Coordination of splicing with other gene expression mechanisms

Question 1

Is the gene expression pathway linear?

Answer 1

No - there is coordination between events, and some occur at the same time.
Diagrams usually imply a false linearity.

Question 2

What is electron microscopy evidence that splicing occurs cotranscriptionally?

Answer 2

We can view pre-mRNA being transcribed from a DNA, protein complexes associating with it, and loops of the pre-mRNA (introns) being removed before the transcription is complete.

Question 3

What is FISH evidence that splicing occurs cotranscriptionally?

Answer 3

Splicing proteins colocalise with sites of active transcription in the nucleus.

Question 4

What is single molecule intron tracking evidence that splicing occurs cotranscriptionally?

Answer 4

Defining the starts and ends of RNA that is being transcribed (associated with Pol II) allows us to track the pre-mRNA lengths from different pol IIs along the gene - if it gets shorter as transcription continues, splicing is occurring. Showed that ≈90% of transcripts had begun to be spliced within 100 nts of transcription initiation.

Question 5

Do splicing and transcription have similar rates?

Answer 5

Yes - spliceosome assembly, splicing catalysis and transcription do have similar rates.

Question 6

What nt range downstream of the 3’ SS does splicing catalysis (step 2) occur?

Answer 6

26 - 129 nts.

Question 7

Does step 1 of splicing occur before pol II has reached the 3’ SS?

Answer 7

Yes! U1 and U2 are already associated. The spliceosome assembles rapidly on nascent RNA.

Question 8

How is RNA pol II activity regulated?

Answer 8

Modification of its C terminal domain (CTD) (tail). The CTD is made up of heptad repeats which are dynamically modified (>100 proteins bind it). Various types of modifications.

Question 9

How do we characterise pol II CTD modifications at different stages of transcription?

Answer 9

ChIP of pol II across the gene unit. Use Abs specific to modifications of specific CTD aas.

Question 10

Which CTD modification is enriched at the TSS?

Answer 10

Serine 5 phosphorylation (S5P).

Question 11

Which CTD modifications are enriched at the polyA site?

Answer 11

• Serine 2 phosphorylation (S2P)
• Threonine 4 phosphorylation (T4P)

Question 12

What does S5P recruit?

Answer 12

• Capping enzymes.
• Early spliceosome components. Evidence for cotranscriptional splicing!

Question 13

What does S2P recruit?

Answer 13

• Cleavage / polyA factors.

Question 14

How can we observe where modifications of pol II occur in relation to its position on the pre-mRNA?

Answer 14

• ChIP seq (low resolution)
• NET seq (high resolution)

Question 15

How do we use NET seq to observe modifications of pol II occur in relation to its position on the pre-mRNA?

Answer 15

• Use CTD modification specific Abs to purify modified pol II.
• Removed bound DNA.
• Sequence pre-mRNA associated with it.
• Also use mass spec to identify proteins associated with these modifications.

Question 16

How are sequential splicing events promoted by RNA pol II?

Answer 16

RNA pol II reorganizes its CTD modifications and interactions during transit along the DNA template to promote the sequential action of distinct RNA-processing events.

Question 17

How is splicing kinetically coupled to transcription?

Answer 17

If the RNA pol II is transcribing quickly, weak splice sites and therefore exons tend to be skipped. This is because they are outcompeted by strong splice sites that have already been transcribed before the weak splice site is recognised.
If the RNA pol II is transcribing slowly, weak splice sites and therefore exons are included.

Question 18

Does speed of RNA pol II impact anything other than spliceosome recruitment?

Answer 18

Yes:
- Regulatory protein recruitment / binding.
E.g. there is alternative splicing of the CFTR gene dependent on pol II speed. When it is fast, negative regulators are not recruited and exon 9 is included, however when it is slow the regulators are recruited and exon 9 is skipped.
- The ability of RNA to fold in different ways is also impacted by pol II speed, which may affect splice site recognition.

Question 19

Why might different genes need different rates of elongation?

Answer 19

They want to optimise alternative splicing in a specific way.

Question 20

Can splicing impact transcription?

Answer 20

Yes:
- RNA pol II pauses due to hyperphosphorylation of CTD at splice sites.
- More time for splicing to occur; promotes inclusion of alternatively spliced exons.
- It is dephosphorylated once the lariat is released and elongation continues.
- Proven by inhibiting splicing; observed no change in CTD modification and uniform elongation rate.

Question 21

How is nucleosome positioning associated with gene structures?

Answer 21

• Nucleosomes are enriched at (internal) exons.
• Nucleosomes are depleted at introns.

Question 22

Which chromatin mark is enriched at exons?

Answer 22

H3K36me3

Question 23

Do we know whether it is chromatin structure influencing exons / introns or vice versa?

Answer 23

No - it could be either.

Question 24

What is thought to be the purpose of nucleosomes at exons?

Answer 24

They slow pol II transcription over exons, which allows more time for splicing factors to bind pre-mRNA.
Aided by CTD modification causing pol II pausing at junctions.

Question 25

What are examples of chromatin modifications impacting splicing decisions?

Answer 25

• H3K9ac in NCAM gene increases elongation rate and promotes exon 18 skipping. H3K9me in NCAM gene reduces elongation rate and promotes exon 18 inclusion.
• H3K36me3 in FGFR2 gene recruits negative regulator proteins that promote exon skipping. H3K4me3 in FGFR2 gene reduces recruitment of negative regulator proteins so promotes exon inclusion.

Question 26

What is an exon junction complex (EJC)?

Answer 26

A complex of proteins that is deposited 20-24 upstream of a splice site that signal an exon-exon junction to a ribosome.

Question 27

What is the function of the EJC?

Answer 27

Quality control of the mRNA.

Question 28

How does the EJC give translation the green light?

Answer 28

After successful pioneer (first round of) translation, the EJCs are cleared from the mRNA. This signals all other ribosomes are free to translate and form a polysome.

Question 29

How does the EJC promote mRNA degradation in the event of a premature termination codon (PTC)?

Answer 29

The UPF1-containing SURF complex is recruited to the PTC. When an EJC is >50 nts downstream of this PTC, it interacts with the SURF complex and triggers a cascade that results in nonsense mediated decay of the mRNA.

Question 30

What are exons containing PTCs called?

Answer 30

NMD exons or poison exons.

Question 31

How is alternative splicing used to regulate translation (molecular switch)?

Answer 31

Introducing a PTC exon inhibits translation.

Question 32

What is an example of alternative splicing being used as a molecular switch?

Answer 32

• PTBP1 is present in non-neuronal cells. It represses inclusion of exon 10 in the related gene PTBP2. This skipping introduces a PTC. When PTBP1 is switched off and exon 10 is included in PTBP2, there is no PTC (frame shift?) and neuron-specific alternative splicing and neuronal differentiation is promoted.
• SR proteins have highly conserved, alternatively spliced PTC exons that are used as switches to control activity of the SR proteins; auto regulation.

Question 33

Which protein is a master regulator of alternatively spliced NMD exons in RBPs?

Answer 33

Rbfox2.

Question 34

What type of regulator is Rbfox2?

Answer 34

Positive and negative!
Promotes inclusion of NMD exons (reduces mRNA) for some genes e.g. Tra2a.
Promotes skipping of NMD exons (increases mRNA) for some genes e.g. Tia1.

Question 35

Why is Rbfox2 called a master regulator?

Answer 35

Switching of Rbfox2 activity causes activity changes in many downstream RBPs (promotes or inhibits their translation) and the genes they act on.