[email protected] Studies on Complex Biological Systems:Sources for Transcriptome and Proteome ComplexityMolecular Studies on Complex Biological Systems:Sources for Transcriptome and Proteome ComplexityOctober 17, 2007 Dr. Stefan Maas, BioS Lehigh U. October 17, 2007 Dr. Stefan Maas, BioS Lehigh [email protected]’’s the molecular difference ?s the molecular difference ?Nature 9/7/[email protected] mountain grasshopper Podisma pedestrisThe fruit flyDrosophila melanogaster18,000 Mb 180 Mb What accounts for the often massive and seemingly arbitrary differences in genome size observed among eukaryotic [email protected] an Expansion in Gene Number drivingEvolution of Higher Organisms?Prokaryotes500 - 7,000UrochordataArthropodaNematoda16,00014,00021,000Unicellular sps.Fungi2,000 – 13,0005,000 – 10,000VertebrataVascular plants30,00025,000 – 60,[email protected] genes to [email protected] splice productGene splicing: Removal of non-coding intronsPROTEINExonExonIntronIntronSMAAS@lehigh.eduSplicingAlternativeMature splice variant IIMature splice variant IAlternative splicing: One gene, several [email protected]@lehigh.edutranscriptiontranscriptionRNA editingRNA editingprepre--mRNAmRNAgenomicgenomicDNADNA5’5’5’5’AA--toto--I editingI editing3’3’3’3’5’5’3’3’AAIsplicing + translationsplicing + [email protected] of EditingEffects of Editing!!Adenosine converted to InosineAdenosine converted to Inosine!!Interpreted as Interpreted as GuanosineGuanosine!!Expand the proteomeExpand the proteomeAdenosine [email protected] of EditingConsequences of [email protected] substrates ofMammalian substrates ofAA--toto--I preI pre--mRNA editingmRNA editingGluR-B CAG/CIGQ/R100GluR-B,-C,-D AAG/AIG R/G 60-80GluR-5,-6 CAG/CIG Q/R 40-80GluR-6 AUU/IUU I/V80GluRGluR--B CAG/CB CAG/CIIGGQ/Q/RR100100GluRGluR--B,B,--C,C,--D AAG/AD AAG/AIIG R/G R/GG6060--8080GluRGluR--5,5,--6 CAG/C6 CAG/CIIG Q/G Q/RR4040--8080GluRGluR--6 AUU/6 AUU/IIUU I/UU I/VV8080Gene codon amino acid editing [%]UAC/UIC Y/C80UAC/UUAC/UIIC Y/C Y/CC80805-HT2CAUA/IUA I/V40-90Serotonin- AAU/AIU N/S35-40receptor AUU/IUU I/V45-7555--HTHT2C2CAUA/AUA/IIUA I/UA I/VV4040--9090SerotoninSerotonin--AAU/AAAU/AIIU N/U N/SS3535--4040receptorreceptorAUU/AUU/IIUU I/UU I/VV4545--7575SMAAS@lehigh.eduGluRGluR--66prepre--mRNAmRNAM1M1M2M2M3M3AUUAUUUACUACCAGCAGIIUUUUUUIICCCCIIGGunediteduneditededitededited5‘3‘NCI I //VVDiversity through RNA editingDiversity through RNA editingYY//CCQ Q //RRGluRGluR--66M1M2SMAAS@lehigh.eduGluRGluR--66prepre--mRNAmRNAM1M1M2M2M3M35‘3‘NCI I //VV10 %10 %5 %5 %10 %10 %5 %5 %5 %5 %65 %65 %Diversity through RNA editingDiversity through RNA editingYY//CCQ Q //RRGluRGluR--66M1M2I YI YI YICIICCVYVVYYI YI YI YV CV CV CICIICCVYVVYYV CV CV CQQQQQQQQQRRRQQQRRRRRRRRRunediteduneditedfully editedfully editedAUUAUUUACUACCAGCAGIIUUUUUUIICCCCIIGGunediteduneditededitededitedSMAAS@lehigh.edu1 536 variants1 536 variantsparalytic pre-mRNAalternativealternativesplicingsplicing5’ 3’Even more diversityEven more diversityconstitutive exonalternative [email protected] 032 192 variants1 032 192 variantsparalytic pre-mRNAalternativealternativesplicingsplicing5’ 3’Even more diversityEven more diversityconstitutive exonRNARNAeditingediting••editing site••••••••••••••••••••••alternative [email protected] M2 M3 M4NCGluR-BcytosolQ/RQ/R--site editing of glutamate receptorsite editing of glutamate receptorsubunit subunit GluRGluR--BBM Q/Q/RR Q GIImRNAF-UUU AUG CAG CAA [email protected] M2 M3 M4NCGluR-BcytosolpostsynapticpostsynapticneuronneuronGluCa2+>99.9% RRRQGluQ/RQ/R--site editing of glutamate receptorsite editing of glutamate receptorsubunit subunit GluRGluR--BBsynapsesynapseM Q/Q/RR Q GIImRNAF-UUU AUG CAG CAA [email protected] (%)Postnatal day(Higuchi, Maas, Single, Hartner et al., 2000, Nature 406, 78-81)RNA editing enzyme deficient miceADAR2-/-/GluR-B+/[email protected]/-/GluR-BR/RADAR2-/-/GluR-B+/+ADAR2-/-/GluR-B+/RSurvival (%)Postnatal dayRNA editing enzyme deficient mice:Rescue by GluR-B point mutation(Higuchi, Maas, Single, Hartner et al., 2000, Nature 406, 78-81)[email protected]:Question:""Could too much or too little Could too much or too little RNA editing cause disease or RNA editing cause disease or alter the progression of known alter the progression of known diseases?diseases?SMAAS@lehigh.eduGlioblastomaGlioblastomamultiformemultiforme(GBM)(GBM)http://www.thejohnphilpthompsonfoundation.org/[email protected] 100 1009069857976838871020406080100cortexcontrols Glioblastoma tissuesPercent editing (%)Q/R-siteQ/R+4 siteGlioblastoma tissuesGlioblastoma tissues12345 67Q/R site editing in normal human brainQ/R site editing in normal human brainand gliomasand gliomasControlsControlsAC1AC1premalignanttumorWM1WM1WM2WM2cortex(Maas et al., 2001, PNAS 98, 14687-92)[email protected]+>99.9% RRRQGluRNA editing of ion channel RNA editing of ion channel GluRGluR--BBsynapsesynapseM Q/Q/RR Q GIImRNAF-UUU AUG CAG CAA [email protected] editing and cancerRNA editing and cancerToo little editing:Too little editing:##Malignant growth,Malignant growth,##Activation of Activation of oncogenesoncogenes/repression of /repression of tumor suppressor genestumor suppressor genes[Glioblastoma (GluR-B)]Too much editing:Too much editing:##activation of activation of oncogenesoncogenesor or ##inhibition of tumor suppressor genesinhibition of tumor suppressor genes[BC10, leukemia (PTPN6), pancreatic cancer (prox1); hepatoma (APOBEC1), neurofibromatosis (NF1), Wilm’s tumor (WT1)][email protected] RNA editing and human diseasesI RNA editing and human diseases$$GlioblastomaGlioblastomamultiformemultiforme$$Amyotrophic Lateral Amyotrophic Lateral Sclerosis (ALS)Sclerosis (ALS)$$Suicidal