a301 152a  (Bethyl)

Journal: The Journal of Biological Chemistry

Article Title: The U1 antisense morpholino oligonucleotide (AMO) disrupts U1 snRNP structure to promote intronic PCPA modification of pre-mRNAs

doi: 10.1016/j.jbc.2023.104854

Figure Lengend Snippet: Effect of U1 AMO on the recruitment of core intronic PAS processing factors onto chromatin and pre-mRNA. A , in vitro cleavage/polyadenylation and in vitro cleavage assays using HeLa NE and RNA substrates derived from intronic PAS of nr3c1 gene. Three repeats of MS2 sequences were added to the 5′ end of the PAS RNA to facilitate the process of protein purification. As negative control, a mutant PAS RNA harboring a point mutation in the core hexamer AAUAAA was prepared simultaneously. The PAS RNA sequences are listed in Table S4 . The polyadenylated or cleaved RNA products are indicated with red arrows . B , silver staining of protein complexes purified from HeLa NE using the indicated PAS RNAs. Potential intronic PAS processing factors, which are indicated with red arrows , could be purified by wt but not m1 PAS RNAs. C , validation of mass spectrometric results by Western blotting analysis using antibodies against indicated proteins. The input is 2% of the total lysates. D , metagene plots of CPSF160, WDR33, and CSTF77 chromatin immunoprecipitation (ChIP-Seq) reads in control and U1 AMO–treated HeLa cells for all actively expressed genes (n = 13,217), intronic PCPAed genes (n = 5937), and other nonintronic PCPAed genes (n = 7280). Control represents control AMO–treated HeLa cells, and U1 represents U1 AMO–treated HeLa cells. E , metagene plots of WDR33 and CSTF64 iCLIP-Seq reads in control and U1 AMO–treated HeLa cells for all actively expressed genes (n = 13,217), intronic PCPAed genes (n = 5937), and other nonintronic PCPAed genes (n = 7280). For intronic PCPAed genes, a second metagene plot was made for each ChIP-Seq/iCLIP-Seq by replacing the default TES (transcription end site) with intronic PCPA sites. Control represents control AMO–treated HeLa cells, and U1 represents U1 AMO–treated HeLa cells. F , a schematic diagram summarizing the key finding in our study. The details are described in the main body of the text. iCLIP-Seq, individual-nucleotide resolution CrossLinking and ImmunoPrecipitation sequencing; PCPA, premature cleavage and polyadenylation.

Article Snippet: The primary antibodies (WDR33 [A301–152A] and CstF64 [A301–092A]) were purchased from Bethyl.

Techniques: In Vitro, Derivative Assay, Protein Purification, Negative Control, Mutagenesis, Silver Staining, Purification, Western Blot, Chromatin Immunoprecipitation, ChIP-sequencing, Immunoprecipitation, Sequencing

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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: Primary antibodies used for ChIP included RNAPII Ser5P (61,986, Active Motif); RNAPII Ser2P (61,984, Active Motif); PCF11(A303-706A, Bethyl); CPSF160(A301-580A, Bethyl); WDR33(A301-152A, Bethyl); CstF77(A301-094A, Bethyl); CPSF100(A301-583A, Bethyl); CPSF30(A301-585A, Bethyl); Fip1(A301-462A, Bethyl); CFIm68(A301-356A, Bethyl); These libraries were sequenced on the NovaSeq platform, and all ChIP-seq data processing and analysis were performed according to the ENCODE ChIP-seq pipeline ( https://www.encodeproject.org/data-standards/chip-seq/ ).

Techniques: ChIP-sequencing, Mobility Shift, Recombinant