Download - Exon selection factor
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Exon selection factor
Exon selection factor
U2 snRNPU1 snRNP
Intron 1
Overview of mRNA Splicing
Exon 1 AGGU Exon 2A 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)
AG
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Beta globin splice mutations:creation of a new acceptor site
NORMAL: INTRON 1 EXON 2TATTGGTCTATTTTCCCACCCTTAG GCTG
MUTATION:
TATTAGTCTATTTTCCCACCCTTAGGCTG
TATTAG TCTATTTTCCCACCCTTAGGCTG
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%
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
Last exon
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Nonsense Mediated DecayOrigin 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 codonPremature 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 DecayClinical 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-DTFII-B
TFII-ATFII-F
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-30 start of transcription +30
RNA polymerase is recruited to the promoter.
The core promoter
TFII-DTFII-B
TFII-ATFII-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-DTFII-B
TFII-ATFII-F
RNA pol IITFII-H
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…...(Tyr-Ser-Pro-Thr-Ser-Pro-Ser)52COOH
CTD: a pol II switchCTD: The COOH Terminal Domain of the RNA pol II large subunit
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…...(Tyr-Ser-Pro-Thr-Ser-Pro-Ser)52COOH
TFIIH
…...(Tyr-Ser-Pro-Thr-Ser-Pro-Ser)52COOH
PO3 phosphorylated Ser 5 of the repeats
Other kinases
More phosphorylation of the CTD
CTD: a pol II switchCTD: 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 cappingBinds components involved in RNA splicingBinds components involved in 3’ end formation
CTD: a pol II switchCTD: 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
CREBFos Jun CREB
AP-1 (Fos-Jun binding site)Regulation bygrowth factors,stress, and varioustransmembranesignals
CRE (Cyclic AMPResponse Element)Regulation by cAMP,and by Ca+. Interacts withcore promoter (through CREB Binding Protein)and modifies chromatin structure (through HAT).
SP-1 SiteProvides basalunregulatedtranscriptionalactivity. Manygenes have multiple SP-1 sites
Core PromoterBinds generaltranscriptionalmachinery
TATAAA
Positions of these elementsare relatively unimportant
HAT Histone Acetyl Transferase
HRENR NR
HRE (HormoneResponse Element)Regulation by hormonessuch 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
PO4
G
cAMP Inctive Protein Kinase A
PO4
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