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Mapping the transcriptional landscape of the budding yeast

Mapping the transcriptional landscape of the budding yeast. Lior David Stanford Genome Technology Center, Department of Biochemistry Stanford University School of Medicine. Contributors. Wolfgang Huber group. Ron Davis group. Lars Steinmetz group. Joern Toedling Lee Bofkin. Curtis Palm

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Mapping the transcriptional landscape of the budding yeast

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  1. Mapping the transcriptional landscape of the budding yeast Lior David Stanford Genome Technology Center, Department of Biochemistry Stanford University School of Medicine

  2. Contributors Wolfgang Huber group Ron Davis group Lars Steinmetz group Joern Toedling Lee Bofkin Curtis Palm Ted Jones Marina Granovskaia Raquel Kuehn Michelle Nguyen

  3. Genechip S. cerevisiae Tiling Array 4 bp tile over the complete genome Designed by Lars and Ron; Manufactured by Affymetrix

  4. cDNA samples preparation Rich Media (YPD) Total RNA Poly(A) RNA Exponential growth First strand cDNA Genomic DNA Hybridization

  5. Probe signal normalization

  6. Making sense of probe signals Along-chromosome plots for each strand (available in a database) http://www.ebi.ac.uk/huber-srv/queryGene/index.html

  7. How much is transcribed? Saccharomyces Genome Database (SGD) http://www.yeastgenome.org 5654 ORFs Verified or uncharacterized Expressed above background in rich media 5104 (90%) Binomial test, FDR = 0.001 Genes that are not transcribed function in: Meiosis, sporulation, mating, sugar transport, vitamin metabolism (Gene ontology enrichment, P < 3 x 10-9)

  8. A closer look Unprecedented, strand specific resolution Splicing High accuracy UTRs

  9. UTR mapping UTR lengths for2044 ORFs 68 nucleotides median On average 3’ UTRs are longer than 5’ UTRs No correlation between 3’ and 5’ lengths 91 nucleotides median

  10. GO categories with exceptional UTR length Ontology Biological process Molecular function Cellular component UTR length correlates with function and localization UTR length correlates with possible need for regulation

  11. Transcriptional architectures 921 ORFs were divided into at least two segments MET7 - folylpolyglutamate synthetase, catalyzes extension of the glutamate chains of the folate coenzymes

  12. Transcription over active promoters Several hundreds of adjacent transcripts cases SER3 3-phosphoglycerate dehydrogenase, catalyzes the first step in serine and glycine biosynthesis SRG1 - SER3 regulatory gene Martens et al. Nature 429 (2004)

  13. Categorization of segments All segments Segments overlapping annotation New transcription Isolated Antisense

  14. Non-annotated Isolated transcription 1.7 kb in size Highly conserved Not likely to code for a long protein 126 confirmed by RT-PCR in both total and poly(A) cDNA samples

  15. Conservation of novel isolated Across four yeast species (S. cerevisiae, S. paradoxus, S. mikatae and S. bayanus ) Little sequence conservation Lack of protein coding signature However, lack of conservation does not mean lack of function!

  16. Antisense transcription SPO22 Meiosis-specific protein CBF1 (TF) Chromosome cycle, important for growth in rich media GO of Genes with antisense Cell wall, transcriptional regulation, meiotic cell cycle……

  17. Antisense and UTR length 3’ UTRs have more antisense than 5’ UTRs UTRs with antisense are longer than UTRs without Similar correlations for miRNAs in Drosophila SAGE tags, indirect experimental evidence

  18. Database http://www.ebi.ac.uk/huber-srv/queryGene/index.html Analyses package – “davidTiling” http://www.bioconductor.org/download/experiments/

  19. The question Transcription in eukaryotes goes far beyond protein coding genes Transcription in yeast – Where? At what level? What is the nature of transcribed regions?

  20. Probe signal normalization RNA intensity of probe i on array j Normalized RNA signal Geometric mean of DNA hybridization intensity of probe i

  21. How much is transcribed? 2. Per base pair analysis 11,412,997 bp Unique genomic sequence 75.2% Base pairs currently annotated on either strand (SGD) 84.5% Base pairs detected as transcribed by the array (YPD only) Newly detected transcription From the transcribed base pairs only 16.2%

  22. YCK2 GIM3 PCR product Bi-cistronic transcripts Several segments contained ORFs of more than one protein-coding gene YCK2 casein kinase I, involved in cytokinesis GIM3 tubulin binding, involved in microtubule biogenesis

  23. Categorization of segments All segments Segments overlapping annotation New transcription Isolated Antisense

  24. Length and expression levels of segments

  25. Polyadenylated and non-polyadenylated Many ncRNAs were captured in a poly(A) oligo-dT sample

  26. Deletions of novel isolated transcripts 47 isolated regions Phenotyping in YPD Three strains show a growth defect Contains TF-bs for ACT1, an essential gene

  27. Deletions of novel isolated transcript

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