DECODING OF EXON SPLICING

PATTERNS IN THE HUMAN RUNX1-RUNX1T1 FUSION GENE

Vasily V. GrinevAssociate ProfessorDepartment 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 partnersin 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 PRODUCTSOF 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 products63 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 exonsof the fusion gene RUNX1-RUNX1T1

empirical data― power-law― truncated power-law― exponential― log-normal― Yule-Simon― stretched exponential― Poison 

Set of exonsof 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 equalin importance for the prediction

of the ECI value

Compendium of the sequence features permits to predict the values of the ECI

by regression random forestswith a high accuracy

A complex relationship betweenthe sequence features

and the ECI value

GENES OF SPLICING FACTORS DIFFERENTIALLY EXPRESSED IN t(8;21)-POSITIVE AML BLASTS

The RBFOX3 gene is not expressedor 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 GeneRulerTM 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 GeneRulerTM 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 sensitivityof 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. RamanouskayaAlina V. VaitsiankovaIlia M. Ilyushonak

Alexandr A. MigasOlga A. MishkovaOlga V. Aleinikova

Petr V. NazarovLaurent Vallar

Aksana D. Kirsanava

Natalia Jr. Siomava

MANY THANKS TO THE MEMBERS OF OUR TEAM:

THANK YOU FOR ATTENTION!

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