pccr_ppt

Transcript

1. Experimental validation of long-range RNA interactions in murine/human genes 1 Petrova Marina 12 Apr. 2022
2. RT-PCR 2 • phf20l1 Phf20l1 (fw+rv_mm_all) NIH3T3 P6 LNA2 LNA1 200 100 150 300 75 50 100nM 25nM 5nM cLNA con 100nM 25nM 5nM MQ 196bp 106bp 5 6 7 5 7
3. RT-PCR 3 • cask Cask (fw+rv_mm_all) NIH3T3 P4 LNA2 LNA1 200 100 150 300 75 50 100nM 25nM 5nM cLNA con 100nM 25nM 5nM MQ 149bp 82bp 18 19 20 18 20
4. LNA Treatment of NIH3T3# 4 • phf20l1 • Isoform fold change = Isoform ratio(lna)/ Isoform ratio (con) 0 1 2 3 5nM 25nM 100nM treatment fc lna1 phf20l1 Isoform_fold_change 2 4 6 8 5nM 25nM 100nM treatment fc lna2 phf20l1 Isoform_fold_change
5. LNA Treatment of NIH3T3# 5 • cask • Isoform fold change = Isoform ratio(lna)/ Isoform ratio (con) 0 2 4 6 5nM 25nM 100nM treatment fc lna1 cask Isoform_fold_change 2 4 6 8 5nM 25nM 100nM treatment fc lna2 cask Isoform_fold_change
6. Minigene construction 6 1. PCR to create a ‘mega-primer’ 2. PCR replication of the plasmid DpnI digestion
7. Brd2 (human) 7 Scale chr6: RIC-seq 1 kb hg19 32,942,000 32,942,500 32,943,000 32,943,500 32,944,000 32,944,500 32,945,000 PCCR dG <= -20 kcal/mol icSHAPE in Hek chromatin RIC-seq UCSC Genes (RefSeq, GenBank, CCDS, Rfam, tRNAs & Comparative Genomics) 100 vertebrates Basewise Conservation by PhyloP loop out eCLIP self peaks 100 vertebrates conservation by PhastCons RNA bridges eCLIP self peaks id=728649,dG=-20.8 id=728672,dG=-24.4,qval=0.66 id=728693,dG=-24 id=728697,dG=-21.6 id=728700,dG=-22.5 id=728710,dG=-25.3,qval=0.68 id=728727,dG=-20.5,qval=0.01 id=728732,dG=-23.8 id=728744,dG=-20.2 id=728745,dG=-20,qval=0.43 id=728746,dG=-20.4,qval=0.36 id=728750,dG=-23.2,qval=0.57 id=728753,dG=-20.3,qval=0.38 id=728759,dG=-20.7 id=728762,dG=-20.2 id=728773,dG=-25.4,qval=0.7 id=728776,dG=-21.9,qval=0.51 id=728779,dG=-29.1 id=728784,dG=-21.4 id=728786,dG=-25.8,qval=0.32 id=728787,dG=-20.7 id=728795,dG=-21.7,qval=0.11 id=728796,dG=-24 BRD2 BRD2 BRD2 BRD2 BRD2 BRD2 icSHAPE
8. LNA Treatment of A549# rep1 8 • brd2 2 3 2 2a 3 171bp 79bp Brd2 A549 P1 LNA2 LNA1 200 100 150 300 75 50 100nM 25nM 5nM cLNA con 100nM 25nM 5nM MQ
9. LNA treatment of A549# 9 • brd2 • Isoform fold change = Isoform ratio(lna)/ Isoform ratio (con) 0 2 4 6 8 5nM 25nM 100nM treatment fc lna1 brd2 Isoform_fold_change 1.0 1.5 2.0 2.5 5nM 25nM 100nM treatment fc lna2 brd2 Isoform_fold_change
10. Minigene mutagenesis 10 P P PCR with phosphorilated back-to-back primers DpnI digestion ligation
11. Minigene mutagenesis 11 GC UGA UGCGGCCCC GGGG CC CG CGA GGGG CC CG GCUGCGGCCCC CGA GGGG CC CG ACUGCGGCCCC CGA GGGG CC CG ACUGCGGCCCC wt m1 m2 m1m2
12. Minigene mutagenesis 12 brd2_all_iso Brd2_minigene 400 500 300 200 100 700 150 75 50 25 pRK5.brd2 pRK5 m1 m2 m1m2 MQ 2 3 2 2a 3 171bp 79bp
13. Minigene mutagenesis 13 0.9 1.2 1.5 1.8 2.1 m1 m2 m1m2 treatment fc mutant brd2 Isoform_fold_change
14. Formula 14 RE = E tar C q exp - C q con E ref C q exp - C q con RIV = 2 C q con·log(Econ)/log2 - C q var·log(Evar)/log2
15. Future plans 15 • Minigene construction for phf20l1 + cask • Targets screening & mutagenesis