DECODING OF EXON SPLICING PATTERNS IN THE HUMAN RUNX1-RUNX1T1 FUSION GENE Vasily V. Grinev Associate Professor Department of Genetics Faculty of Biology Belarusian State University Minsk, Republic of Belarus

MOLECULAR ANATOMY OF THE TRANSLOCATION t(8;21)(q22;q22) RUNX1T1 gene structure chromosome 8 chromosome 21 RUNX1 gene structure Chromosome 8 and chromosome 21 are partners in non-homological reciprocal translocation t(8;21)(q22;q22)

MOLECULAR ANATOMY OF THE TRANSLOCATION t(8;21)(q22;q22) der 8 der 21 RUNX1-RUNX1T1 fusion gene structure The main outcome of the translocation t(8;21)(q22;q22) is fusion gene RUNX1-RUNX1T1 Domains and key interaction partners of the fusion protein RUNX1-RUNX1T1

DIVERSITY OF RNA PRODUCTS OF THE FUSION GENE RUNX1-RUNX1T1 Exon graph-based model of organization of the fusion gene RUNX1-RUNX1T1 For fusion gene RUNX1-RUNX1T1, there is a lot of identified transcripts: 135 full-length RNA products 63 expressed sequence tags

POWER-LAW BEHAVIOR OF THE LOCAL COMBINATORICS OF THE RUNX1–RUNX1T1 EXONS ECI (exon combinatorial index) is a quantitative measure of local exon combinatorics and it means a number of unique splicing events that involve an exon. List of competitive statistical models: 1) power-law distribution; 2) truncated power-law distribution with an exponential cut-off; 3) exponential distribution; 4) log-normal distribution; 5) Yule-Simon distribution; 6) stretched exponential distribution (complementary cumulative Weibull distribution); 7) Poison distribution. Set of exons of the fusion gene RUNX1-RUNX1T1 empirical data ―power-law ―truncated power-law ―exponential ―log-normal ―Yule-Simon ―stretched exponential ―Poison Set of exons of the whole human transcriptome

SEQUENCE FEATURES OF THE EXONS AND FLANKING INTRONS Some example of sequence features of flanking introns Genomic elements for extraction of sequence features

SEQUENCE FEATURES OF THE EXONS AND FLANKING INTRONS Some example of sequence features of exons

DIFFERENT “ATTRACTIVENESS” OF EXONS FOR ALTERNATIVE SPLICING IS ASSOCIATED WITH SEQUENCE-RELATED FEATURES Sequence features are not equal in importance for the prediction of the ECI value Compendium of the sequence features permits to predict the values of the ECI by regression random forests with a high accuracy A complex relationship between the sequence features and the ECI value

GENES OF SPLICING FACTORS DIFFERENTIALLY EXPRESSED IN t(8;21)-POSITIVE AML BLASTS The RBFOX3 gene is not expressed or expressed under the threshold of detection by RT-PCR in normal hematopoietic cells but this gene is expressed in leukemia cells There is a significant (according to Mann–Whitney U test) differential expression of the splicing factors genes in leukemia cells in comparison with normal hematopoietic cells The lanes on the upper electrophoregram: Fermentas GeneRuler TM 100 bp DNA Ladder Plus (1), amplification of cDNA of the TBP gene from Kasumi-1 cells (2), amplification of cDNA of the RBFOX3 gene from normal PBMNC (3, 5), BMMNC (7, 9), CD34+HPSC(11, 13) and from Kasumi-1 cells (15) and amplification of cDNA of the RBFOX3 gene from respective RT−negative controls (4, 6, 8, 10, 12, 14, 16). The lanes on the bottom electrophoregram: Fermentas GeneRuler TM 100 bp DNA Ladder Plus (1), amplification of cDNA of the RBFOX3 gene from the bone marrow samples of nine children with t(8;21)-positive AML (2, 4, 6, 8, 10, 12, 14, 16, 18) and respective RT−negative controls (3, 5, 7, 9, 11, 13, 15, 17, 19).

ACTIVITY OF THE NMD SYSTEM IN CHILDREN t(8;21)-POSITIVE AML CELLS IS DEREGULATED Expression of NMD genes is significantly increased or decreased in leukemia cells in comparison with normal hematopoietic cells

EXONS WITH HIGH ECI VALUES ARE “HOT POINTS” OF THE RUNX1–RUNX1T1 MRNA SPLICING In silico modeling supports a strong sensitivity of splicing of RUNX1–RUNX1T1 transcripts to skipping of exons with high ECI values (A) Skipping of exons that were listed in the descending order of their ECI values: experimentally verified transcripts (on the top), predicted transcripts (on the bottom). (B) This picture is similar to (A), but exons were excluded from splicing process in the ascending order of values of their ECI. Legend: — diversity of transcripts — average size (in number of exons) of transcripts — average length (in number of nucleotides) of transcripts — average length of ORF — portion of transcripts containing PTC

MORE DETAILS IN:

Tatiana V. Ramanouskaya Alina V. Vaitsiankova Ilia M. Ilyushonak Alexandr A. Migas Olga A. Mishkova Olga V. Aleinikova Petr V. Nazarov Laurent Vallar Aksana D. Kirsanava Natalia Jr. Siomava MANY THANKS TO THE MEMBERS OF OUR TEAM:

THANK YOU FOR ATTENTION! HETEROZYGOATS Just allele uneven