Figure 4.

Identifying the RBPs of ACTB mRNA in cells inducibly expressing dPspCas13b-BioID2/TurboID/APEX2-NES

(A) Plasmids used in this figure. NES: nuclear export sequence; EGFP: enhanced green fluorescent protein; P2A: P2A self-cleaving peptide; HA: haemagglutinin tag.(B) HEK293T cells were co-transfected with PspCas13b or PspCas13b-APEX2/BASU/BioID2/TurboID and ACTB Psp-crRNA 4 to detect the mRNA level of ACTB after 48 hours.(C) Gray value analysis and western blot to measure the protein expression level of PspCas13b, PspCas13b-BioID2, PspCas13b-TurboID, PspCas13b-BASU and PspCas13b-APEX2.(D) Western blot to test the inducible expression ability and biotinylation activity of four cell lines inducibly expressing dPspCas13b- BioID2/TurboID/BASU/Apex2-NES. Different concentrations of doxycycline (Dox) were added to induce expression. Cells were collected together 27 hours after addition of doxycycline. For BioID2, biotin was added to the culture medium at a final concentration of 50 uM for 18 hours before harvesting cells; for TurboID, biotin was added at a final concentration of 500uM for 10 minutes before harvesting cells; for BASU, biotin was added at a final concentration of 200uM for 2 hours before harvesting cells; for APEX2, biotin-phenol was added at a final concentration of 500 uM for 30 minutes followed by a 1-minute exposure to 1 mM H₂O₂ before harvesting cells.(E) Timeline to capture the proteins that interact with ACTB mRNA using three cell lines inducibly expressing dPspCas13b-BioID2/TurboID/Apex2-NES. min: minute. h: hour.(F) Western blot to detect the expression level and biotinylation activity of cells collected from (E).(G) Volcano plots of ACTB mRNA-associated proteins identified by CBRPP in HEK293T cells inducibly expressing dPspCas13b-BioID2-NES. The significantly enriched proteins (P-value < 0.05 and fold change > 4) are shown as black dots (n = 3 for the ACTB Psp-crRNAs group and the non-targeting Psp-crRNAs group). Red dots represent known RBPs of ACTB mRNA in StarBase v2.0 database. Four previously uncharacterized RBPs of ACTB mRNA identified and validated in this study are shown as orange dots. FC: fold change, ACTB/NT: the ACTB Psp-crRNAs group/the non-targeting Psp-crRNAs group.(H) Left: RT-qPCR to measure the mRNA level of ACTB pulled down by anti-FLAG antibody in HEK293T cells transfected with indicated plasmids. EV: empty vector. Right: western blot to confirmed that proteins are specifically pulled down by anti-FLAG antibody not IgG. Results showed that some proteins found by CBRPP could significantly enrich ACTB mRNA compared with empty vector (EV) control.For B, C and H: n = 3, mean ± SD, two-sided Student’s t-test, ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns: not significant.