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The cause and consequence of alternative splicing in maize and across species
- 1. The cause and consequence of alternative splicing in maize and across species Wenbin Mei University of California, Davis CGM July 5, 2017
- 2. Acknowledgement University of Florida Committee: Dr. Brad Barbazuk (PhD Advisor) Dr. Pam Soltis Dr. Douglas Soltis Dr. Sixue Chen Dr. Mark Settles Funding Support: Alumni fellowship, UF CLAS Dissertation Fellowship Department of Biology, UF GSC, UF Iowa State University Dr. Patrick Schnable Kansas State University Dr. Sanzhen Liu University of Nebraska Dr. James Schnable University of Minnesota Twin Cities Dr. Nathan Springer Dr. Steven Eichten University of Georgia Dr. Bob Schmitz Dr. Chad Niederhuth Dr. Barbazuk’s lab: Ruth Davenport Lucas Boatwright Guanqiao Feng Brandon Walts Christy Gault Jason Brant Nathan Catlin Jerald Noble Leandro Gomide Srikar Chamala Stela Palii Tales Sidronio Oakland University Dr. Shailesh Lai Maize genetics at UF Dr. Karen Koch Dr. Donald McCarty
- 3. What is alternative splicing ? DNA Pre-mRNA mRNAs Proteins
- 4. A) B) What is alternative splicing ? Keren et al. 2010. Nature Review Genetics Syed et al. 2012. Trend in Plant Science
- 5. How do we define differential expression of isoforms. 1 2 3 4 For example: 3 possible isoforms: I. all exons used (1-4) II. Exon 3 skipped III. All exons used and intron between 2 and 3 retained Expression in Sample 1 Expression in Sample 2 DE isoform ? Scenario 1 Isoform I 2 0 YES (relative isoform ratios different) Isoform II 4 1 Isoform III 3 9 Scenario 2 Isoform I 2 4 NO (relative isoform ratios equal) Isoform II 3 6 Isoform III 4 8 Scenario 3 Isoform I 2 0 YES (relative isoform ratios different) Isoform II 3 6 Isoform III 0 3
- 6. Why alternative splicing is important ? • In animals Sex determination Heart Development CancerMyotonic dystrophy Nature Review Genetics 2011 Genome Medicine 2015 Science 2015 Nature Communication 2016
- 7. Why alternative splicing is important ? • In plants Eckardt. 2002. Plant Cell Staiger and Brown. 2013. Plant Cell
- 8. Why alternative splicing is important ? • In adaptation Mallarino et al. 2017 Molecular Ecology
- 9. Questions qGenetic Regulation ? qConserved Function ? A Comprehensive Analysis Of Alternative Splicing In Paleopolyploid Maize Mei W, Liu S et al. Frontiers in Plant Science. 2017 Mei W et al. Conserved Alternative Splicing Across Monocots. 2017 bioaxiv doi: https://doi.org/10.1101/120469
- 10. Genetic Regulation ? A Comprehensive Analysis Of Alternative Splicing In Paleopolyploid Maize
- 11. Background about maize Springer and Stupar. 2007. Genome Research Schnable et al. 2009. Science Candela and Hake 2008. Nature Review Genetics
- 12. 0 100000 200000 300000 400000 500000 600000 700000 800000 900000 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 Know Splice Junction Novel Splice Junction (FGS) Difficulty to assemble the isoforms using short reads Mei et al. Unpublished Data
- 13. Short Reads Transcriptome Assembly Strategy PASA alt splicing combine data from different source RNA-Seq data Cufflinks assembly Trinity genome guided assembly StringTie assembly EST data Cleaning 1) Filtering Splice Junction Entropy Score >= 2 2) Intron retention ratio >= 10% 3) Intron retention coverage >= 90% 4) Fraction isoform ratio >= 5% at least in one tissue 5) FPKM value >= 1 at least in one tissue Filtering Isoforms from different perspectives Cleaned alt splicing isoforms and alt splicing events Mei et al. 2017. Frontiers in Plant Science
- 14. Pipeline identify new isoforms in maize W22 genome MAKER-P My pipeline Mei et al. Unpublished Data
- 15. AS in maize and sorghum Mei et al. 2017. Frontiers in Plant Science
- 16. LSC error correction GMAP with 90% length aligned and 95% identity Clustering 2,480 isoforms 2,480 isoforms 8,862 splice junction 8,850 (99.8%) Exist in RNA-Seq splice junction 8,766 (98.9%) assemble into transcripts 2,480 isoforms 1,052 isoforms were identical to our assembled transcripts 1,307 isoforms were contained in our transcripts 120 potentially novel isoforms 2,360 (95.2%) isoforms were within or identical to our transcripts Validation AS isoforms based on long reads Mei et al. 2017. Frontiers in Plant Science
- 17. Parameters to compare AS across tissues Intron Retention Ratio = ΣFPKM IR Isoform/ (ΣFPKM IR Isoform + ΣFPKM Intron Skip Isoform) Percentage Splice In (PSI) = ΣFPKM Splice In/ (ΣFPKM Splice In+ ΣFPKM Exon Skip) Mei et al. 2017. Frontiers in Plant Science
- 18. Leaf Seed Seed Leaf Root Root Seedling Endosperm Embryo Embryo Endosperm Ear Tassel Seedling Ear Tassel Sam Sam Amount of Data # of differential splicing loci Splicing is different from gene expression Mei et al. 2017. Frontiers in Plant Science
- 19. Dynamics alternative splicing during seed development Mei et al. 2017. Frontiers in Plant Science
- 20. Stress response alternative splicing Mei et al. 2017. Frontiers in Plant Science
- 21. Intron retention in the 3’ UTR region 414 571631 differential splicing genes Cold vs. control Heat vs. control zm-SC30 Mei et al. 2017. Frontiers in Plant Science
- 22. Chapter 1: Identifying AS different between B73 and Mo17 Mei et al. 2017. Frontiers in Plant Science
- 23. cis-geno = B73 Intron cis-geno = Mo17 Example of cis-regulatory Splicing (2 loci) Mei et al. 2017. Frontiers in Plant Science
- 24. Example of trans-regulatory Splicing (2 loci) Mei et al. 2017. Frontiers in Plant Science
- 25. binomial model with overdispersion control in glm full model: c(not_intron, intron) ~ cis + trans + cis * trans Method to detect sQTL Mei et al. 2017. Frontiers in Plant Science
- 26. At least 35% splice junction regulated in cis- Mei et al. 2017. Frontiers in Plant Science
- 27. trans- sQTL and allelic interaction between marker and splice junction Mei et al. 2017. Frontiers in Plant Science
- 28. Chapter 1: Comparison of AS in maize subgenomes and their orthologues in sorghum Mei et al. 2017. Frontiers in Plant Science
- 29. Conserved Function ? Conserved Alternative Splicing Across Monocots
- 30. Clamydomonas Green Plants Physcomitrella Land Plants Vascular Plants Selaginella Gymnosperm Seed Plants Amborella Flowering Plants Arabidopsisthaliana Eudicot Monocot AfricanOilPalm Banana Maize Sorghum FoxtailMillet Grasses Brachypodium Rice γ β α σ ρ τ Mα Mβ Mγ p Tetraploidy Hexaploidy Mei et al. 2017. Bioaxiv
- 31. Strategy to detect conserved alternative splicing Species A Species B Species A Species A Species B Species B Exon Skip Intron Retention Alt Donor Alt Acceptor Alternate Terminal Exon Species A Species B A) E) C) B) D) Species A Species B 300 bp 300 bp 300 bp 300 bp 300 bp 300 bp 300 bp 300 bp 300 bp 300 bp splice anchor sequence tags (SAST) Mei et al. 2017. Bioaxiv
- 32. Strategy to detect conserved alternative splicing Species 1 Species 2 Species 3 Species 10… AS ASAS AS Intron Retention SAST Alt_acceptor SAST Alt_donor SAST Exon Skip SAST ATE SAST OrthoFinder tblastx Clustering Conserved ASMei et al. 2017. Bioaxiv
- 33. Conserved AS across angiosperm lineages AS type Total Amborella gene counts Arabidopsis gene counts African oil palm gene counts Banana gene counts Rice gene counts Brachypodium gene counts Foxtail mille gene counts Sorghum gene counts Maize gene counts AltA 6,152 444 222 197 409 867 931 1051 985 1046 AltD 2,925 207 105 72 187 427 414 473 491 549 AltTE 539 32 13 30 44 64 71 73 116 96 ExonS 1,249 116 43 68 113 180 173 163 201 192 IntronR 12,071 863 675 1019 974 1591 2238 1218 897 2596 Total (%) 19,235 1413 (18.3) 944 (12.0) 1269 (20.9) 1565 (17.4) 2579 (23.1) 3102 (33.2) 2440 (36.2) 2236 (32.7) 3687 (25.7) γ β α τ p Mα Mβ Mγ σ ρ Tetraploidy Hexaploidy Mei et al. 2017. Bioaxiv
- 34. Syntenic regions have more conserved AS 0% 25% 50% 75% 100% 0% 25% 50% 75% 100% 0% 25% 50% 75% 100% 0% 25% 50% 75% 100% 0% 25% 50% 75% 100% A) B) C) D) E) Maize Sorghum Rice Foxtail Millet Brachypodium Figure 3 Mei et al. 2017. Bioaxiv
- 35. Ka/Ks upsteam and downstream splice junction Mei et al. 2017. Bioaxiv
- 36. Conserved AS in SR protein subfamilies RS RS2Z SR A) B) Mei et al. 2017. Bioaxiv
- 37. Protein-protein network of Arabidopsis genes with evidence of conserved AS with Amborella and at least one species of monocot R2R3-MYB SR, RS, RS2Z Mei et al. 2017. Bioaxiv
- 38. Sequence conservation in RNA-binding KH domain protein with an conserved exon skipping across species Brachypodium Bradi3g56540 Amborella AmTr_v1.0_scaffold00061.77 Arabidopsis AT4G26480 African oil palm p5_sc00068.V1.gene28 Rice LOC_Os02g49080 Foxtail millet Si018005m Maize GRMZM2G029029 Sorghum Sobic.004G256600 Banana GSMUA_Achr1T16430_001 Figure 7 Mei et al. 2017. Bioaxiv
- 39. Research Questions qGenetic Regulation ? Yes, there are genetic contribution to the AS, genotype specific splicing, cis- and trans-, AS in subgenomes et al. Development and stress response are two major areas with dyanmics splicing change qConserved Function ? Yes, but not that high across species. Splicing turnover quickly. Syntenic genes enriched in conserved AS, splicing network connect transcriptional factors.