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.

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

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

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

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

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

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

• 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)

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

DNA with TATA box binding protein

DNA

Protein

The TATA binding protein binds to the TATA box

The core promoter

-30 start of transcription +30

TATA binding protein

-30 start of transcription +30

The TATA binding protein is one subunit of a large complex: TFII-D.

The core promoter

TFII-D

-30 start of transcription +30

Several other complexes bind to TFII-D.

The core promoter

TFII-DTFII-B

TFII-ATFII-F

-30 start of transcription +30

RNA polymerase is recruited to the promoter.

The core promoter

TFII-DTFII-B

TFII-ATFII-F

RNA pol II

-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

…...(Tyr-Ser-Pro-Thr-Ser-Pro-Ser)52COOH

CTD: a pol II switchCTD: The COOH Terminal Domain of the RNA pol II large subunit

…...(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

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

From Orphanadies & Reinberg (2002) Cell 108:439-51

The CTD ties elongation to capping, splicing and 3’-end formation

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

Binding of a leucine zipper protein to DNA

HAT

Serine 133

Phosphorylation of CREB and the CREB binding protein (CBP)

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