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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
For fusion gene RUNX1-RUNX1T1,
there is a lot of identified transcripts:
135 full-length RNA products
63 expressed sequence tags
Exon graph-based model of organization of the fusion gene RUNX1-RUNX1T1
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.
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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
Genomic elements for extraction of sequence features
Some example of sequence features of flanking introns
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
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).
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
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
(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
In silico modeling supports a strong sensitivity
of splicing of RUNX1–RUNX1T1 transcripts to
skipping of exons with high ECI values
MORE DETAILS IN:
MANY THANKS TO THE MEMBERS OF OUR TEAM:
Alexandr A. Migas
Olga A. Mishkova
Olga V. Aleinikova
Petr V. Nazarov
Laurent Vallar
Tatiana V. Ramanouskaya
Alina V. Vaitsiankova
Ilia M. Ilyushonak
Natalia Jr. Siomava
Aksana D. Kirsanava
THANK YOU FOR ATTENTION!
HETEROZYGOATS
Just allele uneven