Studying Alternative Splicing
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1 Studying Alternative Splicing Meelis Kull PhD student in the University of Tartu supervisor: Jaak Vilo CS Theory Days Rõuge 27
2 Overview Alternative splicing Its biological function Studying splicing Technology Computational tasks Tissue-specific alternative splicing Jan 27, 27 CS Theory Days, Rõuge 27 2
3 The Basis of Life on Earth Jan 27, 27 CS Theory Days, Rõuge 27 3
4 Folded DNA Unfolding DNA Transcription pre-mrna Splicing mrna Translation Poly-peptide Protein folding Protein RNA Splicing Jan 27, 27 CS Theory Days, Rõuge 27 4
5 Splicing..GUAUCGUGGAAGGACUCAUGGUAUGAGAGCUGGGGAAUG....GGACUGAGGCUCCCACCUUUCUCAUCCAAGACUGGCUCC....UCCCUGCCGGGGCUGCGUGCAACCCUGGGGUUGGGGGUU....CUGGGGACUGGCUUUCCCAUAAUUUCCUUUCAAGGUGGG....GAGGGAGGUAGAGGGGUGAUGUGGGGAGUACGCUGCAGG....GCCUCACUCCUUUUGCAGACCACAGUCCAUGCCAUCACU.. Jan 27, 27 CS Theory Days, Rõuge 27 5
6 Splicing..GUAUCGUGGAAGGACUCAUGGUAUGAGAGCUGGGGAAUG....GGACUGAGGCUCCCACCUUUCUCAUCCAAGACUGGCUCC....UCCCUGCCGGGGCUGCGUGCAACCCUGGGGUUGGGGGUU....CUGGGGACUGGCUUUCCCAUAAUUUCCUUUCAAGGUGGG....GAGGGAGGUAGAGGGGUGAUGUGGGGAGUACGCUGCAGG....GCCUCACUCCUUUUGCAGACCACAGUCCAUGCCAUCACU.. (A C)(A U)GGU(A G)(A U)G(A U) (C U)(A C G U)(C U)U(A C G)A(C U){-2}(A G)(C U)AG Jan 27, 27 CS Theory Days, Rõuge 27 6
7 EXON Splicing..GUAUCGUGGAAGGACUCAUGGUAUGAGAGCUGGGGAAUG....GGACUGAGGCUCCCACCUUUCUCAUCCAAGACUGGCUCC....UCCCUGCCGGGGCUGCGUGCAACCCUGGGGUUGGGGGUU.. INTRON..CUGGGGACUGGCUUUCCCAUAAUUUCCUUUCAAGGUGGG....GAGGGAGGUAGAGGGGUGAUGUGGGGAGUACGCUGCAGG.. I..GCCUCACUCCUUUUGCAGACCACAGUCCAUGCCAUCACU.. I EXON Jan 27, 27 CS Theory Days, Rõuge 27 7
8 Splicing..GUAUCGUGGAAGGACUCAUGGUAUGAGAGCUGGGGAAUG....GGACUGAGGCUCCCACCUUUCUCAUCCAAGACUGGCUCC....UCCCUGCCGGGGCUGCGUGCAACCCUGGGGUUGGGGGUU.. INTRON..CUGGGGACUGGCUUUCCCAUAAUUUCCUUUCAAGGUGGG....GAGGGAGGUAGAGGGGUGAUGUGGGGAGUACGCUGCAGG....GCCUCACUCCUUUUGCAGACCACAGUCCAUGCCAUCACU.. EXON I EXON.GUAUCGUGGAAGGACUCAUGACCACAGUCCAUGCCAUCACU. Jan 27, 27 CS Theory Days, Rõuge 27 8
9 Splicing Jan 27, 27 CS Theory Days, Rõuge 27 9
10 Alternative Splicing..GUAUCGUGGAAGGACUCAUGGUAUGAGAGCUGGGGAAUG....GGACUGAGGCUCCCACCUUUCUCAUCCAAGACUGGCUCC....UCCCUGCCGGGGCUGCGUGCAACCCUGGGGUUGGGGGUU....CUGGGGACUGGCUUUCCCAUAAUUUCCUUUCAAGGUGGG....GAGGGAGGUAGAGGGGUGAUGUGGGGAGUACGCUGCAGG....GCCUCACUCCUUUUGCAGACCACAGUCCAUGCCAUCACU.. Jan 27, 27 CS Theory Days, Rõuge 27
11 Alternative Splicing..GUAUCGUGGAAGGACUCAUGGUAUGAGAGCUGGGGAAUG....GGACUGAGGCUCCCACCUUUCUCAUCCAAGACUGGCUCC....UCCCUGCCGGGGCUGCGUGCAACCCUGGGGUUGGGGGUU....CUGGGGACUGGCUUUCCCAUAAUUUCCUUUCAAGGUGGG....GAGGGAGGUAGAGGGGUGAUGUGGGGAGUACGCUGCAGG....GCCUCACUCCUUUUGCAGACCACAGUCCAUGCCAUCACU.. Jan 27, 27 CS Theory Days, Rõuge 27
12 Alternative Splicing..GUAUCGUGGAAGGACUCAUGGUAUGAGAGCUGGGGAAUG....GGACUGAGGCUCCCACCUUUCUCAUCCAAGACUGGCUCC.. INTRON..UCCCUGCCGGGGCUGCGUGCAACCCUGGGGUUGGGGGUU....CUGGGGACUGGCUUUCCCAUAAUUUCCUUUCAAGGUGGG....GAGGGAGGUAGAGGGGUGAUGUGGGGAGUACGCUGCAGG.. EXON I EXON.GUAUCGUGGAAGGACUCAUGGUGGGGAGGGAGGUAGAGGGG..UGAUGUGGGGAGUACGCUGCAGGGCCUCACUCCUUUUGCAG. Jan 27, 27 CS Theory Days, Rõuge 27 2
13 Alternative Splicing Jan 27, 27 CS Theory Days, Rõuge 27 3
14 Alternative Splicing Jan 27, 27 CS Theory Days, Rõuge 27 4
15 Overview Alternative splicing Its biological function Studying splicing Technology Computational tasks Tissue-specific alternative splicing Jan 27, 27 CS Theory Days, Rõuge 27 5
16 Origins of Splicing Prokaryotes - splicing occurs but there is no general machinery for performing it Eukaryotes - have got spliceosome - a protein complex specialised in carrying out splicing Jan 27, 27 CS Theory Days, Rõuge 27 6
17 Jan 27, 27 CS Theory Days, Rõuge 27 7
18 Biological Function of Alternative Splicing Protein Diversity - AS enables the cell to produce more than one protein from the same gene (no need for larger genome) Fast Protein Evolution - new splicing variants emerge by replacing only a few nucleotides (DNA letters). Jan 27, 27 CS Theory Days, Rõuge 27 8
19 Alternative Splicing in Action There can be different splice variants in Embryos and Adults (development) Brain and Muscle (anatomy) Normal and Cancer (pathology) Studying alternative splicing helps to decrypt anatomy, development, and pathology Jan 27, 27 CS Theory Days, Rõuge 27 9
20 Regulation of Alternative Splicing Neurons Non-neuronal cells Jan 27, 27 CS Theory Days, Rõuge 27 2
21 Our Task Computationally predict cases of anatomy-, pathology-, or developmentspecific splicing Jan 27, 27 CS Theory Days, Rõuge 27 2
22 Overview Alternative splicing Its biological function Studying splicing Technology Computational tasks Tissue-specific alternative splicing Jan 27, 27 CS Theory Days, Rõuge 27 22
23 Technology How was splicing performed in a cell? Let us find out the final result of splicing Take out molecules of spliced mrna Find out the sequences of nucleotides Compare it with the original mrna (=DNA) Scan the genome to find out the matching parts Matching parts - Exons, Regions between matches - Introns Jan 27, 27 CS Theory Days, Rõuge 27 23
24 Example (CS Theory Days, right?)..guaucguggaaggacucaugguaugagagcuggggaaug....ggacugaggcucccaccuuucucauccaagacuggcucc....ucccugccggggcugcgugcaacccugggguuggggguu....cuggggacuggcuuucccauaauuuccuuucaagguggg....gagggagguagaggggugauguggggaguacgcugcagg....gccucacuccuuuugcagaccacaguccaugccaucacu.. GGCAGGAUUCAACAGCCCAUUCGAUCCC Jan 27, 27 CS Theory Days, Rõuge 27 24
25 Example GGCAGGAUUCA..GUAUCGUGGAAGGACUCAUGGUAUGAGAGCUGGGGAAUG....GGACUGAGGCUCCCACCUUUCUCAUCCAAGACUGGCUCC....UCCCUGCCGGGGCUGCGUGCAACCCUGGGGUUGGGGGUU....CUGGGGACUGGCUUUCCCAUAAUUUCCUUUCAAGGUGGG....GAGGGAGGUAGAGGGGUGAUGUGGGGAGUACGCUGCAGG....GCCUCACUCCUUUUGCAGACCACAGUCCAUGCCAUCACU.. ACAGCCCAUUCGAUCCC GGCAGGAUUCA ACAGCCCAUUCGAUCCC - 6 mismatch Jan 27, 27 CS Theory Days, Rõuge 27 25
26 Example GGCAGGAUUCA..GUAUCGUGGAAGGACUCAUGGUAUGAGAGCUGGGGAAUG....GGACUGAGGCUCCCACCUUUCUCAUCCAAGACUGGCUCC....UCCCUGCCGGGGCUGCGUGCAACCCUGGGGUUGGGGGUU....CUGGGGACUGGCUUUCCCAUAAUUUCCUUUCAAGGUGGG....GAGGGAGGUAGAGGGGUGAUGUGGGGAGUACGCUGCAGG....GCCUCACUCCUUUUGCAGACCACAGUCCAUGCCAUCACU.. ACAGCCCAUUCGAUCCC GGCAGGAUUCA ACAGCCCAUUCGAUCCC - 6 mismatch Jan 27, 27 CS Theory Days, Rõuge 27 26
27 Example GGCAGGAUUCAAC..GUAUCGUGGAAGGACUCAUGGUAUGAGAGCUGGGGAAUG....GGACUGAGGCUCCCACCUUUCUCAUCCAAGACUGGCUCC....UCCCUGCCGGGGCUGCGUGCAACCCUGGGGUUGGGGGUU....CUGGGGACUGGCUUUCCCAUAAUUUCCUUUCAAGGUGGG....GAGGGAGGUAGAGGGGUGAUGUGGGGAGUACGCUGCAGG....GCCUCACUCCUUUUGCAGACCACAGUCCAUGCCAUCACU.. AGCCCAUUCGAUCCC GGCAGGAUUCA ACAGCCCAUUCGAUCCC - 6 mismatch GGCAGGAUUCAAC AGCCCAUUCGAUCCC - 9 mismatch Jan 27, 27 CS Theory Days, Rõuge 27 27
28 Result of Alignment Chromosome: 2 Gene: ENSG7988 (FLJ25323) Location: Mapping: g( ) g( ) g( ) g( ) g( ) e(..86) e( ) e( ) e( ) e( ) Jan 27, 27 CS Theory Days, Rõuge 27 28
29 Overview Alternative splicing Its biological function Studying splicing Technology Computational tasks Tissue-specific alternative splicing Jan 27, 27 CS Theory Days, Rõuge 27 29
30 Combine all mrna produced from the same gene Jan 27, 27 CS Theory Days, Rõuge 27 3
31 Find all splice sites Jan 27, 27 CS Theory Days, Rõuge 27 3
32 Jan 27, 27 CS Theory Days, Rõuge Encode the mrna alignments...
33 Include mrna annotation data Anatomical site: nervous system -> central nervous system -> brain -> brain stem -> medulla oblongata Developmental stage: adult -> 27 years Pathological state: normal Jan 27, 27 CS Theory Days, Rõuge 27 33
34 Find alternative splicing... dermal... normal nervous... normal dermal... cancer dermal... normal nervous... normal... cancer nervous... cancer dermal... normal... normal... cancer muscle... normal muscle... normal dermal... normal dermal... cancer dermal... normal nervous... cancer dermal... normal dermal... normal nervous... normal nervous... dermal... normal dermal... cancer nervous... normal dermal... normal nervous... dermal... normal nervous Jan 27, 27 CS Theory Days, Rõuge 27 34
35 Find alternative splicing... dermal... normal nervous... normal dermal... cancer dermal... normal nervous... normal... cancer nervous... cancer dermal... normal... normal... cancer muscle... normal muscle... normal dermal... normal dermal... cancer dermal... normal nervous... cancer dermal... normal dermal... normal nervous... normal nervous... dermal... normal dermal... cancer nervous... normal dermal... normal nervous... dermal... normal nervous Jan 27, 27 CS Theory Days, Rõuge 27 35
36 Find alternative splicing... dermal... normal nervous... normal dermal... cancer dermal... normal nervous... normal... cancer nervous... cancer dermal... normal... normal... cancer muscle... normal muscle... normal dermal... normal dermal... cancer dermal... normal nervous... cancer dermal... normal dermal... normal nervous... normal nervous... dermal... normal dermal... cancer nervous... normal dermal... normal nervous... dermal... normal nervous Intron Exon Intron Exon Intron Exon dermal Jan 27, 27 CS Theory Days, Rõuge cancer 5 2 dermal cancer 4 other 2 3 normal 8 dermal normal 5
37 Jan 27, 27 CS Theory Days, Rõuge 27 37
38 Jan 27, 27 CS Theory Days, Rõuge 27 38
39 Jan 27, 27 CS Theory Days, Rõuge 27 39
40 Summary Alternative splicing is important for protein diversity and evolution Studying splicing involves text algorithms Our contribution is a method for predicting tissue-specific alternative splicing Experiments are yet to be carried out (by others) Jan 27, 27 CS Theory Days, Rõuge 27 4
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