Journal: bioRxiv
Article Title: U1 AMO (antisense morpholino oligo) disrupts U1 snRNP structure to promote intronic premature cleavage and polyadenylation (PCPA)
doi: 10.1101/2023.02.24.529985
Figure Lengend Snippet: (A) Meta-gene plots of CPSF160, WDR33 and CSTF77 ChIP-seq reads in control and U1 AMO treated HeLa cells for all actively expressed genes (n=13217), intronic PCPAed genes (n=5937), and other non-intronic PCPAed genes (n=7280). For intronic PCPAed genes, a second meta-gene plot was made for each ChIP-seq by replacing the default TES (transcription end sites) with intronic PCPA site. If multiple PCPA sites were detected for a given gene, the one showing the most significant PCPA was chosen to create the plot. (B) Meta-gene plots of WDR33 and CSTF64 iCLIP-seq reads in control and U1 AMO treated HeLa cells for all actively expressed genes (n=13217), intronic PCPAed genes (n=5937), and other non-intronic PCPAed genes (n=7280). For intronic PCPAed genes, a second meta-gene plot was made for each ChIP-seq by replacing the default TES (transcription end sites) with intronic PCPA site. (C) IGV track screen shots showing CPSF160/WDR33/CSTF77 ChIP-seq(s), WDR33/CSTF64 iCLIP-seq(s) and 3’-seq results for basp1 gene in control and U1 AMO treated HeLa cells. (D) IGV track screen shots showing CSTF64 iCLIP-seq and 3’-seq results for eed gene in control and U1 AMO treated HeLa cells. (E) Gel mobility shift assay using recombinant GST-CSTF64-RRM (RNA recognition motif) (0, 1, 2, 5, 10, 15, 20 μM) and indicated PAS RNAs (detectable amount, approximately 0.1 uM). The PAS RNA sequences are listed in Supplemental Table 7. (F) Measurement of the processing efficiencies of eed intronic PAS RNAs and two mutants using pPASPORT system. Experiments were performed three times and the standard deviations are shown in error bars. Student’s t-test was performed to examine the significance of the difference. *P<0.05.
Article Snippet: The primary antibodies [WDR33 (A301-152A) and CstF64 (A301-092A)] were purchased from Bethyl.
Techniques: ChIP-sequencing, Control, Mobility Shift, Recombinant
Bethyl is a verified supplier 
