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
Does not require ATP hydrolysis

3. Self-splicing in pre-rRNA in Tetrahymena : T. Cech et al. 1981
Exon 1
Exon 2
Intron 1 +
pre-rRNA
Spliced exon
Intron circle
Intron linear
Nuclear extract
GTP + -
Products of splicing were resolved by gel electrophoresis:
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 mechanismOH
Intron 1 U U P P U A
Exon 1
Exon 2 P P G U
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 C’s to CCCCC

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

8. Active sites in Group I intron self-splicing

9. Domains of the Group I intron ribozyme

10. Exon exchange via trans-splicing
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. Phosphotransfers for Group I vs. Group II & pre-mRNA2’ G HO 3’
Exon 1
Exon 2
Exon 1
Exon 2 A 2’ G OH OH 2’ A
Exon 1+2
Exon 1+2 + G + OH
Group II and pre-mRNA
Group I

16. 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

17. 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

18. Splicing of pre-mRNA, step 1

19. Splicing of pre-mRNA, step 2

20. 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.

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

22. Analysis reveals a lariate structure in inter-mediate

23. 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

24. 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

25. Sm proteins may form ring around snRNAs
ANGUS I. LAMOND
Nature 397, (1999)
RNA splicing: Running rings around RNA

26. 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, (1999)
RNA splicing: Running rings around RNA

27. 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.

28. Spliceosome assembly and catalysisHO 2’
U2 snRNP A HO 2’ U1
snRNP
Exon 1 A HO 2’
Exon 2 U5
snRNP
U4/U6 U6 U4
Sm proteins
snRNAs
Other proteins A G U O H
U4? A O 2’ G U U6 U2 H U1 U5
Spliceosome
Exons 1+2 2’ A

29. Catalysis by U6/U2 on branch oligonucleotide in vitro
Figure 1 Base-pairing interactions in the in vitro-assembled complex of
U2–U6 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, (2001)
Splicing-related catalysis by protein-free
snRNAs
SABA VALADKHAN &
JAMES L. MANLEY

30. Biochemical results for U6/U2 reaction on Branch oligonucleotide
Nature 413, (2001)
Splicing-related catalysis by protein-free
snRNAs
SABA VALADKHAN &
JAMES L. MANLEY

31. 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

32. 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

33. 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.

34. 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.