splicing. movie removed to simplify downloading. you can download the movie separately

Splicing

Movie removed to simplify downloading.

You can download the movie separately.

Figure 14.3

mouse globin mRNA PRECURSOR RNA hybridized to cloned gene (genomic).

mouse globin MATURE mRNA hybridized to cloned gene (genomic).

4

mRNA Splicing• rRNA and tRNA are also sometimes spliced; however,

these are not the current topic.

• Splice sites are strongly conserved

• Shapiro and Senapathy examined 3700 splice sites. Immediately before 3' splice site is pyridine rich and free of AG dinucleotide.

• Invariant consensus is GU at 5' donor site and AG at 3' acceptor site. This is usually referred to as the GT/AG rule.

MAMMALLIAN EXON |-------INTRON-------------------| EXON5’---------AG GUAAGU-------------YNCURAC-YnNAG G---------3’ | | | 5’ SPLICE SITE * 3’ SPLICE SITE

YEAST EXON |-------INTRON-------------------| EXON5’----------- GUAUGU----------------UACUAA-YAG ---------3’ | | | 5’ SPLICE SITE * 3’ SPLICE SITE

Exon 1 pGU-----------A---------------AGp Exon2OH

A---------------AGp Exon2UGpExon 1-OH

A---------------OHUGpExon 1-p-Exon 2

Splicing is a 2 step trans-esterification

Figure 14.4

Transesterification 1.

First the 5' transesterification occurs and it generates 2'-5' phosphodiester bond. The 2' attachment point is a 2' hydroxyl of an A nucleotide within the intron. This position is called the branch point.

The branch point is 17 - 40 nucleotides upstream of the 3' splice site.

Transesterification 2.

Second to occur is the 3' splice site cleavage, with simultaneous exon ligation. Intron leaves the complex as a lariat.

The number of phophodiesters is conserved. No energy is lost or consumed.

Structure of the lariat U5'|P|3'G5'|P|2'A A3'-P-5'-G-3'-OH3'-P-5'3'-P-5'

5' most nucleotide of intron3' most nucleotide ofintron

Notice the 2' to 5' phosphodiestion bond at the branch point.

Spliceosome does splicing

This is a very large macromolecular complex. Spliceosomes are about 25 nm X 50 nm. It assembles on the mRNA. Assembly consumes ATP.

Weaver page 404

9

• Sharp & coworkers

• L1----IVS----L2

• intron is 231n

• DO is an antiSNURP sera

• ME is the control sera

• Panel C is a Southern blot of the 10% acrylamide 8M urea gel probed with a L1L2 fragment.

4% poly-acrylamidegel 8M urea

10% poly- acrylamide gel 8 M urea

Very hard to see band

10

Thin layer chromatography to demonstrate that A is the branchpoint

RNase T1 cuts after guanylate residues.RNase T1 cuts only single stranded RNA.

Is the branched nucleotide attached to the 5’ end of the intron?

Snurps

• snRNPs pronounced snurps– small nuclear ribonucleoprotein particle

• The particle contains– small nuclear RNAs = snRNAs

– small nuclear ribonucleoproteins

pg 407

5’ splice site also called the donor site

3’ splice site also called the acceptor site

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Branchpoint

• Yeast consensus: UACUAAC

• Mammallian:

• U47NC63U53R72A91C47

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Importance of the branchpoint

Figure 14.8 4th edition

Figure 14.12

Base pairing is requried but is it sufficient?

Base pairing is requried but is it sufficient?

Base pairing is requried but is it sufficient?

Clearly base pairing with U1 is not all that is required.

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Recognition of mammalian pre-mRNA intron sequences by

snRNPs

Spliceosome CycleFig 14.28

27

SR proteins

• Serine (S) and Arginine (R) rich proteins that help to identify exons

• Involved in alternative splicing

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snRNAs • U1 - recognition (bp) of the 5’ splice site (donor site).

• U2 - branch point recognition (bp) & bps to U6 snRNA

• U5 binds ends of exons

• U4 binds U6 and holds it untile U6 is needed in a splicing reaction

• U6 - bps to 5’ splice site and U2 snRNA & U4 snRNA

• 3’ splice site should be 18-40n downstream of branch point. Slu7 and U2AF use branch point to help recognize 3’ splice site.

Group II self splicing

Self splice and non-self splicing

Group II mitochondria

Figure 14.22

U p AU p AG p U

U p AG p U

U p A

p G OHU-OHG p U

U p Ap G p A

U p UG

U p Ap G p A

G p A

G p AU-OH

Degrade

Group I intronsrRNA

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RNAP II CTD

• Experiment: CTD-GST stimulates splicing in vitro. GST does not.

• CTD binds snRNPs and splicing proteins.

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35

Exon definition - intron definition

• Intron definition is sufficient to identify ends of introns.– For some transcripts the splicing machinery

identifies the ends of introns without help from CTD.

• Exon definition is needed to successfully identify the ends of introns– Here CTD helps to identify the ends of the

EXONS.– These types of transcripts are not spliced if the

exons are not whole.35

Figure 14.37. This topic begins on page 427.

QuickTime™ and aAnimation decompressor

are needed to see this picture.

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U1SNRP

U1SNRP

U2SNRP

U2AF

U2SNRP

U2AF

SXL SXL

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Regulation of splicing

• Negative - SR protein binds and hides a required sequence

• Positive - a crummy sequence (eg branch point) is enhanced with the help of a protein (eg U2AF).

40

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endend