Journal: Journal of Molecular Cell Biology

Article Title: U1 snRNP proteins promote proximal alternative polyadenylation sites by directly interacting with 3′ end processing core factors

doi: 10.1093/jmcb/mjac054

Figure Lengend Snippet: SNRNP70 promotes the proximal APA sites by binding with CPSF6. ( A ) Schematic diagram of the tethering assay. The 3×BoxB and ±50 bp sequence of PAS signal were inserted into a bicistronic reporting system containing two luciferases ( Renilla and Firefly ). λN-tag was fused to N-terminus of SNRNP70. ( B ) Tethering assay reveals that SNRNP70 can promote the proximal APA sites by binding to upstream of them. The sequences of the proximal APA sites of two genes (CTNNBIP1 and WAPAL) were cloned into the bicistronic reporter. The reporters were co-transfected into HEK293T cells with λN, SNRNP70, and λN-SNRNP70, respectively. Data were represented as mean ± SD, n = 3. P -values were calculated with the student t -test. * P < 0.05; ** P < 0.01; *** P < 0.001. ( C ) SNRNP70 can rescue the repression of CPSF6 on the proximal APA sites. The bicistronic reporters were co-transfected with CPSF6, λN-SNRNP70, and empty vector control, respectively. Data were represented as mean ± SD, n = 4. P -values were calculated with the student t -test. * P < 0.05; *** P < 0.001; **** P < 0.0001. ( D ) Proposed models for SNRNP70 to promote the proximal APA sites. Top: SNRNP70 and CPSF6 independently promote the proximal and distal APA sites, respectively; bottom: SNRNP70 recruites CPSF6 and then promotes the proximal APA sites. ( E ) Gentical interaction analysis shows that SNRNP70 promotes the proximal APA sites by recruting CPSF6 to upstream of them. The bicistronic reporters and siRNA (si-NC/si-CPSF6) were co-transfected into HEK293T cells with λN, SNRNP70, and λN-SNRNP70, respectively. While tethering SNRPN70 to upstream of proximal poly(A) site could promote it, this ability was almost lost with knockdown of CPSF6. Data are mean ± SD, n = 6. P -values were calculated with the Student t -test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; n.s., no significance. Two-way ANOVA test also shows significant promotive effect by the interaction between SNRNP70 and CPSF6 ( P = 0.014 for CTNNBIP1 gene and P = 0.0037 for WAPAL gene). ( F ) The arginines in LC1 domain (231–306) of SNRNP70 were mutated into alanines. ( G ) Co-IP results show that SNRNP70-Mut pulled down less CPSF6 than SNRNP70-WT did. ( H ) SNRNP70 can competitively bind to CPSF6 with FIP1L1 but not CPSF5 or CPSF7. Co-IP of FIP1L1, CSPF5, and CPSF7 with CPSF6 was performed using antibody against CPSF6 with gradient expression of FLAG-SNRNP70 in HEK293T cells. ( I ) HEK293T cells were transfected with MYC, MYC-SNRNP70-WT, and MYC-SNRNP70-Mut, respectively. Co-IP results show that the SNRNP70 mutant loses its ability to disturb the interaction of CPSF6 with FIP1L1.

Article Snippet: The antibodies used in western blotting, co-IP, and immunofluorescence are as follows: MYC-tag (Sigma, M4439), FLAG-tag (Sigma, F1804), SNRPA (Abcepta, AW5557), SNRPC (Abcepta, AW5526), SNRNP70 (Santa Cruz Biotechnology, sc-390899), SNRPD2 (Abcam, ab198296), CPSF6 (Novus, NBP1-85676), PABPN1 (Abclonal, A1735), FIP1L1(Novus, NBP1-85064), CPSF5 (Proteintech, 66335-1-Ig), CPSF7 (Proteintech, 55195-1-AP), GAPDH (Proteintech, 60004-1-Ig), IgG-rabbit (Cell Signaling Technology, 2729S), HRP-linked-mouse (Cell Signaling Technology, 7076S), HRP-linked-rabbit (Cell Signaling Technology, 7074S), Alexa Fluor TM 568 labeled-rabbit (Invitrogen, A-11011), Alexa Fluor TM 488 labeled-mouse (Invitrogen, A-11001).

Techniques: Binding Assay, Sequencing, Clone Assay, Transfection, Plasmid Preparation, Control, Knockdown, Co-Immunoprecipitation Assay, Expressing, Mutagenesis