Maas101707 - Molecular Studies on Complex Biological...

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Unformatted text preview: Molecular Studies on Complex Biological Systems: Sources for Transcriptome and Proteome Complexity lehigh.edu SMAAS @ October 17, 2007 Dr. Stefan Maas, BioS Lehigh U. What's the molecular difference ? @ lehigh.edu SMAAS Nature 9/7/2006 What accounts for the often massive and seemingly arbitrary differences in genome size observed among eukaryotic organisms? The fruit fly Drosophila melanogaster The mountain grasshopper Podisma pedestris lehigh.edu 180 Mb 18,000 Mb SMAAS @ Is an Expansion in Gene Number driving Evolution of Higher Organisms? Vertebrata Urochordata Arthropoda Nematoda Fungi 30,000 16,000 14,000 21,000 2,000 13,000 Vascular plants 25,000 60,000 Unicellular sps. lehigh.edu SMAAS 5,000 10,000 @ Prokaryotes 500 - 7,000 From genes to proteins DNA RNA Protein lehigh.edu SMAAS @ Gene splicing: Removal of non-coding introns Exon Intron mature splice product lehigh.edu SMAAS @ PROTEIN Alternative splicing: One gene, several proteins! Alternative Splicing Mature splice variant I Mature splice variant II @ lehigh.edu SMAAS SMAAS @ lehigh.edu n RNA editing 5' A 3' transcription genomic DNA 5' A 3' pre-mRNA A-to-I editing 5' I 3' splicing + translation lehigh.edu SMAAS @ Effects of Editing Adenosine Inosine ! ! lehigh.edu ! Adenosine converted to Inosine Interpreted as Guanosine Expand the proteome SMAAS @ Consequences of Editing @ lehigh.edu SMAAS Mammalian substrates of A-to-I pre-mRNA editing Gene codon amino acid editing [%] GluR-B GluR-B GluR-B,-C,-D GluR-B,-C,-D GluR-5,-6 GluR-5,-6 GluR-6 GluR-6 5-HT2C 5-HT2C SerotoninSerotoninreceptor lehigh.edu CAG/CIG CAG/CIG AAG/AIG AAG/AIG CAG/CIG CAG/CIG AUU/IUU AUU/IUU UAC/UIC UAC/UIC AUA/IUA AUA/IUA AAU/AIU AAU/AIU AUU/IUU AUU/IUU Q/R Q/R R/G R/G Q/R Q/R I/V I/V Y/C Y/C I/V I/V N/S N/S I/V I/V 100 60-80 60-80 40-80 40-80 80 80 40-90 40-90 35-40 35-40 45-75 45-75 SMAAS @ Diversity through RNA editing GluR-6 pre-mRNA M1 5` M2 M3 3` AUU UAC IUU UIC I/V Y/C CAG C IG Q/R M2 C unedited edited GluR-6 N M1 @ lehigh.edu SMAAS Diversity through RNA editing GluR-6 pre-mRNA M1 5` M2 M3 3` AUU UAC IUU UIC I/V Y/C CAG C IG Q/R M2 Q Q Q Q Q Q R Q Q R R R C unedited edited GluR-6 N M1 I I V I V I V Y C Y Y C C Y unedited 10 % 5% 5% 10 % 5% @ lehigh.edu SMAAS V C fully edited 65 % Even more diversity paralytic pre-mRNA 5' 3' constitutive exon alternative splicing alternative exon lehigh.edu SMAAS @ 1 536 variants Even more diversity paralytic pre-mRNA 5' alternative splicing 3' constitutive exon RNA editing alternative exon editing site lehigh.edu SMAAS @ 1 032 192 variants Q/R-site editing of glutamate receptor subunit GluR-B mRNA F M N I Q/R Q G -UUU AUG CAG CAA GGA- lehigh.edu cytosol C SMAAS @ M1 M2 M3 M4 GluR-B Q/R-site editing of glutamate receptor subunit GluR-B mRNA F M N Q R I synapse -UUU AUG CAG CAA GGA- Q/R Q G Glu Ca2+ Glu lehigh.edu cytosol C postsynaptic neuron SMAAS @ M1 M2 M3 M4 >99.9% R GluR-B RNA editing enzyme deficient mice Survival (%) ADAR2-/-/GluR-B+/+ @ lehigh.edu Postnatal day (Higuchi, Maas, Single, Hartner et al., 2000, Nature 406, 78-81) SMAAS RNA editing enzyme deficient mice: Rescue by GluR-B point mutation Survival (%) ADAR2-/-/GluR-BR/R ADAR2-/-/GluR-B+/R ADAR2-/-/GluR-B+/+ @ lehigh.edu Postnatal day (Higuchi, Maas, Single, Hartner et al., 2000, Nature 406, 78-81) SMAAS Question: " Could too much or too little RNA editing cause disease or alter the progression of known diseases? @ lehigh.edu SMAAS Glioblastoma multiforme (GBM) @ lehigh.edu http://www.thejohnphilpthompsonfoundation.org/GlioblastomaMultiforme_1_.jpg SMAAS Q/R site editing in normal human brain and gliomas 100 100 100 100 90 69 85 Pe rce nt e diting (%) 79 83 76 88 71 80 60 40 20 0 Q/R-site Q/R+4 site cortex c ortex WM1 WM2 AC1 premalignant 1 2 3 4 5 6 7 lehigh.edu controls tumor Controls Glioblastoma tissues Glioblastoma tissues SMAAS @ (Maas et al., 2001, PNAS 98, 14687-92) RNA editing of ion channel GluR-B mRNA F M I Q/R Q G synapse ADAR2 -UUU AUG CAG CAA GGA- unedited Glu-R lehigh.edu postsynaptic neuron SMAAS Glu Q Ca2+ Glu R @ >99.9% R RNA editing and cancer Too little editing: # Malignant growth, # Activation of oncogenes/repression of tumor suppressor genes [Glioblastoma (GluR-B)] Too much editing: lehigh.edu # activation of oncogenes or # inhibition of tumor suppressor genes [BC10, leukemia (PTPN6), pancreatic cancer (prox1); hepatoma (APOBEC1), neurofibromatosis (NF1), Wilm's tumor (WT1)] SMAAS @ A-to-I RNA editing and human diseases $ Glioblastoma multiforme => GluR-B underediting due to ADAR2 deficiency (Maas et al PNAS 2001) $ Amyotrophic Lateral => GluR-B Q/R-site underediting in motor neurons Sclerosis (ALS) (Kawahara et al Nature 2004) $ lehigh.edu Suicidal Depression => Change in Serotonin receptor editing (Gurevich et al., Neuron 2002) SMAAS @ Hyperediting phenotypes: Lupus erythematosis, inflammatory lung disease Question: How many genes are subject to RNA editing in the human genome? @ lehigh.edu % Investigate using the complete human genome sequence as an important tool. SMAAS The "Smoking Gun" of A-to-I RNA editing -TTT ATG CAG CAA GGA-UUU AUG CAG CAA GGA-UUU AUG CI G CAA GGA- genomic DNA unedited edited mRNA Reverse transcription -TTT ATG CAG CAA GGA- lehigh.edu + -TTT ATG CGG CAA GGA- SMAAS cDNA @ In silico evidence of RNA editing Athanasiadis et al., PLoS Biology 2004 @ lehigh.edu SMAAS Experimental validation lehigh.edu SMAAS Athanasiadis et al., PLoS Biology 2004 @ Number of edited sites exceeds predicted numbers Analysis of 103,723 human cDNA sequences 17, 406 contain one or more Alu @ lehigh.edu SMAAS Athanasiadis et al., PLoS Biology 2004 Transposable elements in the human genome @ lehigh.edu SMAAS Alu-mediated RNA foldback structures @ lehigh.edu SMAAS Levanon et al. EMBO Rep. 2005 Experimental validation lehigh.edu SMAAS Athanasiadis et al., PLoS Biology 2004 @ Tissue origin of Alus determines editing extent lehigh.edu SMAAS @ Athanasiadis et al., PLoS Biology 2004 Distance between inverted Alu pairs lehigh.edu SMAAS @ Athanasiadis et al., PLoS Biology 2004 Functions (?) " None " Accelerated evolution " Alu biology " prevent RNAi lehigh.edu " RNA transport, stability, translation SMAAS @ SMAAS @ lehigh.edu Spectrum of edited human RNAs GluRs 5HT2C etc ADAR2 int simple repeats LINE pairs MIR pairs Alu pairs | | | | | | | | | | | | | | | | | | | | | | | | | | | | | ds character site selectivity inosine content lehigh.edu Prevalence ? SMAAS @ Average for each class Summary - Conclusions We do know: ! Complexity is generated by processes of alternative splicing and RNA editing ! RNA editing regulates the function of genes through recoding and probably through other mechanisms ! We can use genomic and transcriptomic sequence information to search for RNA variations We don't know: ! What is the total impact of RNA editing on complexity ? How is it regulated in vivo ? ! Are there other molecular phenomena that contribute to complexity ? ! What is all the non-coding RNA (and DNA) doing ? @ lehigh.edu SMAAS ...
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This note was uploaded on 03/09/2008 for the course BIOS 95 taught by Professor Varies during the Spring '08 term at Lehigh University .

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