Studying the impact of secondary structure on alternative splicing in the MARK2 and HMGXB4 genes.

Transcript

1. Studying the impact of secondary structure on alternative splicing in the MARK2 and HMGXB4 genes. 1 Petrova Marina 18 Oct. 2023
2. MARK2 ASO treatment of A549# 2 164bp 137bp 16 17 18 16 18 0.0 0.1 0.2 0.3 0.4 0.5 con clna 5nM 25nM 100nM psi mark2 ASO1 0.0 0.1 0.2 0.3 0.4 0.5 con clna 5nM 25nM 100nM psi mark2 ASO2
3. MARK2 mutagenesis scheme 1 3 GUCUCGCUGAGCGGCU CGGGGCGACUCGCCGA wt m1 m2 m1m2 CAGAGCGACUCGCCGA CGGGGCGACUCGCCGA GUCUCGCUGAGCGGCU GCCCCGCUGAGCGGCU CAGAGCGACUCGCCGA GCCCCGCUGAGCGGCU
4. MARK2 minigene transfection of A549# 4 0.0 0.1 0.2 0.3 wt m1 m2 m1m2 psi psi Mark2 Quantitative (top) and qualitative (bottom) assessment of m1, m2, and m1m2 mutants using qRT-PCR and RT-PCR.
5. MARK2 mutagenesis scheme 2 5 GUCUCGCUGAGCGGCU CGGGGCGACUCGCCGA wt m1 m2 m1m2 GAGAGCGACUCGCCGU CGGGGCGACUCGCCGA GUCUCGCUGAGCGGCU CCCCCGCUGAGCGGCA GAGAGCGACUCGCCGU CCCCCGCUGAGCGGCA
6. MARK2 mutagenesis scheme 3 6 GUCUCGCUGAGCGGCU CGGGGCGACUCGCCGA wt m1 m2 m1m2 GUGUCGCUCACCGGGU CGGGGCGACUCGCCGA GUCUCGCUGAGCGGCU CGCGGCGAGUGGCCCA GUGUCGCUCACCGGGU CGCGGCGAGUGGCCCA
7. HMGXB4 7 HMGXB4 is a member of the HMG protein family, known for its involvement in DNA processes such as transcription, DNA repair, and chromatin structure. We predicted long-distance RNA interactions within the HMGXB4 gene around Exon 2: Scale chr22: 2 kb hg19 35,654,000 35,654,500 35,655,000 35,655,500 35,656,000 35,656,500 35,657,000 35,657,500 35,658,000 35,658,500 35,659,000 PCCR dG <= -30 kcal/mol UCSC Genes (RefSeq, GenBank, CCDS, Rfam, tRNAs & Comparative Genomics) 100 vertebrates conservation by PhastCons Contacts with handles in [-100;100] from PCCR in I/O orientation 100 vertebrates Basewise Conservation by PhyloP id=567685,dG=-30.4,qval=0.55 id=567714,dG=-53.9,qval=0.59 HMGXB4 HMGXB4 HMGXB4 H1_r1_id_1256949 hNPC_r1_id_783741 H1_r1_id_1256950 H1_r1_id_1256951 H1_r2_id_1333392 GM12878_r2_id_2148308 HeLa_r1_id_529495 HeLa_r1_id_529496 neuron_r1_id_1856300 IMR90_r1_id_920377 IMR90_r1_id_920377 IMR90_r1_id_920377 K562_r1_id_1155921 100 Vert. Cons
8. HMGXB4 8 The formation of RNA secondary structure triggers exclusion of exon 2. Skipping of this exon leads to the selection of an alternative translation initiation codon in exon 5, resulting in the production of a shorter HMGXB4 protein. Exon skipping events leading to alternative translation start sites and shorter protein isoforms are relatively rare!
9. Examples of alternative initiation exons. 9 Exon skipping events leading to alternative translation start sites and shorter protein isoforms are relatively rare: One example of an isoform with alternative initiation exons in the NFATC1: NFATc1-α – short isoform induced upon T-cell activation. Short isoform may have distinct localization patterns and functions.
10. HMGXB4 ASO treatment of A549# 10 300bp 200bp NT C 5 25 100 AON1, nM neg 1 2 3 1 3 300bp 200bp NT C 5 25 100 AON2, nM neg 1 2 3 1 3
11. 0.5 0.6 0.7 0.8 0.9 0 Ctrl 5 25 100 psi ASO2 0.5 0.6 0.7 0.8 0.9 0 Ctrl 5 25 100 psi ASO1 HMGXB4 ASO treatment of HEK293T# 11 The response of HMGXB4 exon 2 splicing to the addition of ASO1 and AON2. 0 (non- treated control); C (control AON); 5, 25, and 100 are ASO1 and AON2 concentrations (nM).
12. HMGXB4 mutagenesis 12 HMGXB4 minigene mutagenesis scheme wt m1 m2 m1m2 AGCUGGCAGGGGCUGGGAG ACGACG CUC GGUU CCU UU UCGACCG UGC UGC UCGACCGUCCCCGACCCUCACGACG UCGACCGUUCCUGGUUCUCACGACG AGCUGGCA UGCUGC AGCUGGCAAGGACCAAGAGUGCUGC AG G G G AG CU G G G G UCGACCGUCCCCGACCCUCACGACG AGCUGGCAAGGACCAAGAGUGCUGC
13. HMGXB4 minigene transfection of A549# 13 Quantitative assessment of m1 and m1m2 mutants using qRT-PCR. The m1 mutation destabilized the structure, leading to an increase in exon 2 inclusion ratio. 0.80 0.85 0.90 0.95 1.00 wt m1 m1m2 psi HMGXB4