Depleting SRSF1 or inhibiting its repeat-RNA sequestration inhibit the nuclear export of C9ORF72 RAN-translated transcripts. ( a , b ) Protein:RNA UV crosslinking assays using purified recombinant proteins and 32P-end-radiolabelled G4C2x5 ( a ) and C4G2x5 ( b ) repeat RNA probes. Proteins are visualized on SDS–PAGE stained with Coomassie blue (left panels) and covalently linked RNA:protein complexes by autoradiography on PhosphoImages (right panels). ( c , d ) RNA immunoprecipitation (RIP) assays. Formaldehyde was added to the medium of live N2A cells co-transfected with G4C2x15, G4C2x38 ( c ), C4G2x15 or C4G2x39 ( d ) and either FLAG control (FLAG Ctrl), FLAG-tagged SRSF1 aa11-196 wild-type (SRSF1) or SRSF1-m4 were subjected to anti-FLAG immunoprecipitation. Purified RNA was analysed by qRT–PCR following normalization to U1 snRNA levels in three biological replicate experiments (mean±s.e.m.; two-way ANOVA with Tukey's correction for multiple comparisons; N (qRT–PCR reactions)=6). ( e ) Western blots of N2A cells co-transfected with G4C2x38 and either Ctrl or SRSF1-RNAi plasmids or with G4C2x38 and either FLAG-tagged SRSF1 aa11-196 wild-type (SRSF1) or SRSF1-m4, subjected to cellular fractionation using hypotonic lysis to yield cytoplasmic fractions. The chromatin remodelling SSRP1 factor is used to check for potential nuclear contamination. ( f ) Cytoplasmic and total G4C2-repeat sense transcript levels were normalized to U1 snRNA levels in three biological replicate experiments prior to plotting as a ratio to account for potential changes in mRNA transcription/stability (mean±s.e.m.; one-way ANOVA with Tukey's correction for multiple comparisons; N (qRT–PCR reactions)=6). ( g ) Western blots of Drosophila expressing G4C2x36 and either Ctrl or SRSF1-RNAi, subjected to cellular fractionation using hypotonic lysis to yield cytoplasmic fractions. Histone H3 is used to check for potential nuclear contamination. ( h ) Cytoplasmic and total G4C2-repeat sense transcript levels were normalized to Tub84b levels in three biological replicate experiments prior to plotting as a ratio to account for potential changes in mRNA transcription/stability (mean±s.e.m.; paired two-tailed t -test; N (qRT–PCR reactions)=3). In contrast to cytoplasmic levels, total levels of hexanucleotide repeat transcripts were not significantly altered upon expression of SRSF1-m4 or depletion of SRSF1 in cells or flies ( Supplementary Fig. 7 ). Statistical significance of data is indicated as follows: NS: non-significant, P ≥0.05; * P