1. Molecular Biology Fourth Edition
Lecture PowerPoint to accompany
Molecular Biology Fourth Edition
Robert F. Weaver
Chapter 14
Messenger RNA Processing I:
Splicing
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

2. This is bhgty the human b-globin qwtzptlrbn gene.
14.1 Genes in Pieces
Consider the sequence of the human b-globin gene as a sentence:
This is bhgty the human b-globin qwtzptlrbn gene.
Two italicized regions make no sense
Contain sequences unrelated to the globin coding sequences surrounding them
Called intervening sequences, IVSs
Usually called introns
Parts of the gene making sense
Coding regions
Exons
Some lower eukaryotic genes have no introns

3. Evidence for Split Genes
Most higher eukaryotic genes coding for mRNA and tRNA are interrupted by unrelated regions called introns
Other parts of the gene, surrounding the introns, are called exons
Exons contain the sequences that finally appear in the mature RNA product
Genes for mRNAs have been found with anywhere from 0 to 362 exons
tRNA genes have either 0 or 1 exon

4. RNA Splicing Introns are present in genes but not in mature RNA
How does the information not find its way into mature RNA products of the genes?
Introns are never transcribed
Polymerase somehow jumps from one exon to another
Introns are transcribed
Primary transcript result, an overlarge gene product is cut down by removing introns
This is correct process
Process of cutting introns out of immature RNAs and stitching together the exons to form final product is RNA splicing

5. Splicing Outline
Introns are transcribed along with exons in the primary transcript
Introns are removed as the exons are spliced together

6. Stages of RNA Splicing
Messenger RNA synthesis in eukaryotes occurs in stages
First stage:
Synthesis of primary transcript product
This is an mRNA precursor containing introns copied from the gene if present
Precursor is part of a pool of large nuclear RNAs – hnRNAs
Second stage:
mRNA maturation
Removal of introns in a process called splicing

7. Splicing Signals
Splicing signals in nuclear mRNA precursors are remarkably uniform
First 2 bases of introns are GU
Last 2 are AG
5’- and 3’-splice sites have consensus sequences extending beyond GU and AG motifs
Whole consensus sequences are important to proper splicing
Abnormal splicing can occur when the consensus sequences are mutated

8. 14.2 Mechanism of Splicing of Nuclear mRNA Precursors
Intermediate in nuclear mRNA precursor splicing is branched – looks like a lariat
2-step model
2’-OH group of adenosine nucleotide in middle of intron attacks phosphodiester bond between 1st exon and G beginning of intron
Forms loop of the lariat
Separates first exon from intron
3’-OH left at end of 1st exon attacks phosphodiester bond linking intron to 2nd exon
Forms the exon-exon phosphodiester bond
Releases intron in lariat form at same time

9. Simplified Mechanism of Splicing
Excised intron has a 3’-OH group
Phosphorus atom between 2 exons in spliced product comes from 3’-splice site
Intermediate and spliced intron contain a branched nucleotide
Branch involves 5’-end of intron binding to a site within the intron

10. Signal at the Branch
Along with consensus sequences at 5’- and 3’-ends of nuclear introns, branchpoint consensus sequences also occur
Yeast sequence invariant: UACUAAC
Higher eukaryote consensus sequence is more variable
Branched nucleotide is final A in the sequence

11. Spliceosomes Splicing takes place on a particle called a spliceosome
Yeast spliceosomes and mammalian spliceosomes have sedimentation coefficients of 40S and 60S
Spliceosomes contain the pre-mRNA
Along with snRNPs and protein splicing factors
These recognize key splicing signals and orchestrate the splicing process

12. snRNPs
Small nuclear RNAs coupled to proteins are abbreviated as snRNPs, small nuclear ribonuclear proteins
The snRNAs (small nuclear RNAs) can be resolved on a gel:
U1, U2, U4, U5, U6
All 5 snRNAs join the spliceosome to play crucial roles in splicing

13. U1 snRNP
U1 snRNA sequence is complementary to both 5’- and 3’-splice site consensus sequences
U1 snRNA base-pairs with these splice sites
Brings the sites together for splicing is too simple an explanation
Splicing involves a branch within the intron

14. Wild-Type and Mutant U1 snRNA
Genetic experiments completed
Base pairing between U1 snRNA and 5’-splice site of mRNA precursor
Is necessary
Not sufficient for binding

15. U6 snRNP
U6 snRNP associates with the 5’-end of the intron by base pairing through the U6 RNA
Occurs first prior to formation of lariat intermediate
Character may change after first step in splicing
Association between U6 and splicing substrate is essential for the splicing process
U6 also associates with U2 during splicing

16. U2 snRNP
U2 snRNA base-pairs with the conserved sequence at the splicing branchpoint
This base pairing is essential for splicing
U2 also forms base pairs with U6
This region is called helix I
Helps orient snRNPs for splicing
5’-end of U2 interacts with 3’-end of U6
This interaction forms a region called helix II
This region is important in splicing in mammalian cells, not in yeast cells

17. Yeast U2 Base Pairing with Yeast Branchpoint Sequence
Source: Adapted from Parker, R., P. G. Sliciano, and C. Guthrie, Recognition of the TACTAAC box during mRNA splicing in yeast involves base pairing to the U2-like snRNA. Cell 49:230, 1987.

18. U5 snRNP
U5 snRNA associates with the last nucleotide in one exon and the first nucleotide of the next exon
This should result in the two exons lining up for splicing

19. U4 snRNP U4 base-pairs with U6 Its role seems to be to bind U6
When U6 is needed in a splicing reaction U4 is removed
U6 gene is split by an mRNA-type intron in at least two yeast species

20. snRNP Involvement in mRNA Splicing
Spliceosomal complex contains:
Substrate U2 U5 U6
The complex ready for the 2nd step in splicing can be drawn as a group II intron at same stage of splicing
Spliceosomal snRNPs substitute for elements at center of catalytic activity of group II introns at same stage of splicing

21. Spliceosome Catalytic Activity
Catalytic center of spliceosome appears to include Mg2+ and a base-paired complex of 3 RNAs:
U2 snRNA
U6 snRNA
Branchpoint region of the intron
Protein-free fragments of these RNAs can catalyze a reaction related to the first step in splicing

22. Spliceosome Assembly and Function
Spliceosome is composed of many components – proteins and RNA
These components assemble stepwise
The spliceosome cycle:
Assembly
Function
Disassembly
By controlling assembly of the spliceosome, a cell can regulate quality and quantity of splicing and so regulate gene expression

23. Spliceosome Cycle
Assembly begins with binding of U1 to splicing substrate forming a commitment complex, a unit committed to to splicing out the intron
U2 joins the complex next, followed by the others
U2 binding requires ATP
U6 dissociates from U4 and displaces U1 at the 5’-splice site
This step is ATP-dependent
Activates the spliceosome
Allows U1 and U4 to be released

24. snRNP Structure All snRNP’s have the same set of 7 Sm proteins
Common targets of antibodies in patients with systemic autoimmune diseases
Sm protein binds to a common Sm site on the snRNAs: AAUUUGUGG
U1 snRNP has 3 specific proteins
70K has an Mr of 52 kD
A has an Mr of 31 kD
C has an Mr of 17.5 kD
Sm proteins form a doughnut-shaped structure with a hole through the middle, like a flattened funnel

25. Sm Site and RNA Five snRNPs participate in splicing
All contain a common set of 7 Sm proteins and several other proteins that are specific to snRNP
Structure of U1 snRNP reveals that the Sm proteins form a doughnut-shaped structure to which the other proteins are attached

26. A Minor Spliceosome
A minor class of introns with variant but highly conserved 5’-splice sites and branchpoints can be spliced with the help of a variant class of snRNAs
Cells can contain minor snRNAs:
U11 performs like U1
U12 acts like U2
U4atac and U6atac perform like U4 and U6 respectively

27. Commitment, Splice Site Selection and Alternative Splicing
snRNPs do not have enough specificity and affinity to bind exclusively and tightly at exon-intron boundaries
Additional splicing factors are needed to help snRNPs bind
Some splicing factors are needed to bridge across introns and exons and so define these RNA elements

28. 3’-Splice Site Selection
Splicing factor Slu7 is required for correct 3’-splicing site selection
Without Slu7, splicing to correct 3’-splice site AG is suppressed and splicing to aberrant AG’s within 30 nt of the branchpoint is activated
U2AF is also required for 3’-splice site recognition
65-kD U2AF subunit binds to polypyrimidine tract upstream of 3’-splice site and 35-kD subunit binds to the 3’-splice site AG

29. Commitment
Commitment to splice at a given site is determined by an RNA-binding protein
This protein binds to splicing substrate and recruits other spliceosomal components
The first component to follow is U1
SR proteins SC35 and SF2/ASF commit splicing on human b-globin pre-mRNA and HIV tat pre-mRNA
Part of the commitment involves attraction of U1 in some cases

30. Bridging Proteins and Commitment
Yeast commitment complex has a branchpoint bridging protein (BBP) binds to:
U1 snRNP protein at the 5’-end of the intron
Mud2p near the 3’-end of the intron
RNA near the 3’-end of the intron
Bridges the intron and could play a role defining intron prior to splicing
Mammalian BBP is SF1, may serve same bridging function

31. Yeast Two-Hybrid Assay

32. Intron-Bridging Protein-Protein Interactions
Branchpoint bridging protein binds to U1 snRNP protein
Comparison of yeast to mammalian complexes is seen at right

33. Role of the RNA Polymerase II CTD
C-terminal domain of the Rpb1 subunit of RNA polymerase II stimulates splicing of substrates that use exon definition
This does not apply to those that use intron definition to prepare for splicing
CTD binds to splicing factors and could assemble the factors at the end of exons to set them off for splicing

34. Alternative Splicing
Transcripts of many eukaryotic genes are subject to alternative splicing
This splicing can have profound effects on the protein products of a gene
Can make a difference between:
Secreted or membrane-bound protein
Activity and inactivity
Products of 3 genes in sex determination pathway of the fruit fly are subject to alternative splicing

35. Sex-Specific Splicing
Female-specific splicing of tra transcript gives:
An active product that causes female-specific splicing of dsx pre-mRNA
This produces a female fruit fly
Male-specific splicing of tra transcript gives:
An inactive product that allows male-specific splicing of dsx pre-mRNA
This produces a male fruit fly

36. Tra and Tra-2
Tra and its partner Tra-2 act in conjunction with one or more other SR proteins to commit splicing at the female-specific splice site on the dsx pre-mRNA
Commitment is probably the basis of most, if not all, alternative splicing schemes

37. Alternative Splicing Patterns
Alternative splicing of the same pre-mRNA gives rise to very different products
Alternative splicing patterns occur in over half of human genes
Many genes have more than 2 splicing patterns, some have thousands

38. Types of Alternative Splicing
Begin transcripts at alternative promoters
Some exons can simply be ignored resulting in deletion of the exon
Alternative 5’-splice sites can lead to inclusion or deletion of part of an exon
Alternative 3’-splice sites can lead to inclusion or deletion of part of an exon
A retained intron can be retained in the mRNA if it is not recognized as an intron
Polyadenylation causes cleavage of pre-mRNA and loss of downstream exons

39. Silencing of Splicing
What stimulates recognition of signals under only some circumstances?
Exons can contain sequences –
Exonic splicing enhancers (ESEs) stimulate splicing
Exonic splicing silencers (ESSs) inhibit splicing

40. Reporter Construct Detects ESS Activity

41. 14.3 Self-Splicing RNAs
Some RNAs could splice themselves without aid from a spliceosome or any other protein
Tetrahymena 26S rRNA gene has an intron, splices itself in vitro
Group I introns are a group of self-splicing RNAs
Another group, Group II introns also have some self-splicing members

42. Group I Introns
Group I introns can be removed in vitro with no help from protein
Reaction begins with attack by a guanine nucleotide on the 5’-splice site
Adds G to the 5’-end of the intron
Releases the first exon
Second step, first exon attacks the 3’-splice site
Ligates 2 exons together
Releases the linear intron
Intron cyclizes twice, losing nucleotides each time, then linearizes a last time

43. Linear Introns

44. Group II Introns
RNAs containing group II introns self-splice by a pathway using an A-branched lariat intermediate, like spliceosome lariats
Secondary structures of the splicing complexes involving spliceosomal systems and group II introns are very similar