3/4 - Alternative Splicing

Question 1

Give the steps of intronic splicing

Answer 1

1. U1 binds to pre-mRNA
2. Other ribozymes bind to make a spliceosome (uses ATP)
3. A snRNP binds to make a pre-catalytic spliceosome
4. ATP and GTP are used and some ribozymes leave to activate complex
5. Lariat formed
6. Lariat excised bound to proteins, which later come off (with ATP) to leave the intron

Question 2

Give the five RNA/protein components of the core human spliceosome

Answer 2

U1: snRNA with Sm protein. Binds with 5’ splice site.

U2: snRNA with Sm protein. Binds with branch site.

U6/U4: snRNAs with Sm and LSm proteins

US: snRNA with Sm protein

These are pretty conserved, with most having homologs in yeast and presumably other eukaryotes

Question 3

What is the 5’ splice site in humans?

Answer 3

CAG-GURAGU

8 base pairs long

Question 4

Give the three parts of the 3’ splice site in humans

Answer 4

• CAG
• Polypyrimidine tract immediately upstream of the 3’ splice site (stretch of U residues)
• Branchpoint sequence (YNYURAC)

Question 5

Describe the two ways that a spliceosome can interact with the transcript.

Answer 5

• RNA-RNA base pairing (eg. hydrogen bonds with U1, U2, U4 etc.)
• Protein-RNA interactions

Question 6

What is the function of introns?

Answer 6

No good answer.

Some introns are involved in regulation of gene expression, but these cases tend to be species specific.

Question 7

Why are there introns at all?

Answer 7

• Introns thought to have arisen from parasites, self splicing group II introns etc.
• They could have invaded the nucleus of the ancestral eukaryotes from endosymbiotic organelles, such as the mitochondria.
• Group II introns are removed by a mechanism similar to what’s carried out by the spliceosome.

Question 8

Why do introns still exist?

Answer 8

• After their arisal (likely parasitic) their removal became very complex, dependent on many tightly connected steps, with which eukaryotic genomes got ‘stuck’ with them forever.
• Some introns have regulatory roles (eg. enhancing eukaryotic gene expression), making them important elements of the genome.

Question 9

How do splicing factors find and define the introns?

Answer 9

Recognition of splice sites:

FOrmation of commitment complex (E/early complex)

• Binding of U1 and SF factors to the 5’ SS
• Binding of BP, Py and 3’ SS recognizing factors (BBP, U2AF65 and U2AF35, respectively)
• SR and other factors interact, bridging together the two regions.

Question 10

What is intron definition? How does this differ in genes with numerous and very long introns (eg. animals)?

Answer 10

The type of recognition where the elements (eg. U1, U2, BBP, U2AF65 and U2AF35) are defined by interactions across the intron at specific sites.

In animals with longer and more introns, the splice sites are defined by interactions across the exon (exon definition - think of human fibronectin gene experiment)

Question 11

What did experiments with a 3’exon ‘mini’ version of the human fibronectin gene (normally a very long gene with many introns) reveal?

Answer 11

Some parts of the exonic sequence are important for successful splicing!

This and other studies showed that exons can contain sequences that favour either the inclusion or exclusion of that exon in the mature mRNA. Likewise, it was found that sequences that enhance or limit a particular type of splicing are also located inside introns.

The combined effect of these elements (and the protein factors that bind to them) determines the outcome of the process, namely, which exons will be present in the final product (enter alternative splicing)

Question 12

Give three methods for splicing enhancers

Answer 12

Splicing enhancers act like glue to attach the spliceosome to the pre-mRNA

• Anchoring proteins through stabilization of early components of the spliceosome
• Anchoring snRNPs through stabilization of early snRNPs in spliceosome
• Steric hindrance: blocking binding of silencers to nearby silencer sequences

Question 13

Give four methods for splicing silencing

Answer 13

Splicing silencers sequences prevent access to the splicing machinery:

• Steric hindrance: binding of a single PTB molecule to the branchpoint prevents binding of U2AF and binding of hnRNPA1 prevents U1 snRNP access
• Steric hindrance: Binding of multiple PTB molecules or hnRNP A1 molecules blocks access of splicing factors
• Looping: Splicing repressors can form loops through protein-protein or protein-RNA interactions to block splicing
• Blocking exon definitions: Splicing repressors can bind to ESS silencer elements and prevent interaction across exon during spliceosome assembly

Question 14

List the type of sequences that the following proteins bind to:

• RBP
• SR
• A/B

Answer 14

RBP: Intronic splicing silencers (ISS) or intronic splicing enhancers (ISE)

SR: Exonic splicing enhancers

A/B: (hnRNP proteins) bind to exonic silencers

Question 15

Describe the Nova and Fox families of RNA binding proteins

Answer 15

• Binding of Nova to exons and flanking upstream introns inhibits the inclusion of the alternative exon
• Nova binding to the downstream flanking intronic sequences promotes the inclusion of the alternative exon
• Fox binding upstream intronic sequence inhibits inclusion of the alternative exon
• Fox binding to the downstream intronic sequence promotes the inclusion of the alternative exon

Question 16

What four features affect how pre-mRNAs are decoded by the spliceosome? (list from low recognition to high recognition)

Answer 16

Weak exons

• Poor splice sites
• High concentration of splicing silencers

Strong exons

• Good splice sites
• High concentration of splicing enhancers

The combination of good and bad factors affect how easily the spliceosome will recognize the exon

Question 17

What are the three main types of alternative pre-mRNA splicing?

Answer 17

Exon skipping
- Whole exons are either skipped or pliced into mRNA

Alternative exon size
- Exons can also come in different sizes by using different splice sites

Intron retention
- Within genomic DNA and in unspliced pre-mRNA, exons are separated from each other by introns and splicing usually joins the exon together. HOwever, whole introns can sometimes be retained in mRNAs (ie. remain unspliced)

Question 18

List 7 different types of alternative splicing

Answer 18

• Exon skipping
• Alternative 3’ ss
• Alternative 5’ ss
• Intron retention
• Mutually exclusive exons
• Alternative promoters
• Alternative poly(A)

Question 19

Why is intron retention not very common in animals

Answer 19

Most common in yeast and plants but not metazoa.

• Occurs with small genomes/small introns
• Introns are usually much bigger than exons and nucleotide sequences of introns are not subject to restriction or pressure like coding sequences. Retaining an intron is going to kill the coding potential of a mRNA and produce a nonfunctional protein (mostly due to early stop codon)
• Especially when the intron is longer (more chance of accidental stop codon!)

Question 20

How can the splicing pattern of a gene by disturbed by mutations?

Answer 20

• The identity or strength of a splicing signal can be changed
• The new patter can become fixed or not depending on many factors (eg. phenotype effect of mutation, population structure and dynamics leading to genetic drift or not)
• Transition from one splicing pattern to another over evolution or in another tissue happens by the strengthening/creation or weakening of splice sites

Question 21

What are the two mechanism may lead to a constitutive exon to become an alternative exon?

Answer 21

Mutations

• Mutations that lead to weak recognition of exon and result in exon skipping
• Mutation that leads to a new 5’ or 3’ splice site
• Mutations that disrupt intron/exon silencers/enhancers

Secondary structure
- Usually formed between two alu elements in opposite orientation, can interrupt exon recognition

Question 22

What are the possible results of mutations affecting constitutive exon usae? Possible mutation sites?

Answer 22

Results: exon skipping or intron retention

Sites: SSs, polypyrimidine tract, branch point and enhancers

Question 23

What are the possible results of mutations that affect the ratio of alternatie exons?

Answer 23

Mutations will result in the change of the relative rates of inclusion of exon. May result in phenotypic change or not.

Question 24

What is SVA-insertion mediated altered splicing?

Answer 24

Retrovirus gene elements can have elements which the host can use a different way than in the virus (eg. as a pseudoexon)

Question 25

Give an example of alternative splicing being used as a functional way to regulate expression

Answer 25

When RPL30 is abundant, alternative splicing kicks in to block splicing (the protein blocks the splice site)

Alternatively, there can be regulation of molecular pathways by alternative splicing. Eg. signalling pathways (RAS, MAPK etc.) can activate splicing factors to change the splicing pattern of exons.

Question 26

True or false? The complexity of splicing is higher in humans

Answer 26

True, especially in the brain

The facilitation of alternative splicing in humans may explain partially the human brain’s complexity and size

Question 27

How is alternative splicing important in the nervous system of vertebrates?

Answer 27

Non-neuronal cells: Proteins prevent recognition of N1 region of a gene (not expressed)

Neurons: N1 is included because exon definition allows it (includes it)

Question 28

What determines the outcome of RNA splicing of a particular gene in a particular moment?

Answer 28

It is the combination of active splicing proteins available WHEN and WHERE transcription occurs

Splicing reactions are spatially localized, nuclear proteins are not homogenously distributed and splicing proteins and many factors and enzymes involved in transcription and RNA processing are found in distinct domains or speckles.

To be able to affect splicing, factors must be available where the reaction is happening.

Phosphorylation by kinases and dephosphorylation by phosphatases are the most frequently used mechanisms for controlling the activity and localizations of SFs

Question 29

How can RNA polymerase II affect alternative splicing?

Answer 29

Polymerase moves at a particular speed (processivity). With normal RNA polymerase you get normal size RNA, but slow enzyme will give more inclusion of exons = bigger RNA

Slowed transcription allows for

• Weak signals to be seen and acted upon
• Negative and positive regulators to bind an exonic or intronic splicing enhancers or silencer (eg. PTB can bind with certain methylation pattern - not directly kinetic though)

Acetylation can cause fast transcription, methylation slows it.