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Exon selection factor Exon selection factor U2 snRNPU1 snRNP Intron 1 Overview of mRNA Splicing Exon 1 AGGU Exon 2 A AGG Factors such as U1 and U2 snRNP identify splice sites Exons are identified by RNA sequences within the exons that are recognized by exon selection factors.
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Beta globin splice mutations are one cause of beta thalassemia EXON1 INTRON1 PHENOTYPE AG GT AGT CONSENSUS GCCAG GTTGGTAT NORMAL GCCAG ATTGGTAT 0 (no beta chains) GCCAG TTTGGTAT 0 (no beta chains) GCCAG GTTGTTAT + (some beta chains) GCCAG GTTGCTAT + (some beta chains) GCCAG GTTGGCAT + (some beta chains) AGAG
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Beta globin splice mutations: creation of a new acceptor site NORMAL: INTRON 1 EXON 2 TATTGGTCTATTTTCCCACCCTTAG GCTG MUTATION: TATTAGTCTATTTTCCCACCCTTAGGCTG Normal site used 10% of the time: normal protein from these RNAs New site used 90% of the time: no protein from these RNAs (note the shift in reading frame). 10%10% 10% 90% 100% Net result: this allele shows a 90% reduction in β-globin production 19 nucleotides
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Nonsense Mediated Decay Exon/Exon junction Normal stop codon is downstream or <50 bases upstream from splice junction Premature stop codon >50 bases upstream from splice junction >50 bases mRNA Decay Translation A. B. Last exon
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Nonsense Mediated Decay Origin of premature stop codons - Improper splicing - intron retained - frameshift - Mutation Possible consequences of premature stop codons: - non functional protein - formation of amyloid - loss of a regulatory region from a protein that regulates growth cancer
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Nonsense Mediated Decay Exon/Exon junctions Normal Stop Codon 1st round of Translation Spliceosomes Protein complexes (Exon-junction-complexes; EJC) Nucleus Cytoplasm More translation Complexes removed by ribosome transit
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Nonsense Mediated Decay Exon/Exon junctions Normal stop codon Premature stop codon mRNA Decay 1st round of Translation Spliceosomes Protein complexes (Exon-junction-complexes; EJC) Nucleus Cytoplasm Stopped ribosome Complex not removed
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Some drugs that affect the accuracy of codon recognition by ribosomes (such as gentamicin) decrease Nonsense mediated decay. Treatment with these drugs allows a low level of expression from genes with premature stop codons. Possible treatment for several disorders including some alleles of cystic fibrosis. Reference: Holbrook et al Nature Genetics 36:801-808 (2004) Nonsense mediated Decay Clinical applications (in trials)
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For most (but not all) promoters, a complex of proteins is assembled around the TATA box, located about 25-30 b.p. upstream from the start site. The consensus sequence of the TATA box is TATAAA The core promoter -30 start of transcription +30 TATA box
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DNA with TATA box binding protein DNA Protein
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The TATA binding protein binds to the TATA box The core promoter -30 start of transcription +30 TATA binding protein
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-30 start of transcription +30 The TATA binding protein is one subunit of a large complex: TFII-D. The core promoter TFII-D
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-30 start of transcription +30 Several other complexes bind to TFII-D. The core promoter TFII-D TFII-B TFII-A TFII-F
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-30 start of transcription +30 RNA polymerase is recruited to the promoter. The core promoter TFII-D TFII-B TFII-A TFII-F RNA pol II
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-30 start of transcription +30 The factor TFII-H plays a key role in initiating transcription by phosphorylating the C-terminal domain of the large subunit of RNA pol II. The core promoter TFII-D TFII-B TFII-A TFII-F RNA pol II TFII-H
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…...(Tyr-Ser-Pro-Thr-Ser-Pro-Ser) 52 COOH CTD: a pol II switch CTD: The COOH Terminal Domain of the RNA pol II large subunit
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…...(Tyr-Ser-Pro-Thr-Ser-Pro-Ser) 52 COOH TFIIH …...(Tyr-Ser-Pro-Thr-Ser-Pro-Ser) 52 COOH PO 3 phosphorylated Ser 5 of the repeats Other kinases More phosphorylation of the CTD CTD: a pol II switch CTD: The COOH Terminal Domain of the RNA pol II large subunit ATP ADP ATP ADP TFIIH controls the start of transcription
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Unphosphorylated CTD: Involved in initiation: Binding of initiation factors Phosphorylated CTD: Involved in elongation & RNA processing Binds components involved in RNA capping Binds components involved in RNA splicing Binds components involved in 3’ end formation CTD: a pol II switch CTD: The COOH Terminal Domain of the RNA pol II large subunit
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From Orphanadies & Reinberg (2002) Cell 108:439-51 The CTD ties elongation to capping, splicing and 3’-end formation
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A model promoter TGACTCA GACGTC GGGCGG SP-1 CREB Fos Jun CREB AP-1 (Fos-Jun binding site) Regulation by growth factors, stress, and various transmembrane signals CRE (Cyclic AMP Response Element) Regulation by cAMP, and by Ca +. Interacts with core promoter (through CREB Binding Protein) and modifies chromatin structure (through HAT). SP-1 Site Provides basal unregulated transcriptional activity. Many genes have multiple SP-1 sites Core Promoter Binds general transcriptional machinery TATAAA Positions of these elements are relatively unimportant HAT Histone Acetyl Transferase HRE NR HRE (Hormone Response Element) Regulation by hormones such as estrogen which enter the cell
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Binding of a leucine zipper protein to DNA
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HAT Serine 133 Phosphorylation of CREB and the CREB binding protein (CBP)
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Plasma membrane Nuclear membranes Hormone receptor Adenylate cyclase ATP Active Protein Kinase A PO 4 G cAMP Inctive Protein Kinase A PO 4 Active pKA enters the nucleus and phosphorylates CREB on Serine 133 Hormone G-protein Core promoter Phosphorylation of CREB: - stimulates interactions with several core promoter proteins - induces binding of HAT and acetylation of histones Signaling mediated by cAMP and protein kinase A
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