohort, the SMN2 copy number for each infant is indicated by the color as indicated in the key by each graph. For the healthy cohort, the SMN1 copy number for each infant is indicated by the color as indicated in the key by each graph.
Supplier Page from Enzo Life Sciences, Inc. for SMN1 (human), (recombinant) (His-tag)