Fig 1: Transcription-dependent regulation of 7SK/hnRNP/Smn complexes.a Western blot analysis of proteins co-immunoprecipitated by anti-Larp7 from NSC-34 cells treated with DMSO or actinomycin D (ActD) for 1 h. b Co-immunoprecipitation of Larp7 by anti-Smn from NSC-34 cells treated with DMSO or ActD for 1 h. c, d Co-immunoprecipitation of proteins by anti-Mepce from NSC-34 cells treated with DMSO or ActD for 1 h. For Western blots of hnRNP R and Cdk9, a shorter exposure of the inputs was chosen. e Co-immunoprecipitation of Larp7 by anti-Smn from cultured mouse primary motoneurons treated with DMSO or ActD for 6 h. f Co-immunoprecipitation of proteins by anti-SMN from HEK293TN cells treated with DMSO or ActD for 1 or 6 h. Source data are provided as a Source Data file.
Fig 2: Smn interacts with 7SK/hnRNP complexes in the nucleus and cytosol.a Western blot analysis of NSC-34 subcellular fractions. Cyt, cytosol; Nuc, nuclear soluble proteins and organelles; Chr, chromatin-associated proteins; T, total lysate. b Quantification of Western blot signals in a. Data are mean ± s.d. (n = 3). c Quantification of 7SK RNA levels by qPCR. Data are mean ± s.d. (n = 4). d Western blot analysis of proteins co-immunoprecipitated by anti-Larp7 from subcellular fractions of NSC-34 cells. Fractions were pretreated with RNase as indicated. e Same as in d but with anti-Smn. Source data are provided as a Source Data file.
Fig 3: Reduced snRNP production upon transcriptional inhibition.a Western blot analysis of Smn and Larp7 protein levels in NSC-34 cells treated with DMSO or ActD for the indicated durations. b Quantification of Larp7 protein levels in a. Data are mean ± s.d.; ****P = 0.0001; n.s., not significant; two-way ANOVA with Sidak’s multiple-comparisons test (n = 3). c Co-immunoprecipitation of Larp7 by anti-Smn from NSC-34 cells treated with DMSO or ActD for the indicated durations. d Quantification of Larp7 co-purification by anti-Smn in c. Data are mean ± s.d.; **P = 0.01; ***P = 0.001; ****P = 0.0001; n.s., not significant; two-way ANOVA with Sidak’s multiple-comparisons test (n = 3). e Co-immunoprecipitation of Mepce by anti-Smn from NSC-34 cells treated with DMSO or ActD for 6 h. f Co-immunoprecipitation of Mepce and Smn by anti-SmB/B’ from NSC-34 cells treated with DMSO or ActD for 6 h. Note that the immunosignal at 25 kDa for mouse-IgG immunoprecipitation is non-specific from the antibody light chain. g qPCR analysis of total snRNA levels in NSC-34 cells treated with DMSO or ActD for the indicated durations. Data are mean ± s.d.; *P = 0.05; **P = 0.01; ***P = 0.001; ****P = 0.0001; two-way ANOVA with Sidak’s multiple-comparisons test (n = 3). h qPCR analysis of snRNAs co-precipitated by anti-SmB/B’ from NSC-34 cells treated with DMSO or ActD for the indicated durations. Data are mean ± s.d.; *P = 0.05; ****P = 0.0001; two-way ANOVA with Sidak’s multiple-comparisons test (n = 3). i Agarose gel electrophoresis of biotinylated U2 snRNA. j In vitro snRNP assembly assay. Biotinylated U2 snRNA was incubated with cytosolic extracts from NSC-34 cells exposed to DMSO or ActD for 6 h. Reactions were assembled with or without exogenous ATP. Biotinylated U2 snRNPs were pulled down with streptavidin beads and SmB/B’ was analyzed by Western blot. k Quantification of in vitro U2 snRNP assembly assay in j. Data are mean ± s.d.; **P = 0.01; ***P = 0.001; ****P = 0.0001; two-way ANOVA with Tukey’s multiple-comparisons test (n = 3). Source data are provided as a Source Data file.
Fig 4: Depletion of individual components of the Smn complex alters its binding to Larp7.a Western blot analysis of proteins co-immunoprecipitated by anti-Larp7 from control and SmB/B’ knockdown (shSnrpb) NSC-34 cells. b Quantification of Western blot signals in a. Data are mean ± s.d.; *P = 0.05; ***P = 0.001; ****P = 0.0001; unpaired two-tailed t-test (n = 3). c Co-immunoprecipitation of proteins by anti-Larp7 from control or Gemin2 knockdown (shGemin2) NSC-34 cells. d,e Co-immunoprecipitation of proteins by anti-Larp7 from control or Smn knockdown (shSmn) NSC-34 cells. f Co-immunoprecipitation of proteins by anti-SmB/B’ from brains of Smn+/+;SMN2 or Smn-/-;SMN2 mice. Note that the immunosignal at 25 kDa for mouse-IgG immunoprecipitation is non-specific from the antibody light chain. g Model of Smn interactions with 7SK/hnRNP R. Source data are provided as a Source Data file.
Fig 5: Model depicting the role of MEPCE in the 7SK snRNP complex and possible pathomechanism underlying the MEPCE nonsense variant. (a) In eukaryotes, synthesis of precursor mRNAs by RNA polymerase II (RNAP II) is essentially regulated by many factors (bottom figure). During the transcriptional process, RNAP II is paused proximal to the promoter by negative elongation factors (light and dark red ellipses). The release of RNAP II is mediated by phosphorylation through the positive transcription elongation factor b (P-TEFb) consisting of Cyclin-T1 (CT1) and the cyclin-dependent kinase 9 (CDK9). P-TEFb in turn is under control of the inhibitory 7SK small nuclear ribonucleoprotein (snRNP) complex (top figure). The 7SK snRNP core complex consists of the 7SK snRNA that is permanently bound to MEPCE and LARP7. MEPCE stabilizes the 7SK snRNA by 5' cap methylation, and LARP7 protects the 7SK snRNA by binding its 3' end. P-TEFb inhibition and incorporation in the 7SK snRNP complex is ensued by HEXIM1/2 dimers. Upon extra- and/or intracellular stimuli, P-TEFb is released from this complex and recruited to the paused RNAP II. Here, the CDK9 subunit of P-TEFb phosphorylates negative elongation factors which then dissociate from the complex or are inactivated. These steps are necessary for the release of paused RNAP II. In addition, P-TEFb phosphorylates specific serines in the C-terminus of RNAP II to stimulate the elongation of transcription. (b) The MEPCE nonsense mutation leads to a decrease in MEPCE protein amount by ~50% in patient-derived cells that is accompanied by depletion of 7SK snRNA and LARP7 protein. Consequently, disintegration of the 7SK snRNP complex likely leads to enhanced release and activation of P-TEFb, followed by hyperphosphorylation of RNAP II’s C-terminal domain. We postulate that the P-TEFb equilibrium is shifted toward free P-TEFb in patient cells leading to preferred transition of RNAP II from the paused to the productively elongating state and dysregulated expression of P-TEFb-regulated genes.
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