Figure 5. Biochemical, qPCR, and RNAseq analysis of NEXT and variants.

(A) EMSA plots comparing NEXT and NEXT^ΔHD binding to 3′ A₅U₅A₁₀-tailed RNA duplex substrate. Data points represent mean ± SD from three technical replicates.

(B) Bar graphs of dissociation constants K_D (mean ± SEM) and Hill coefficients h (mean ± SEM) obtained from fitting the EMSA data in (B) to Hill equation.

(C) Graph of initial strand displacement rate (v₀) at varying protein concentration (plotted as equivalent molar concentration of MTR4) for WT NEXT, NEXT with ZCCHC8 HD deleted (NEXT^ΔHD), heterodimeric NEXT^WT/EQ containing wild-type MTR4 and MTR4 E253Q mutant, and a stoichiometric mixture of wild-type NEXT^WT/WT and helicase-dead NEXT^EQ/EQ. Assays were performed using 3′ A₅U₅A₁₀-tailed RNA duplex substrate. Data points are shown as mean ± SD from three separate reactions.

(D) Bar graphs of V_max (mean ± SEM) obtained by fitting the data in (A) to equation v₀ = V_max*[E]/(K_1/2 + [E]*/(1+[E]/K′_1/2)).

(E) EMSA plots comparing NEXT with RBM7 F13A/F52A mutations (NEXT^FAFA) and NEXT with ZCCHC8 ZK deleted (NEXT^ΔZK) binding to 3′ A₅U₅A₁₀-tailed RNA duplex substrate.

(F) Bar graphs of K_D (mean ± SEM) and h (mean ± SEM) obtained from fitting the EMSA data in (E) to Hill equation.

(G) EMSA plots comparing NEXT, NEXT^FAFA, and NEXT^ΔZK binding to 3′ A₂₀-tailed RNA substrate. Data points in (E) and (G) represent mean ± SD from three technical replicates.

(H) Bar graphs of K_D (mean ± SEM) and h (mean ± SEM) obtained from fitting the EMSA data in (G) to Hill equation.

(I) v₀ at varying protein concentration (plotted as equivalent molar concentration of MTR4) for NEXT, NEXT^FAFA, NEXT^ΔZK, and MTR4. Assays were performed with 3′ A₅U₅A₁₀-tailed RNA substrates.

(J) Bar graphs of V_max (mean ± SEM) obtained by fitting the data in (I) to equation v₀ = V_max*[E]/(K_1/2 + [E]*/(1+[E]/K′_1/2)).

(K) v₀ at varying protein concentration (plotted as equivalent molar concentration of MTR4) for NEXT, NEXT^FAFA, NEXT^ΔZK, and MTR4. Assays performed with 3′ A₂₀-tailed RNA substrates. Data points for (I) and (K) are shown as mean ± SD from three separate reactions.

(L) Bar graphs of V_max (mean ± SEM) obtained by fitting the data in (K) to equation v₀ = V_max*[E]/K_1/2 + [E]*/(1+[E]/K′_1/2)) for NEXT^ΔZK or v₀ = V_max [E]/K_1/2 for WT NEXT and NEXT^FAFA.

(M) Bar graph of relative RNA levels (mean ± SD) obtained from qPCR analysis of PROMPTS in parental HAP1 cells, ZCCHC8 CRISPR knockout HAP1 cells (HAP1 Z8 KO), and stable clones of ZCCHC8 knockout HAP1 cells complemented with GFP-tagged ZCCHC8 (GFP-Z8), ZCCHC8 with HD deleted (GFP-Z8^ΔHD) or ZCCHC8 with ZK deleted (GFP-Z8^ΔZK). Individual data points from three biological replicates are shown as solid circles. Data were normalized relative to RNA levels in parental line. Statistical analysis was performed using two-tailed t test. P values are indicated by *p < 0.05, **p <0.01, ***p <0.001; ****p < 0.0001.

(N) Density profiles of RNA seq (−) strand reads around protein-coding genes and long intergenic RNA transcript transcription start sites (TSSs) within 3 kb upstream and downstream of PROMPTS.

(O) Read density plot upstream and downstream of eRNAs TSSs. Both (+) and (−) strand reads are shown.

(P) Read density 500 bp upstream and 1 kb downstream of 3′ ends (EAG) of all snRNAs. Each sample in Figures 5O-5Q is labeled and colored uniquely. See also Figure S8.