RNA Rearrangements in the Spliceosome-extensive U4/U6 interaction is replaced with a U2/U6 structure-U1 displaced at 5’ splice site by U6

RNA Rearrangment at 5’ Splice Site

GUAUGU5' exon

CAUUCAU1

••• ••

5' splice site

5'

GUAUGU5' exon

GACACAU6 47

• • •5'

3

Why Splicing?

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Why Splicing?

-ancient or new?-functional units (W. Gilbert)-diversity of function, evolution-alternative splicing

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Exon Shuffling

GF GF P

GF K K P

PKK

K K K K K P

K PGF

prothrombin

plasminogen

plasminogen-activator

urokinase

factor VII

Sequence Conservation Within Introns

Yeast

Human

5’ Splice Site

Sequence Conservation Within Introns

3’ Splice SiteBranch Region

Yeast

Human

Drosophila Doublesex (dsx) RNA

3 4 5

female

male

1

smooth muscle

2 3 4

non-smooth muscle

α-tropomyosin

3

neuronal

1N 2N 4

-non neuronal

Src

7b

embryonic

EIIIB 8a

most adult

Fibronectin EIIIB

Tissue and Developmental Regulation

Dscam

115 exons

20 constitutive, 95 alternatively spliced38,016 possible proteins

Exon 412 alternative

Exon 648 alternative

Exon 633 alternative

Exon 172 alternative

Alternative Splicing and Proteome Complexity

Mechanisms of Alternative Splicing

-activation/repression-intron sequences: splice sites, pyrimidine tract,branch region-exon sequences (purine rich enhancers)

Recall:Selection of 5’ Splice Site and Branch

GUAUGU5' exon

CAUUCAU1

••• ••

5' splice site

5'

UACUAC 3' exon

AUGAUGU2

• • • • • 5'

A

•34

UACUAAC 3' exon

3' splice siteBBP

SR Proteins

MSYGRPPPDVEGMTSLKVDNLTYRTSPDTLRRVFEKYRRVGDVYIPRDRYTKESRGFAFVRFHDKRDAEDAMDAMDGAVLDGRELRVQMARYGRPPDSHHSRRGPPPRRYGGGGYGRRSRSPRRRRRSRSRSRSRSRSRSRSRYSRSKSRSRTRSRSRSTSKSRSARRSKSKSSSVSRSRSRSRSRSRSRSPPPVSKRESKSRSRSKSPPKSPEEEGAVSS

-human SC35, 24 C terminal SR repeats

-S100 extract is deficient in pre-mRNA splicing-S100 + SR splices-SR proteins are phosphoproteins (SRPK kinase)

SRRRMRRMN C

OH O P

O

O

O

-

-

RNA Recognition Motif (RRM)

PTB RRM-3 Sxl RRM-1

Activation

-downstream activation of 5’ splice site-adenovirus E1A, also Tra/Tra2

Mol. Cell. Biol. (1999) 19, 7347

Pu

9G8 U1

“Enhancers”:

-stimulate 5’ or 3’ splice site usage

vs

“Enhancers”:

-stimulate 5’ or 3’ splice site usage

vs

“Enhancers”:

-within exons-proximal to splice sites-conserved

introns exons

weak

strong

5' exon

3' exon

Science 297, 1007 (2002)

Repression

-formation of RNA secondary structure (blocking site access); relievedby ATP dependent helicases-binding of trans factors

Drosophila sxl Gene

male

female

sxl Regulation of Drosophila splicing-sxl competition with U2AF on dsx (also polypyrimidine tract binding protein-PTB; neuronal specific events)

female

male

malesxl

femaleU2AF

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