Splicing RNA: Mechanisms

Splicing of Group I and II introns

• Introns in fungal mitochondria, plastids, Tetrahymena pre-rRNA

• Group I– Self-splicing– Initiate splicing with a G nucleotide– Uses a phosphoester transfer mechanism – Does not require ATP hydrolysis.

• Group II– self-splicing– Initiate splicing with an internal A– Uses a phosphoester transfer mechanism– Does not require ATP hydrolysis

Self-splicing in pre-rRNA in Tetrahymena : T. Cech et al. 1981

Exon 1 Exon 2Intron 1 Exon 1 Exon 2 Intron 1

+

pre-rRNASpliced exon

Intron circleIntron linear

pre-rRNANuclear extract

GTP

+ + + +- + - +- + + -

•Products of splicing were resolved by gel electrophoresis:

Additional proteinsare NOT needed forsplicing of this pre-rRNA!

Do need a G nucleotide (GMP, GDP, GTP or Guanosine).

Self-splicing by a phosphoester transfer mechanism

Exon 1

Exon 2

Intron 1

Exon 1 Exon 2

Intron 1+

PP

P P

G

OHU

UG

A P

U

P

U

P

P

G

OH

G

AP

N15 N16

G AN15

P OH

Circular intron+

A catalytic activity in Group I intron

• Self-splicing uses the intron in a stoichiometric fashion.

• But the excised intron can catalyze cleavage and addition of C’s to CCCCC

Group I intron catalyzes cleavage and nucleotide addition

5'pCCCCCC-OH+

2 pCCCCC-OH pCCCC-OH + pCCCCCC-OH

GGGAGG 5'

3'GOH

5'pCCCCC-OHGGGAGG 5'

3'GOHC-

5'pCCCCC-OH

GGGAGG 5'

3'GOH

GGGAGG 5'

GOHC-3'

5'pCCCC-OH

The intron folds into a particular 3-D structure

• Has active site for phosphoester transfer

• Has G-nucleotide binding site

Active sites in Group I intron self-splicing

GGGAGG

GGGAGG 5'

G

GGGAGG5'

3'GOH

GGGAGG

G

+

G414

G-OH

3'

CUCUCU5'

G-binding site

Substrate binding site

IGS

ex1

ex2

414

3'1st transfer

CUCUCU5'

ex1

G

OH

ex2

ex1

ex2+

414

2nd transfer

UUUACCUG

3rd transfer

414

5' G UUUACCU

Domains of the Group I intron ribozyme

RNAs that function as enzymes

• RNase P

• Group I introns

• Group II introns

• rRNA: peptide bond formation

• Hammerhead ribozymes: cleavage

• snRNAs involved in splicing

Hammerhead ribozymes

• A 58 nt structure is used in self-cleavage

• The sequence CUGA adjacent to stem-loops is sufficient for cleavage

CUGAG

ACCGGGGCC

AAA

ACUC G

AGU C

ACCACUGGUG

U

Bond that is cleaved.

5'3'

CUGA is required for catalysis

Design hammerhead ribozymes to cleave target RNAs

CUGAG

ACCGGGGCC

AAA

ACUC

GU A

A

GAGU C

ACCACUGGUG

U

Bond that is cleaved.

5'

3'

substrate strand

enzyme strand

Potential therapy for genetic disease.

Mechanism of hammerhead ribozyme

• The folded RNA forms an active site for binding a metal hydroxide

• Abstracts a proton from the 2’ OH of the nucleotide at the cleavage site.

• This is now a nucleophile for attack on the 3’ phosphate and cleavage of the phosphodiester bond.

Phosphotransfers for Group I vs. Group II & pre-mRNA

2’

GHO

3’Exon 1 Exon 2

OH

G

OH

Exon 1+2G

AHO

2’

Exon 1 Exon 2

OH

A

Exon 1+22’ A

++

Group IGroup II and pre-mRNA

Splicing of pre-mRNA

• The introns begin and end with almost invariant sequences: 5’ GU…AG 3’

• Use ATP to assemble a large spliceosome

• Mechanism is similar to that of the Group II fungal introns:

– Initiate splicing with an internal A

– Uses a phosphoester transfer mechanism for splicing

Initiation of phosphoester transfers in pre-mRNA

• Uses 2’ OH of an A internal to the intron

• Forms a branch point by attacking the 5’ phosphate on the first nucleotide of the intron

• Forms a lariat structure in the intron

• Exons are joined and intron is excised as a lariat

• A debranching enzyme cleaves the lariat at the branch to generate a linear intron

• Linear intron is degraded

Splicing of pre-mRNA, step 1

Splicing of pre-mRNA, step 2

Investigation of splicing intermediates

5' 3'

RNARNase H

5'

oligodeoxyribonucleotide

+

|||||

In vitro splicing reaction: nuclear extracts + ATP+ labeled pre-mRNAResolve reaction intermediates and products on gels.Some intermediates move slower than pre-mRNA.Suggest they are not linear.Use RNase H to investigate structure of intermediate.RNase H cuts RNA in duplex with RNA or DNA.

RNase H + oligonucleotides complementary to different regions give very different products

5'3'

precursor RNA

exon 1 intron exon 2

1 2 3 4

splicing reaction

exons joined in a linear molecule excised intron, non-linear molecule

Map of positions of

oligodeoxyribonucleotides

that annealed to different

regions of the excised

intron. This is not the

structure of the excised

intron.

+

5'

5'

5'

3'

3'

3'

3'

3'

+

5'

RNase H

Analysis reveals a

lariate structure in inter-mediate

5'3'

precursor RNA

exon 1 intron exon 2

1 2 3 4

splicing reaction

excised intron, non-linear molecule

Map of positions of

oligodeoxyribonucleotides

that annealed to different

regions of the excised

intron.

+

5'

5'

3'

3'

3'

3'

3'

+

5'

After annealing with

the oligo, the

heteroduplexes were

treated with RNase H

Answer:

exons joined in a linear molecule

1

2

34

GU

AG

A

oligo 1

oligo 2

oligo 3

oligo 4

Involvement of snRNAs and snRNPs

• snRNAs = small nuclear RNAs• snRNPs = small nuclear ribonucleoprotein

particles• Antibodies from patients with the autoimmune

disease systemic lupus erythematosus (SLE) can react with proteins in snRNPs– Sm proteins

• Addition of these antibodies to an in vitro pre-mRNA splicing reaction blocked splicing.

• Thus the snRNPs were implicated in splicing

snRNPs

• U1, U2, U4/U6, and U5 snRNPs– Have snRNA in each: U1, U2, U4/U6, U5– Conserved from yeast to human– Assemble into spliceosome– Catalyze splicing

• Sm proteins bind “Sm RNA motif” in snRNAs– 7 Sm proteins: B/B’, D1, D2, D3, E, F, G– Each has similar 3-D structure: alpha helix

followed by 5 beta strands– Sm proteins interact via beta strands, may form

circle around RNA

Sm proteins may form ring around snRNAs

ANGUS I. LAMOND Nature 397, 655 - 656 (1999)RNA splicing: Running rings around RNA

Predicted structure of assembled Sm proteins

ANGUS I. LAMOND Nature 397, 655 - 656 (1999)RNA splicing: Running rings around RNA

Channel for singlestrand of RNA

4th beta strand of one Sm proteininteracts with 5th beta strand of next.

Assembly of spliceosome

• The spliceosome is a large protein-RNA complex in which splicing of pre-mRNAs occurs.

• snRNPs are assembled progressively into the spliceosome.– U1 snRNP binds (and base pairs) to the 5’ splice site– U2 snRNP binds (and base pairs) to the branch point– U4-U6 snRNP binds, and U4 snRNP dissociates– U5 snRNP binds

• Assembly requires ATP hydrolysis• Assembly is aided by various auxiliary factors and

splicing factors.

Spliceosome assembly and catalysis

Exon 1

AHO

2’

Exon 2

AHO

2’

U1 snRNP A

HO2’

U2 snRNP

U5 snRNP U4/U6

snRNP

U6 U4

AG

U

OH

Exons 1+2

2’ A

Sm proteins

snRNAs

Other proteins

U4?

AO2’G

U

U6

U6

U2

HU1

U5Spliceosome

Catalysis by U6/U2 on branch oligonucleotide in vitro

Figure 1 Base-pairing interactions in the in vitro-assembled complex ofU2–U6 and the branch oligonucleotide (Br). Shaded boxes mark the invariantregions in U6 and previously established base-paired regions are indicated.Dashed lines connect psoralen-crosslinkable nucleotides (S.V. and J.L.M.,unpublished data). The circled residues connected by a zigzag can becrosslinked by ultraviolet light. The underlined residues in Br constitute theyeast branch consensus sequence. Asterisks denote the residues involved inthe covalent link between Br and U6 in RNA X (see text). Arrowheads pointto residues involved in a genetically proven interaction in yeast22. Numbersindicate nucleotide positions from the 5' ends of full-length human U2 and U6.

Nature 413, 701 - 707 (2001) Splicing-related catalysis by protein-freesnRNAs SABA VALADKHAN & JAMES L. MANLEY

RNA editing

• RNA editing is the process of changing the sequence of RNA after transcription.

• In some RNAs, as much as 55% of the nucleotide sequence is not encoded in the (primary) gene, but is added after transcription.

• Examples: mitochondrial genes in trypanosomes and Leishmania.

• Can add, delete or change nucleotides by editing

Addition of nucleotides by editing

• Uses a guide RNA that is encoded elsewhere in the genome

• Part of the guide RNA is complementary to the mRNA in vicinity of editing

• U nt at the the 3’ end of the guide RNA initiates a series of phosphoester transfers that result in insertion of that U at the correct place.

• More U’s are added sequentially at positions directed by the guide RNA

• Similar mechanism to that used in splicing

What is a gene?• Making a correctly edited mRNA requires one

segment of DNA to encode the initial transcript and a different segment of DNA to encode each guide RNA.

• Thus making one mRNA that uses 2 guide RNAs requires 3 segments of DNA - is this 3 genes or 1 gene?

• Loss-of-function mutations in any of those 3 DNA segments result in an nonfunctional product (enzyme), but they will complement in trans in a diploid analysis!

• This is an exception to the powerful cis-trans complementation analysis to define genes.

Mammalian example of editing

• Apolipoprotein B in the intestine is much shorter than apolipoprotein B in the liver.

• They are encoded by the same gene.

• The difference results from a single nt change in codon 2153:

• CAA for Gln in liver, but UAA for termination of translation in intestine

• The C is converted to U in intestine by a specific deaminating enzyme, not by a guide RNA.