Figure 3. SNORD90 down-regulates NRG3.

(A) Full sequence of mature SNORD90 transcript with highlighted regions labeled predicted binding site (B.S.) 1 and predicted B.S. 2, which are predicted to bind to NRG3. Schematic representation of NRG3 pre-mRNA transcript indicating regions on NRG3 where SNORD90 is predicted to bind. The color of the bracket corresponds to predicted B.S.-1 or predicted B.S.-2. (red nucleotide indicates mismatch, orange nucleotide indicates G-T wabble pair). (B) Expression of SNORD90 (left) and NRG3 (right) in human NPCs after transfection with SNORD90 expression vector (SNORD90 O.E., n=6), seed scramble expression vector (seed scramble O.E., n=6), and full scramble expression vector (full scramble O.E., n=6). Non-transfected NPCs under normal culture conditions as control (n=6) (C) Expression of SNORD90 (left) and NRG3 (right) after transfecting human NPCs with an antisense oligonucleotides (ASO) designed to knock-down SNORD90 (SNORD90 K.D., n=6) or scrambled ASO (scramble K.D., n=6). Non-transfected NPCs under normal culture conditions as control (n=6). (D) Schematic representation of co-transfection of SNORD90 over-expression vector without target blockers (top) and with target blockers (bottom). Target blockers designed to bind to regions on pre-NRG3 where SNORD90 is predicted to bind, consequently blocking SNORD90 from interacting with those regions on NRG3 (bottom). (E) NRG3 expression following SNORD90 expression vector co-transfected with a scrambled blocker (+scramble blocker, n=6), target blockers one site at a time (+Blocker 1, n=6;+Blocker 2, n=6, and +Blocker 3, n=6), or all three blockers simultaneously (+Blocker1-to-3, n=6). Non-transfected NPCs under normal culture conditions as control (n=6). All bar plots represent the mean with individual data points as dots. Error bars represent S.E.M. One-way ANOVA with Bonferroni post-hoc. **p<0.01, ****p<0.0001.

Figure 3—figure supplement 1. SNORD90 over-expression and knock-down in human NPC culture.

(A) Sequences of each primary transcript used for over-expression experiment in human NPC culture. SNORD90 O.E. (top), seed scramble O.E. (middle), and full scramble O.E. (bottom). Red indicates scrambled sequence and black indicates wild-type sequence. Yellow highlight sequence indicates where forward and reverse primers bind for qPCR quantification. Over-expression of SNORD90 WT transcript and seed scramble will be detected by our primers however full scramble will not. (B) Image of human NPC culture 48 hr post-transfection. White indicates the expression of GFP to confirm successful transfection. (C) Schematic representation of expression vectors used to over-expression primary transcripts (A) in human NPC culture (B). (D) Western blot quantification of NRG3 protein levels following transfection of SNORD90 (SNORD90 O.E., n=5), or full scramble (full scramble O.E., n=5) expression vectors in human NPC culture. Non-transfected NPCs under normal culture conditions as control (n=5). Samples were equally distributed between two separate gels and randomly assigned a well within each gel (right). (E–J) Expression of ENG (E), ST6GALNAC3 (F), ARHGAP29 (G), LDLRAD4 (H), EXOC6B (I), and SPOCK3 (J) in human NPCs after transfection with SNORD90 expression vector (SNORD90 O.E., n=6), seed scramble expression vector (seed scramble O.E., n=6), and full scramble expression vector (full scramble O.E., n=6). Non-transfected NPCs under normal culture conditions as control (n=6). (K) Schematic diagram indicating where each antisense oligonucleotides (ASO) is designed to target and cleave SNORD90. (L) Each of the four ASOs (ASO1, n=6; ASO2, n=6; ASO3, n=6; ASO4, n=6) were separably transfected into human NPCs to determine which achieved the most significant knock-down of SNORD90 expression. Scrambled ASO (scramble, n=6) and non-transfected NPCs under normal culture conditions as control (control, n=6). ASO4 was selected (subsequently referred to as SNORD90 K.D.). (M–R) Expression of ENG (M), ST6GALNAC3 (N), ARHGAP29 (O), LDLRAD4 (P), EXOC6B (Q), and SPOCK3 (R) after transfecting human NPCs with an ASO designed to knock-down SNORD90 (SNORD90 K.D., n=6) or scrambled ASO (scramble K.D., n=6). Non-transfected NPCs under normal culture conditions as control (n=6). (S) Same as (E–J) but for pre-NRG3 expression. (T) Same as (M–R) but for pre-NRG3 expression. (U) Expression of pre-NRG3 following SNORD90 expression vector was co-transfected with scrambled blocker (+scramble blocker, n=6), target blockers one site at a time (+Blocker 1, n=6;+Blocker 2, n=6, and +Blocker 3, n=6), or all three sites together (+Blocker1-to-3, n=6). Non-transfected NPCs under normal culture conditions as control (n=6). All bar plots represent the mean with individual data points as dots. Error bars represent S.E.M. One-way ANOVA with Bonferroni post-hoc. *p<0.05, **p<0.01, ***p<0.001.