Fig. 3: Npl3 is critical for U1 snRNP recruitment and phosphorylation of Npl3 activates Prp28’s ATPase activity.
From: Activation of Prp28 ATPase by phosphorylated Npl3 at a critical step of spliceosome remodeling
a p-Npl3 is associated with pre-mRNA in an ATP-independent manner. Splicing reactions (lanes 1–5) were done in splicing extracts at 0, 0.02, 0.05, 0.2, or 2 mM ATP and a portion was subjected to immunoprecipitation with either anti-Prp28 antibody (lanes 6–10), anti-p-Npl3 antibody (lanes 11–15), or pre-immune serum (lanes 16–20), respectively. Relative loadings are 1:10 for splicing reactions alone (lanes 1–5) vs. immunoprecipitated reactions (lanes 6–20). The experiment was repeated three times with similar results. b ChIP analysis showed that Npl3 is important for U1 snRNP recruitment. npl3∆, npl3-deleted strain. Error bars are ± SEM; n = 3 biological repeats; ****P < 0.0001, *P < 0.05 (P = 0.0356), unpaired two-tailed t-test. c ChIP analysis revealed that U1 snRNP’s departure is delayed in the prp28-E326Δ3 strain. Error bars are ± SEM.; n = 3 biological repeats; *P < 0.05 (P = 0.031), ****P < 0.0001, ***P < 0.001 (P = 0.0008), unpaired two-tailed t-test. d, e Prp28 ATPase assay. Purified Prp28, Prp28-AAAD, Prp28-E326∆3, p-Npl3, and Npl3 were assayed for their ATPase activity in various combinations using [α-32P]-ATP as a substrate. The products were separated by thin-layer chromatography for phosphoimager quantitation. Error bars are ± SEM; n = 3 biological repeats. f ChIP analysis showed that blocking S411 residue’s phosphorylation in Npl3 (npl3-S411A) resulted in U1 snRNP accumulation in the spliceosome during co-transcriptional splicing. Error bars are ± SEM.; n = 6 biological repeats; ****P < 0.0001, **P < 0.01 (P = 0.0017), unpaired two-tailed t-test. Source data are provided as a Source Data file.
