pccr_ppt

Transcript

1. Experimental validation of long-range RNA interactions in murine/human genes 1 Petrova Marina 12 Apr. 2022
2. 2 • phf20l1 196bp 106bp 5 6 7 5 7 ASO Treatment of NIH3T3# Phf20l1 (fw+rv_mm_all) NIH3T3 P6 LNA2 LNA1 200 100 150 300 75 50 100nM 25nM 5nM cLNA con 100nM 25nM 5nM MQ
3. 3 • cask 149bp 82bp 18 19 20 18 20 ASO Treatment of NIH3T3# Cask (fw+rv_mm_all) NIH3T3 P4 LNA2 LNA1 200 100 150 300 75 50 100nM 25nM 5nM cLNA con 100nM 25nM 5nM MQ
4. ASO 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. ASO 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. Minigene construction 7 1. PCR generated insert Restriction enzyme digestion
8. Cask minigene mutagenesis 8 P P PCR with phosphorilated back-to-back primers DpnI digestion ligation
9. Cask minigene mutagenesis 9 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
10. Minigene mutagenesis 10 • cask 166bp 97bp 18 19 20 18 20 cask_all_iso cask_minigene_p1 pRK5.cask pRK5 m1 m2 m1m2 MQ m2 75 35 50 100 150 200 300 25
11. Minigene mutagenesis 11 • cask 166bp 97bp 18 19 20 18 20 cask_all_iso cask_minigene_p2 pRK5.cask pRK5 m1 m2 m1m2 MQ m2 75 35 50 100 150 200 300 25
12. Minigene mutagenesis 12 • cask 166bp 97bp 18 19 20 18 20 cask_all_iso cask_minigene_p3 pRK5.cask pRK5 m1 m2 m1m2 MQ m2 75 35 50 100 150 200 300 25
13. Cask minigene mutagenesis 13 0.00 0.25 0.50 0.75 1.00 con m1 m2 m1m2 treatment per_in cask Inclusion_percent
14. Brd2 (human) 14 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
15. ASO treatment of A549# rep1 15 • 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
16. ASO treatment of A549# 16 • 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
17. Minigene mutagenesis 17 P P PCR with phosphorilated back-to-back primers DpnI digestion ligation
18. Minigene mutagenesis 18 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
19. Minigene mutagenesis 19 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 • brd2
20. Minigene mutagenesis 20 0.9 1.2 1.5 1.8 2.1 m1 m2 m1m2 treatment fc mutant brd2 Isoform_fold_change
21. New target genes (from 27.12.21) 21
22. ralgps2 22 • id85009. chr1: 178860928_178862183 Scale chr1: RNA_bridges_eCLIP tRNA Genes 1 kb hg19 178,861,000 178,861,500 178,862,000 178,862,500 PCCR dG <= -15 kcal/mol icSHAPE in Hek chromatin RIC-seq UCSC Genes (RefSeq, GenBank, CCDS, Rfam, tRNAs & Comparative Genomics) 100 vertebrates conservation by PhastCons 100 vertebrates Basewise Conservation by PhyloP loop out eCLIP self peaks RNA bridges eCLIP self peaks Transfer RNA Genes Identified with tRNAscan-SE HeLa-contacts HeLa-junctions HepG2-contacts HepG2-junctions id=85009,dG=-24.9,qval=0.05 RALGPS2 RALGPS2 id_140328,count=2 id_140333,count=1 id_140335,count=2 id_166009,count=1 id_166058,count=2 id_166061,count=2 id_166063,count=2 id_166064,count=1 id_166065,count=1 id=127454,count=2 id=127455,count=1 id=127456,count=1 icSHAPE 100 Vert. Cons
23. ASO treatment of A549# 23 • ralgps2 204bp 126bp ralgps2 A549 P1 LNA2 LNA1 200 100 150 300 75 50 100nM 25nM 5nM cLNA con 100nM 25nM 5nM MQ 13 15 13 14 15
24. ptpru 24 • id18157. chr1:29633580_29633827 Scale chr1: RNA_bridges_eCLIP tRNA Genes 100 bases hg19 29,633,550 29,633,600 29,633,650 29,633,700 29,633,750 29,633,800 29,633,850 PCCR dG <= -15 kcal/mol icSHAPE in Hek chromatin RIC-seq UCSC Genes (RefSeq, GenBank, CCDS, Rfam, tRNAs & Comparative Genomics) 100 vertebrates conservation by PhastCons 100 vertebrates Basewise Conservation by PhyloP loop out eCLIP self peaks RNA bridges eCLIP self peaks Transfer RNA Genes Identified with tRNAscan-SE HeLa-contacts HeLa-junctions HepG2-contacts HepG2-junctions id=18153,dG=-18.5,qval=0.52 id=18154,dG=-16.5,qval=0.38 id=18155,dG=-17.6,qval=0.38 id=18156,dG=-19.1,qval=0.46 id=18157,dG=-27.5,qval=0.3 id=18158,dG=-19.1,qval=0.24 id=18159,dG=-21.1 id=18160,dG=-19.7,qval=0.31 PTPRU PTPRU PTPRU PTPRU id_43706,count=1 id_46668,count=1 id_46669,count=2 icSHAPE 100 Vert. Cons
25. camk2b 25 • id775954. chr7:44271930_44272918 Scale chr7: RNA_bridges_eCLIP tRNA Genes 500 bases hg19 44,272,000 44,272,500 44,273,000 PCCR dG <= -15 kcal/mol icSHAPE in Hek chromatin RIC-seq UCSC Genes (RefSeq, GenBank, CCDS, Rfam, tRNAs & Comparative Genomics) 100 vertebrates conservation by PhastCons 100 vertebrates Basewise Conservation by PhyloP loop out eCLIP self peaks RNA bridges eCLIP self peaks Transfer RNA Genes Identified with tRNAscan-SE HeLa-contacts HeLa-junctions HepG2-contacts HepG2-junctions id=775954,dG=-26.3 CAMK2B CAMK2B CAMK2B CAMK2B CAMK2B CAMK2B CAMK2B CAMK2B CAMK2B id_1472080,count=1 id_1472118,count=2 id_2034690,count=1 id_2034778,count=2 icSHAPE 100 Vert. Cons
26. ASO treatment of A549# 26 • ptpru_lna2 camk2b_lna1 117bp 97bp camk2b_lna1 A549 P1 200 100 150 300 75 50 ptpru_lna2 100nM 25nM 5nM cLNA con 100nM 25nM 5nM MQ cLNA con MQ 15 17 20 22
27. ppfia3 27 • id457947. chr19:49649365_49649457 Scale chr19: RNA_bridges_eCLIP tRNA Genes 50 bases hg19 49,649,350 49,649,400 49,649,450 PCCR dG <= -15 kcal/mol icSHAPE in Hek chromatin RIC-seq UCSC Genes (RefSeq, GenBank, CCDS, Rfam, tRNAs & Comparative Genomics) 100 vertebrates conservation by PhastCons 100 vertebrates Basewise Conservation by PhyloP loop out eCLIP self peaks RNA bridges eCLIP self peaks Transfer RNA Genes Identified with tRNAscan-SE HeLa-contacts HeLa-junctions HepG2-contacts HepG2-junctions id=457944,dG=-23.3 id=457945,dG=-19.6 id=457946,dG=-15.5,qval=0.24 id=457947,dG=-24.9,qval=0.39 id=457948,dG=-19.6 id=457949,dG=-18.7,qval=0.28 id=457950,dG=-21.1,qval=0.43 id=457951,dG=-28.5 id=457952,dG=-21 E T K E I S W E Q PPFIA3 PPFIA3 PPFIA3 PPFIA3 id_856886,count=1 id_856969,count=1 id_856991,count=1 id_856999,count=2 id_857002,count=1 id_857003,count=2 id_1201343,count=1 id_1201406,count=1 id_1201424,count=2 id_1201428,count=1 id_1201431,count=1 id_1201438,count=1 id_1201449,count=1 id_1201457,count=1 id=1010662,count=1 icSHAPE 100 Vert. Cons
28. cstf2 28 • id917778. chrX:100085762_100085925 Scale chrX: RNA_bridges_eCLIP tRNA Genes 100 bases hg19 100,085,650 100,085,700 100,085,750 100,085,800 100,085,850 100,085,900 100,085,950 100,086,000 PCCR dG <= -15 kcal/mol icSHAPE in Hek chromatin RIC-seq UCSC Genes (RefSeq, GenBank, CCDS, Rfam, tRNAs & Comparative Genomics) 100 vertebrates conservation by PhastCons 100 vertebrates Basewise Conservation by PhyloP loop out eCLIP self peaks RNA bridges eCLIP self peaks Transfer RNA Genes Identified with tRNAscan-SE HeLa-contacts HeLa-junctions HepG2-contacts HepG2-junctions id=917754,dG=-17.9 id=917755,dG=-16.2 id=917756,dG=-15.6,qval=0.36 id=917767,dG=-19.6,qval=0.52 id=917768,dG=-16,qval=0.35 id=917769,dG=-24.4,qval=0.64 id=917770,dG=-17.5 id=917772,dG=-16.7,qval=0.44 id=917771,dG=-16.2 id=917773,dG=-15.6 id=917775,dG=-17.8 id=917776,dG=-16.3,qval=0.45 id=917777,dG=-16 id=917778,dG=-20.9,qval=0.34 id=917779,dG=-16.2,qval=0.82 id=917780,dG=-18.6,qval=0.29 id=917781,dG=-18.3,qval=0.54 id=917782,dG=-17.1,qval=0.34 id=917784,dG=-17 id=917785,dG=-16.8,qval=0.18 id=917786,dG=-16,qval=0.62 id=917787,dG=-17.3 id=917788,dG=-17.3,qval=0.57 id=917789,dG=-17.3,qval=0.63 id=917790,dG=-17.9,qval=0.6 CSTF2 CSTF2 CSTF2 id_1727359,count=2 id_1727361,count=1 id_1727362,count=1 id=1715340,count=1 id_2400652,count=1 id_2400662,count=2 id_2400666,count=1 id_2400667,count=1 id_2400669,count=1 id_2400671,count=1 id=2084409,count=1 id=2084411,count=1 icSHAPE 100 Vert. Cons
29. ASO treatment of A549# 29 • cstf2_lna2 ppfia3_lna1 136bp 106bp ppfia3_lna1 A549 P1 200 100 150 300 75 50 cstf2_lna2 100nM 25nM 5nM cLNA con 100nM 25nM 5nM MQ cLNA con 23 25 9 11
30. New target genes (from 16.02.22) 30
31. dopey1 31 • id742834. • chr6:83873841_83877270 Scale chr6: RNA_bridges_eCLIP tRNA Genes 1 kb hg19 83,874,500 83,875,000 83,875,500 83,876,000 83,876,500 83,877,000 PCCR dG <= -15 kcal/mol icSHAPE in Hek chromatin RIC-seq UCSC Genes (RefSeq, GenBank, CCDS, Rfam, tRNAs & Comparative Genomics) 100 vertebrates conservation by PhastCons 100 vertebrates Basewise Conservation by PhyloP loop out eCLIP self peaks RNA bridges eCLIP self peaks Transfer RNA Genes Identified with tRNAscan-SE HeLa-contacts HeLa-junctions HepG2-contacts HepG2-junctions id=742830,dG=-18.4,qval=0.86 id=742834,dG=-23,qval=0.23 id=742841,dG=-16.9,qval=0.85 Cluster_1096976;r=6 Cluster_1490970;r=6 DOP1A DOP1A DOP1A DOP1A PGM3 PGM3 PGM3 PGM3 id_1399915,count=2 id_1399924,count=6 id_1399932,count=2 id_1399933,count=1 id_1399934,count=1 id_1399935,count=1 id_1399936,count=2 id=1334148,count=1 id_1943018,count=2 id_1943045,count=4 id_1943074,count=2 id_1943075,count=1 id_1943076,count=2 id_1943077,count=2 id_1943078,count=4 id_1943079,count=3 id_1943080,count=2 id_1943081,count=2 id_1943082,count=2 id_1943083,count=3 id_1943084,count=2 id_1943085,count=1 id_1943086,count=1 id_1943087,count=1 id_1943088,count=2 id_1943089,count=2 id_1943090,count=2 id_1943091,count=2 id_1943092,count=2 id_1943093,count=2 id_1943094,count=2 id_1943095,count=2 id_1943096,count=2 id_1943097,count=2 id_1943098,count=4 id_1943099,count=1 id_1943100,count=1 id_1943101,count=2 id_1943102,count=2 id_1943103,count=1 id=1624162,count=2 id=1624163,count=1 id=1624164,count=1 id=1624165,count=1 id=1624166,count=2 id=1624167,count=4 id=1624168,count=3 id=1624169,count=2 id=1624170,count=2 id=1624171,count=2 id=1624172,count=2 id=1624173,count=1 id=1624174,count=2 id=1624175,count=1 id=1624176,count=1 id=1624177,count=2 id=1624178,count=2 id=1624179,count=2 id=1624180,count=2 id=1624181,count=2 id=1624182,count=1 id=1624183,count=1 id=1624184,count=4 id=1624185,count=1 id=1624186,count=2 id=1624187,count=1 icSHAPE 100 Vert. Cons
32. ASO treatment of A549# rep.1 32 • dopey1 122bp 62bp A549 P1 200 300 150 100 75 50 LNA2 LNA1 100nM 25nM 5nM cLNA con 100nM 25nM 5nM MQ dopey1 38 40 38 39 40
33. ASO treatment of A549# rep.2 33 • dopey1 122bp 62bp 38 40 38 39 40 A549 P2 200 300 150 100 75 50 LNA2 LNA1 dopey1 100nM 25nM 5nM cLNA 100nM 25nM 5nM MQ con
34. ASO treatment of A549# rep.3 34 • dopey1 122bp 62bp 38 40 38 39 40 A549 P3 200 300 150 100 75 50 LNA2 LNA1 dopey1 100nM 25nM 5nM cLNA 100nM 25nM 5nM MQ con
35. lrp8 35 • id39502 chr1:53715015_53715245 Scale chr1: RIC-seq 200 bases hg19 53,714,900 53,715,000 53,715,100 53,715,200 53,715,300 53,715,400 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 Transfer RNA Genes Identified with tRNAscan-SE id=39494,dG=-21.5,qval=0.35 id=39502,dG=-21.8 id=39503,dG=-20.4,qval=0.1 LRP8 LRP8 LRP8 LRP8 LRP8 icSHAPE
36. ASO treatment of A549# rep.1 36 • lrp8 & csnk1g3 139bp 115bp A549 P1 200 300 150 100 75 50 lrp8 LNA2 LNA1 csnk1g3 100nM 25nM 5nM cLNA con 100nM 25nM 5nM con 256 79 12 14 12 13 14
37. ASO treatment of A549# rep.2 37 • lrp8 256bp 79bp 12 14 12 13 14 lrp8 LNA1 cLNA 100nM 25nM 5nM con 200 300 150 100 75 50 A549 P2
38. ASO treatment of A549# rep.3 38 • lrp8 256bp 79bp 12 14 12 13 14 lrp8 LNA1 100nM 25nM 5nM con 200 300 150 100 75 50 A549 P3
39. mark2 39 • id177645 chr11:63673416_63675010 Scale chr11: RIC-seq 1 kb hg19 63,673,500 63,674,000 63,674,500 63,675,000 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 Transfer RNA Genes Identified with tRNAscan-SE HeLa HepG2 id=177586,dG=-20 id=177591,dG=-25.4 id=177596,dG=-21.2,qval=0.87 id=177606,dG=-25.7 id=177605,dG=-23.3,qval=0.31 id=177610,dG=-26.7 id=177615,dG=-23.4,qval=0.46 id=177617,dG=-23,qval=0.42 id=177618,dG=-22.5,qval=0.48 id=177619,dG=-22.1,qval=0.51 id=177623,dG=-24.3 id=177625,dG=-21.3,qval=0.2 id=177628,dG=-23.3 id=177629,dG=-25.9 id=177630,dG=-20.3,qval=0.72 id=177632,dG=-21.5,qval=0.33 id=177636,dG=-23.9,qval=0.32 id=177637,dG=-21.5 id=177638,dG=-20.5 id=177639,dG=-21.5 id=177645,dG=-36.7,qval=0.19 id=177648,dG=-22.6 id=177649,dG=-23.7 id=177655,dG=-21.8,qval=0.49 id=177657,dG=-20.7 id=177664,dG=-20.7,qval=0.61 id=177667,dG=-26.1 id=177668,dG=-28.2 id=177673,dG=-21.4 id=177674,dG=-21.2,qval=0.19 MARK2 MARK2 MARK2 MARK2 MARK2 MARK2 id_66200;count=1 id_66201;count=2 id_72740;count=4 id_66202;count=1 id_72741;count=1 id_66203;count=2 id_66204;count=3 id_72742;count=2 id_66205;count=1 id_72743;count=2 id_66206;count=2 id_72744;count=2 id_72745;count=1 id_72746;count=1 id_66207;count=2 id_72747;count=1 id_72748;count=2 id_72749;count=2 id_66208;count=1 id_72750;count=4 id_66209;count=1 id_66210;count=1 id_66211;count=2 id_72751;count=1 id_64502;count=3 id_64503;count=1 id_42745;count=2 id_58324;count=2 id_40403;count=4 id_42746;count=2 id_58325;count=1 id_64504;count=1 id_58326;count=1 id_64505;count=1 id_42747;count=1 id_40404;count=1 id_58327;count=2 id_40405;count=1 id_58328;count=1 id_40406;count=1 id_40407;count=2 id_64506;count=2 id_64507;count=1 id_40408;count=2 id_64508;count=1 id_42748;count=2 id_64509;count=1 id_64510;count=1 id_40409;count=2 id_42749;count=1 id_64511;count=2 icSHAPE
40. mark2 ASO treatment of A549# rep.1 40 164bp 137bp 16 17 18 16 18 A549 P1 ASO2 ASO1 100nM 25nM 5nM cLNA 100nM 25nM 5nM MQ con 500 100 300 700 50 400 200 150 75 mark2 35x
41. mark2 ASO treatment of A549# rep.2 41 164bp 137bp 16 17 18 16 18 A549 P2 ASO2 ASO1 500 100 300 700 50 400 200 150 75 mark2 35x 100nM 25nM 5nM cLNA 100nM 25nM 5nM MQ con
42. mark2 ASO treatment of A549# rep.3 42 164bp 137bp 16 17 18 16 18 A549 P3 ASO2 ASO1 500 100 300 700 400 200 150 mark2 35x MQ 100nM 25nM 5nM cLNA 100nM 25nM 5nM con
43. mark2 ASO treatment of A549# 43 • mark2 0.0 0.1 0.2 0.3 0.4 0.5 con clna 5nM 25nM100nM treatment per_in lna1 lna2 mark2 Inclusion_percent
44. Brd2_microsatellite 44
45. Brd2_microsatellite 45 Figure 3. The conserved region in intron 2 of BRD2 contains an alternatively spliced exon. A. A cartoon portraying the ~180 bp region in intron 2 of BRD2 that is highly conserved between human, dog, mouse, rat, and opossum, adapted from the ECR Browser. The yellow bar indicates the alternative exon 2a (92 bp) in intron 2. The green column near exon 3 is a microsatellite (GT repeats). A vertical axis cut-off of 75% identity is utilized to visualize only the significant alignments. Note that the level of conservation in intron 2 is higher than in the two adjacent coding exons. B. RT-PCR of human brain RNA using primers located in exon 2 and exon 3 of human BRD2. The upper weaker band contains a 92 bp conserved region in intron 2 (exon 2a), indicating the existence of mRNAs containing the alternatively spliced exon. C. RT-PCR of RNA from constructs containing different length of the GT repeats using primers across exon 2 to exon 3. It shows that the proportion of the alternatively spliced transcript (top band) increases relative to the regularly spliced transcript (lower band) with the shortening of the GT repeats. Shang et al. Page 15 NIH-PA Author Manuscript NIH-PA Aut
46. Mark2_microsatellite 46
47. clk2 47 • id75420 • chr1:155238304_155238655 Scale chr1: tRNA Genes 200 bases hg19 155,238,300 155,238,350 155,238,400 155,238,450 155,238,500 155,238,550 155,238,600 155,238,650 155,238,700 PCCR dG <= -15 kcal/mol icSHAPE in Hek chromatin RIC-seq UCSC Genes (RefSeq, GenBank, CCDS, Rfam, tRNAs & Comparative Genomics) 100 vertebrates conservation by PhastCons 100 vertebrates Basewise Conservation by PhyloP loop out eCLIP self peaks RNA bridges eCLIP self peaks Transfer RNA Genes Identified with tRNAscan-SE HeLa-contacts id=75398,dG=-19.4,qval=0.44 id=75399,dG=-18.9,qval=0.48 id=75400,dG=-16.7,qval=0.37 id=75401,dG=-23.5,qval=0.25 id=75402,dG=-18.5,qval=0.45 id=75403,dG=-21.6,qval=0.57 id=75404,dG=-16.4,qval=0.33 id=75405,dG=-15.5,qval=0.6 id=75406,dG=-21.7,qval=0.69 id=75407,dG=-19.7,qval=0.36 id=75408,dG=-18.4,qval=0.46 id=75409,dG=-19.5 id=75410,dG=-22.7,qval=0.74 id=75411,dG=-19.8,qval=0.31 id=75412,dG=-18.2,qval=0.51 id=75413,dG=-19.3 id=75414,dG=-19.3 id=75415,dG=-20.2 id=75416,dG=-17.8 id=75417,dG=-23.2,qval=0.21 id=75419,dG=-16.1,qval=0.44 id=75420,dG=-17.4 id=75421,dG=-15.7 id=75422,dG=-20.5,qval=0.73 id=75423,dG=-25.9 id=75425,dG=-15.7 id=75426,dG=-17.4 id=75427,dG=-15.8 id=75428,dG=-18.1,qval=0.42 id=75429,dG=-18.8,qval=0.57 id=75430,dG=-15.6 id=75432,dG=-29.5 id=75431,dG=-20.1 id=75433,dG=-19.6,qval=0.38 id=75434,dG=-18.5,qval=0.28 id=75435,dG=-15.1 id=75436,dG=-49 id=75437,dG=-26.8 id=75438,dG=-23.4 id=75439,dG=-28.7,qval=0.22 Cluster_1403480;r=24 Cluster_1601027;r=7 Cluster_1355776;r=3 Cluster_922285;r=13 CLK2 CLK2 CLK2 CLK2 id_122623,count=1 id_122646,count=1 id_122655,count=1 id_122657,count=1 id_122677,count=1 id_122707,count=2 id_122731,count=2 id_122735,count=2 id_122736,count=2 id_122737,count=1 id_122738,count=2 id_122739,count=4 id_122740,count=4 icSHAPE 100 Vert. Cons tRNA Genes icSHAPE in Hek chromatin RIC-seq UCSC Genes (RefSeq, GenBank, CCDS, Rfam, tRNAs & Comparative Genomics) 100 vertebrates conservation by PhastCons 100 vertebrates Basewise Conservation by PhyloP loop out eCLIP self peaks RNA bridges eCLIP self peaks Transfer RNA Genes Identified with tRNAscan-SE HeLa-contacts HeLa-junctions HepG2-contacts id=75410,dG=-22.7,qval=0.74 id=75411,dG=-19.8,qval=0.31 id=75412,dG=-18.2,qval=0.51 id=75413,dG=-19.3 id=75414,dG=-19.3 id=75415,dG=-20.2 id=75416,dG=-17.8 id=75417,dG=-23.2,qval=0.21 id=75419,dG=-16.1,qval=0.44 id=75420,dG=-17.4 id=75421,dG=-15.7 id=75422,dG=-20.5,qval=0.73 id=75423,dG=-25.9 id=75425,dG=-15.7 id=75426,dG=-17.4 id=75427,dG=-15.8 id=75428,dG=-18.1,qval=0.42 id=75429,dG=-18.8,qval=0.57 id=75434,dG=-18.5,qval=0.28 id=75435,dG=-15.1 id=75436,dG=-49 id=75437,dG=-26.8 id=75438,dG=-23.4 id=75439,dG=-28.7,qval=0.22 Cluster_1403480;r=24 Cluster_1601027;r=7 Cluster_1355776;r=3 Cluster_922285;r=13 CLK2 CLK2 CLK2 CLK2 id_122623,count=1 id_122646,count=1 id_122655,count=1 id_122657,count=1 id_122677,count=1 id_122707,count=2 id_122731,count=2 id_122735,count=2 id_122736,count=2 id_122737,count=1 id_122738,count=2 id_122739,count=4 id_122740,count=4 id_122741,count=2 id=113835,count=2 id=113836,count=2 id=113837,count=1 id=113838,count=1 id_142627,count=1 id_142628,count=1 id_142708,count=2 id_142721,count=1 id_142742,count=1 id_142777,count=2 id_142783,count=1 id_142837,count=1 icSHAPE 100 Vert. Cons tRNA Genes 100 vertebrates conservation by PhastCons 100 vertebrates Basewise Conservation by PhyloP loop out eCLIP self peaks RNA bridges eCLIP self peaks Transfer RNA Genes Identified with tRNAscan-SE HeLa-contacts HeLa-junctions HepG2-contacts HepG2-junctions CLK2 CLK2 id_122623,count=1 id_122646,count=1 id_122655,count=1 id_122657,count=1 id_122677,count=1 id_122707,count=2 id_122731,count=2 id_122735,count=2 id_122736,count=2 id_122737,count=1 id_122738,count=2 id_122739,count=4 id_122740,count=4 id_122741,count=2 id=113835,count=2 id=113836,count=2 id=113837,count=1 id=113838,count=1 id_142627,count=1 id_142628,count=1 id_142708,count=2 id_142721,count=1 id_142742,count=1 id_142777,count=2 id_142783,count=1 id_142837,count=1 id_142891,count=1 id_142892,count=1 id_142899,count=2 id_142903,count=1 id_142924,count=1 id_142961,count=1 id_143048,count=1 id_143055,count=1 id_143073,count=1 id_143074,count=2 id_143075,count=6 id_143076,count=2 id_143077,count=4 id_143078,count=2 id_143079,count=4 id_143080,count=1 id_143081,count=4 id_143082,count=1 id_143083,count=2 id_143084,count=1 id_143085,count=2 id_143086,count=2 id_143087,count=1 id=108209,count=1 id=108210,count=2 id=108211,count=1 id=108212,count=2 id=108213,count=2 id=108214,count=2 id=108215,count=2 id=108216,count=1 id=108217,count=2 100 Vert. Cons
48. clk2 48 213bp 125bp clk2 35x A549 HEK HeLa HepG2 IMR 500 100 300 700 50 400 200 150 75 25 3 4 5 3 5 • isoform expression
49. clk2 49 213bp 125bp 3 4 5 3 5 clk2 35x MCF7 Skbr3 Sk_N_MC U87 A549+CH U251 MQ 500 100 300 700 50 400 200 150 75 • isoform expression
50. serf2 50 • id316511 • chr15: 44085401_44085783 Scale chr15: tRNA Genes 200 bases hg19 44,085,300 44,085,400 44,085,500 44,085,600 44,085,700 44,085,800 44,085,900 44,086,000 PCCR dG <= -15 kcal/mol icSHAPE in Hek chromatin RIC-seq UCSC Genes (RefSeq, GenBank, CCDS, Rfam, tRNAs & Comparative Genomics) 100 vertebrates conservation by PhastCons 100 vertebrates Basewise Conservation by PhyloP loop out eCLIP self peaks RNA bridges eCLIP self peaks Transfer RNA Genes Identified with tRNAscan-SE HeLa-contacts id=316446,dG=-21.2 id=316447,dG=-20.9 id=316449,dG=-18.3,qval=0.22 id=316455,dG=-19.5 id=316456,dG=-18.8 id=316457,dG=-17.6 id=316459,dG=-16.6 id=316463,dG=-20.1,qval=0.09 id=316464,dG=-15.2,qval=0.49 id=316465,dG=-21.7,qval=0.23 id=316468,dG=-15.7,qval=0.73 id=316472,dG=-20.9,qval=0.26 id=316474,dG=-29.7 id=316475,dG=-19.2 id=316476,dG=-16.4 id=316477,dG=-16.7 id=316495,dG=-18.1 id=316496,dG=-24.1 id=316497,dG=-17.7 id=316500,dG=-22.7,qval=0.37 id=316501,dG=-23.1,qval=0.24 id=316502,dG=-22.5 id=316503,dG=-17.6,qval=0.68 id=316504,dG=-18.2,qval=0.53 id=316506,dG=-16.5,qval=0.72 id=316511,dG=-20.2,qval=0.14 id=316513,dG=-15.5 id=316516,dG=-19 id=316520,dG=-17.3 id=316521,dG=-34.5,qval=0.48 id=316524,dG=-15.1,qval=0.38 id=316526,dG=-15.2,qval=0.17 id=316530,dG=-15,qval=0.19 id=316531,dG=-19.1,qval=0.42 id=316535,dG=-16.5 id=316538,dG=-16.2,qval=0.28 id=316539,dG=-15,qval=0.61 id=316543,dG=-15.1,qval=0.43 id=316544,dG=-18.9,qval=0.27 id=316547,dG=-20.8,qval=0.21 id=316546,dG=-20.6,qval=0.77 id=316548,dG=-19.2,qval=0.4 id=316549,dG=-15.2,qval=0.29 id=316550,dG=-16 id=316552,dG=-17.6 id=316554,dG=-21.4,qval=0.44 id=316558,dG=-16.8,qval=0.13 id=316559,dG=-18.2,qval=0.78 id=316561,dG=-15.5,qval=0.5 id=316564,dG=-20.6,qval=0.68 id=316565,dG=-29.1 id=316568,dG=-19.9 id=316569,dG=-19 Cluster_270987;r=4 Cluster_220387;r=15 Cluster_1074034;r=8 Cluster_567700;r=2 Cluster_1596234;r=42 RP11-296A16.1 SERF2 SERF2 SERF2 SERF2 SERF2 SERF2 SERF2 MIR1282 id_522033,count=1 id_522038,count=2 id_522072,count=1 id_522086,count=1 id_522089,count=2 id_522096,count=1 id_522105,count=1 id_522114,count=1 id_522116,count=1 id_522122,count=1 id_522123,count=3 id_522124,count=2 id_522127,count=1 id_522142,count=2 id_522143,count=2 id_522144,count=1 icSHAPE 100 Vert. Cons tRNA Genes icSHAPE in Hek chromatin RIC-seq UCSC Genes (RefSeq, GenBank, CCDS, Rfam, tRNAs & Comparative Genomics) 100 vertebrates conservation by PhastCons 100 vertebrates Basewise Conservation by PhyloP loop out eCLIP self peaks RNA bridges eCLIP self peaks Transfer RNA Genes Identified with tRNAscan-SE HeLa-contacts HeLa-junctions HepG2-contacts id=316504,dG=-18.2,qval=0.53 id=316506,dG=-16.5,qval=0.72 id=316511,dG=-20.2,qval=0.14 id=316513,dG=-15.5 id=316516,dG=-19 id=316520,dG=-17.3 id=316521,dG=-34.5,qval=0.48 id=316524,dG=-15.1,qval=0.38 id=316526,dG=-15.2,qval=0.17 id=316530,dG=-15,qval=0.19 id=316531,dG=-19.1,qval=0.42 id=316535,dG=-16.5 id=316538,dG=-16.2,qval=0.28 id=316539,dG=-15,qval=0.61 id=316543,dG=-15.1,qval=0.43 id=316544,dG=-18.9,qval=0.27 id=316547,dG=-20.8,qval=0.21 id=316546,dG=-20.6,qval=0.77 id=316548,dG=-19.2,qval=0.4 id=316549,dG=-15.2,qval=0.29 id=316552,dG=-17.6 id=316554,dG=-21.4,qval=0.44 id=316558,dG=-16.8,qval=0.13 id=316559,dG=-18.2,qval=0.78 id=316561,dG=-15.5,qval=0.5 id=316564,dG=-20.6,qval=0.68 id=316568,dG=-19.9 id=316569,dG=-19 Cluster_270987;r=4 Cluster_220387;r=15 Cluster_1074034;r=8 Cluster_567700;r=2 Cluster_1596234;r=42 RP11-296A16.1 SERF2 SERF2 SERF2 SERF2 SERF2 SERF2 SERF2 MIR1282 id_522033,count=1 id_522038,count=2 id_522072,count=1 id_522086,count=1 id_522089,count=2 id_522096,count=1 id_522105,count=1 id_522114,count=1 id_522116,count=1 id_522122,count=1 id_522123,count=3 id_522124,count=2 id_522127,count=1 id_522142,count=2 id_522143,count=2 id_522144,count=1 id_522145,count=2 id_522146,count=6 id_522147,count=2 id_522148,count=2 id_522149,count=2 id_522150,count=2 id_522151,count=2 id_522152,count=2 id=493734,count=2 id=493735,count=2 id=493736,count=2 id=493737,count=2 id=493738,count=1 id_730887,count=1 id_730949,count=1 id_731036,count=1 id_731050,count=2 id_731071,count=2 id_731074,count=1 id_731076,count=1 id_731078,count=1 id_731081,count=1 id_731082,count=1 id_731083,count=1 id_731183,count=2 id_731191,count=3 icSHAPE 100 Vert. Cons HeLa-junctions HepG2-contacts HepG2-junctions id_522149,count=2 id_522150,count=2 id_522151,count=2 id_522152,count=2 id=493734,count=2 id=493735,count=2 id=493736,count=2 id=493737,count=2 id=493738,count=1 id_730887,count=1 id_730949,count=1 id_731036,count=1 id_731050,count=2 id_731071,count=2 id_731074,count=1 id_731076,count=1 id_731078,count=1 id_731081,count=1 id_731082,count=1 id_731083,count=1 id_731084,count=1 id_731090,count=1 id_731093,count=2 id_731095,count=2 id_731097,count=1 id_731102,count=1 id_731103,count=1 id_731105,count=1 id_731106,count=1 id_731107,count=2 id_731122,count=1 id_731125,count=1 id_731127,count=1 id_731132,count=1 id_731133,count=2 id_731135,count=2 id_731136,count=1 id_731138,count=1 id_731142,count=1 id_731143,count=1 id_731144,count=4 id_731145,count=3 id_731146,count=1 id_731148,count=6 id_731149,count=9 id_731150,count=4 id_731151,count=2 id_731152,count=2 id_731153,count=2 id_731154,count=2 id_731156,count=1 id_731159,count=2 id_731160,count=2 id_731161,count=1 id_731162,count=6 id_731163,count=1 id_731164,count=6 id_731165,count=5 id_731166,count=10 id_731167,count=8 id_731168,count=22 id_731169,count=8 id_731170,count=1 id_731171,count=2 id_731172,count=2 id_731173,count=2 id_731174,count=3 id_731175,count=6 id_731176,count=3 id_731177,count=1 id_731178,count=1 id_731179,count=1 id_731180,count=1 id_731181,count=2 id_731182,count=2 id_731183,count=2 id_731184,count=2 id_731185,count=1 id_731186,count=1 id_731187,count=2 id_731188,count=1 id_731189,count=2 id_731190,count=1 id_731191,count=3 id_731192,count=1 id_731193,count=2 id_731194,count=1 id_731195,count=2 id_731196,count=1 id_731197,count=1 id_731198,count=11 id_731199,count=4 id_731200,count=9 id_731201,count=13 id=623529,count=1 id=623558,count=2 id=623559,count=1 id=623560,count=1 id=623561,count=2 id=623562,count=1 id=623563,count=1 id=623564,count=1 id=623565,count=1 id=623566,count=1 id=623567,count=1 id=623568,count=1 id=623569,count=2 id=623570,count=2 id=623571,count=1
51. serf2 ASO treatment of A549# 51 128bp 105bp ASO1 ASO2 cLNA 100nM 25nM 5nM MQ con 500 100 300 700 50 400 200 150 75 u2surp 35x cLNA MQ con serf2 35x 2 3 2 3
52. sirt2 52 • id452110 (not in a table) • chr19:39388948_39389726 Scale chr19: 500 bases hg19 39,388,800 39,388,900 39,389,000 39,389,100 39,389,200 39,389,300 39,389,400 39,389,500 39,389,600 39,389,700 39,389,800 PCCR dG <= -20 kcal/mol icSHAPE in Hek chromatin RIC-seq UCSC Genes (RefSeq, GenBank, CCDS, Rfam, tRNAs & Comparative Genomics) 100 vertebrates conservation by PhastCons 100 vertebrates Basewise Conservation by PhyloP loop out eCLIP self peaks RNA bridges eCLIP self peaks Transfer RNA Genes Identified with tRNAscan-SE HeLa HepG2 id=452105,dG=-22.3,qval=0.47 id=452110,dG=-25.6 Cluster_1275722;r=3 Cluster_1601104;r=7 Cluster_1249856;r=5 Cluster_183402;r=3 SIRT2 SIRT2 SIRT2 SIRT2 id_349969;count=2 id_349970;count=1 id_349971;count=2 id_349972;count=2 id_385671;count=2 id_385672;count=1 id_385673;count=1 id_349973;count=1 id_349974;count=1 id_385674;count=2 id_385675;count=2 id_349975;count=1 id_349976;count=1 id_349977;count=2 id_349978;count=2 id_245932;count=1 id_245933;count=1 id_245934;count=1 id_273147;count=1 id_273148;count=1 id_245935;count=1 id_245936;count=1 id_245937;count=1 id_245938;count=1 id_181160;count=1 id_173354;count=1 id_245939;count=2 icSHAPE 100 Vert. Cons
53. sirt2 ASO treatment of A549# 53 1 2 3_4 1 3_4 183bp 230bp A549 P1 ASO2 ASO1 100nM 25nM 5nM cLNA 100nM 25nM 5nM MQ con 100 300 700 50 150 sirt2
54. u2surp 54 Scale chr3: RIC-seq 500 bases hg19 142,743,000 142,743,500 142,744,000 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 Transfer RNA Genes Identified with tRNAscan-SE HeLa HepG2 id=620943,dG=-22.7,qval=0.12 id=620971,dG=-21.2 id=620973,dG=-20.8 id=620991,dG=-22.8,qval=0 id=620994,dG=-24.8,qval=0.15 id=621002,dG=-25.6 id=621006,dG=-25.5,qval=0.77 U2SURP U2SURP U2SURP U2SURP id_469068;count=2 id_516886;count=2 id_469069;count=2 id_516887;count=6 id_516888;count=2 id_516889;count=2 id_516890;count=2 id_469070;count=1 id_516891;count=3 id_367700;count=1 id_367701;count=2 id_367702;count=5 id_331181;count=1 id_367703;count=2 id_367704;count=2 id_331182;count=1 id_331183;count=2 id_233439;count=1 id_244399;count=2 id_233440;count=1 id_367705;count=2 id_331184;count=1 id_331185;count=4 id_367706;count=4 id_331186;count=2 id_331187;count=2 id_331188;count=1 id_244400;count=4 id_233441;count=1 icSHAPE • id620991 • chr3: 142742930_142743512
55. u2surp ASO.1 treatment of A549# 55 A549 P1 ASO2 ASO1 100nM 25nM 5nM cLNA 100nM 25nM 5nM MQ con 500 100 300 700 50 400 200 150 75 u2surp_1 35x 175bp 110bp 13 15
56. u2surp ASO.2 treatment of A549# 56 175bp 110bp 13 15 A549 P1 ASO1 100nM 25nM 5nM cLNA MQ con 100 300 700 50 200 u2surp_2 35x
57. u2surp ASO.1+2 treatment of A549# 57 175bp 110bp 13 15 A549 ASO1+1 cLNA MQ con u2surp 75 35 50 100 150 200 300 25 ASO2+2
58. RBP genes (from 20.06.22) 58
59. srrm4 siRNA treatment of IMR-32#.p1.p2.p3 59 0.00 0.25 0.50 0.75 1.00 1.25 con luc sirna1_10 sirna1_100 treatment fold_change p1 p2 p3 srrm4 fold_change
60. ilf2 siRNA treatment of A549#.p1+p3 60 0.00 0.25 0.50 0.75 1.00 1.25 con luc sirna1_10 sirna1_100 sirna2_10 sirna2_100 treatment fold_change p1 p3 ilf2 fold_change
61. qki siRNA treatment of A549#. p1+p3 61 0.00 0.25 0.50 0.75 1.00 1.25 con luc sirna1_10 sirna1_100 sirna2_10 sirna2_100 treatment fold_change p1 p3 qki fold_change
62. Ptbp1 siRNA treatment of A549# 62 0.0 0.5 1.0 1.5 con con_chx luc luc_chx sirna_10 sirna_100 sirna_100_chx treatment ddct p1 p3 p4 p5 ptbp1
63. Ptbp1 siRNA treatment of A549# 63 ptbp1_antibody siRNA_CHX con luc siRNA con_CHX luc_CHX
64. Acsf3 under ptbp1 knockdown 64 • no sheer effect of knockdown on electrophoresis 75 100 150 200 300 acsf3 254+157bp siRNA_CHX con luc 50 siRNA con_CHX luc_CHX MQ 6 6 7 8 8 6 8 6 PTBP1 8 7
65. Acsf3 under ptbp1 knockdown 65 0.01 0.02 0.03 con luc sirna_10 sirna_100 con_chx luc_chx sirna_100_chx treatment per_in p1 p4 acsf3 Inclusion_percent 6 8 6 PTBP1 8 7
66. Rnpep under ptbp1 knockdown 66 rnpep 341+192bp 1_100nM 1_10nM con luc MQ 75 100 150 200 300 50 75 100 150 200 300 iqgap1 rnpep 341+192bp 153+105bp 1_100nM 1_10nM con luc 50 MQ 1_100nM 1_10nM con luc MQ • no sheer effect of knockdown on electrophoresis • primer annealing temperature adjustment
67. Dclk2 under ptbp1 knockdown 67 75 100 150 200 300 dclk2 219+123bp siRNA_CHX con luc 50 siRNA con_CHX luc_CHX MQ 15 17 15 16 PTBP1 • dclk2 gene is expressed in hippocampus where ptbp1 is inhibited 15 17 15 16
68. Dclk2 under ptbp1 knockdown 68 15 17 15 16 PTBP1 0.00 0.25 0.50 0.75 1.00 con con_chx luc luc_chx sirna_100 sirna_100_chx treatment per_in p1 p3 p4 p5 dclk2 Inclusion_percent
69. Iqgap1 under ptbp1 knockdown 69 75 100 150 200 300 iqgap1 153+105bp siRNA_CHX con luc 50 siRNA con_CHX luc_CHX MQ 28 30 28 29 PTBP1 • The expression of iqgap1 is maintained at low levels in healthy brain tissues, but in brain tumors it is increased 28 28 29 30
70. Iqgap1 under ptbp1 knockdown 70 28 30 28 29 PTBP1 0.00 0.05 0.10 0.15 con luc sirna_10 sirna_100 con_chx luc_chx sirna_100_chx treatment per_in p1 p4 p5 p6 iqgap1 Inclusion_percent
71. Sf3b4, Sf3b1 siRNA treatment of A549# 71 75 100 150 200 300 50 brd2 sf3b4.siRNA 100nM sf3b4.siRNA 10nM luc con
72. Sf3b4, Sf3b1 siRNA treatment of A549# 72 brd2 sf3b1.siRNA1 10nM sf3b1.siRNA1 100nM sf3b1.siRNA2 10nM 75 100 150 200 300 50
73. plans 73 • qPCR for acsf3 & rnpep • RT-PCR for iqgap1 with CHX • rnpep & mrrf – primer annealing temperature adjustment • acsf3 & carpin2 – primers ordered • cuta – primers designed