Splicing RNA: Mechanisms

1
Splicing RNA Mechanisms
2
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

3
Self-splicing in pre-rRNA in Tetrahymena T.
Cech et al. 1981
Additional proteins are NOT needed for splicing
of this pre-rRNA!
Do need a G nucleotide (GMP, GDP, GTP or
Guanosine).
4
Self-splicing by a phosphoester transfer mechanism
Intron 1
U
U
P
P
U
A
Exon 1
Exon 2
P
P
U
G
Exon 1
P
P

Intron 1
Exon 2
N15
N16
G
A
P
P
G
P
OH
N15
G
A
OH
P

Circular intron
5
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 Cs to CCCCC
• Fig. 3.3.12

6
Group I intron catalyzes cleavage and nucleotide
addition
7
The intron folds into a particular 3-D structure

• Has active site for phosphoester transfer
• Has G-nucleotide binding site
• Fig. 3.3.13

8
Active sites in Group I intron self-splicing
9
Domains of the Group I intron ribozyme
10
Exon exchange via trans-splicing (Fig. 3.3.14)

• Although splicing is usually in cis, splicing can
  occur in trans (between 2 different RNAs).
• The internal guide sequence is only needed for
  specificity, NOT for catalysis
• Thus any RNA can serve as an IGS
• Can engineer RNAs with an IGS complementary to
  the region 5 to a mutation for exon exchange.
• This a potential approach to therapy for some
  genetic diseases.

11
RNAs that function as enzymes

• RNase P
• Group I introns
• Group II introns
• rRNA peptide bond formation
• Hammerhead ribozymes cleavage
• snRNAs involved in splicing

12
Hammerhead ribozymes

• A 58 nt structure is used in self-cleavage
• The sequence CUGA adjacent to stem-loops is
  sufficient for cleavage

13
Design hammerhead ribozymes to cleave target RNAs
Potential therapy for genetic disease.
14
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.

15
Splicing of pre-mRNA

• The introns begin and end with almost invariant
  sequences 5 GUAG 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

16
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

17
Phosphotransfers for Group I vs. Group II
pre-mRNA
Exon 1
Exon 1
Exon 2
Exon 2
A
2
G
OH
OH
Exon 12
Exon 12

G

OH
Group II and pre-mRNA
Group I
18
Investigation of splicing intermediates
In vitro splicing reaction nuclear extracts
ATP labeled pre-mRNA Resolve 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.
19
RNase H oligonucleotides complementary to
different regions give very different products
20
Analysis reveals a lariate structure in
inter-mediate
21
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

22
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

23
Sm proteins may form ring around snRNAs
ANGUS I. LAMOND Nature 397, 655 - 656 (1999) RNA
splicing Running rings around RNA
24
Predicted structure of assembled Sm proteins
4th beta strand of one Sm protein interacts with
5th beta strand of next.
Channel for single strand of RNA
ANGUS I. LAMOND Nature 397, 655 - 656 (1999) RNA
splicing Running rings around RNA
25
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.

26
Spliceosome assembly and catalysis
27
Catalysis by U6/U2 on branch oligonucleotide in
vitro
Figure 1 Base-pairing interactions in the in
vitro-assembled complex of U2U6 and the branch
oligonucleotide (Br). Shaded boxes mark the
invariant regions 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 be crosslinked by
ultraviolet light. The underlined residues in Br
constitute the yeast branch consensus sequence.
Asterisks denote the residues involved in the
covalent link between Br and U6 in RNA X (see
text). Arrowheads point to residues involved in a
genetically proven interaction in yeast22.
Numbers indicate nucleotide positions from the 5'
ends of full-length human U2 and U6.
Nature 413, 701 - 707 (2001) Splicing-related
catalysis by protein-free snRNAs SABA VALADKHAN
JAMES L. MANLEY
28
Biochemical results for U6/U2 reaction on Branch
oligonucleotide
Nature 413, 701 - 707 (2001) Splicing-related
catalysis by protein-free snRNAs SABA VALADKHAN
JAMES L. MANLEY
29
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

30
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 Us are added sequentially at positions
  directed by the guide RNA
• Similar mechanism to that used in splicing

31
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.

32
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.