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Gene Expression > Splicing and the spliceosome > Flashcards

Splicing and the spliceosome Flashcards

1
Q

What is splicing?

A

The process of the spliceosome removing introns from and joining exons of pre-mRNA to produce mRNA that can be translated. Occurs in the nucleus.

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2
Q

What is the 5’ splice site (5’ SS)?

A

The GU at the 5’ end of the intron to be spliced. The phosphodiester bond preceding this sequence is broken.

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3
Q

What is the 3’ splice site (3’ SS)?

A

The AG at the 3’ end of the intron to be spliced. The phosphodiester bond after this sequence is broken.

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4
Q

What is step 1 of the splicing reaction?

A

The 2’ OH of the branchpoint (BP) adenosine carries out a nucleophilic attack on the 5’ splice site (phosphate before G). An intermediate lariat is formed by the intron where the 5’ G of the intron is attached to the BP A by a 2’ to 5’ phosphodiester bond.

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5
Q

What is step 2 of the splicing reaction?

A

The 3’ OH of exon 1 is available and carries out a nucleophilic attack on the 3’ splice site (phosphate after G). The exons are joined.

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6
Q

What happens to the intron lariat after splicing?

A

It is debranched (linearised) and then degraded.

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7
Q

Which sequences are conserved in introns?

A

5’ SS, 3’ SS, BP nucleotide.

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8
Q

How do introns vary between species?

A
  • Different lengths
  • Different sequences apart from conserved points.
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8
Q

What does greater sequence variability in humans allow?

A

Greater variation in splicing - not so constrained.

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8
Q

What are in vitro splicing assays?

A

We incubate a synthetic substrate (with composition altered e.g. depletion of a splicing factor) with a nuclear extract and observe splicing (via presence of the spliced mRNA - moves further in gel).

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8
Q

How has cryo-EM improved understanding of splicing?

A

Interactions of different splicing proteins and RNA can be characterised in high resolution using samples from in vitro splicing assays. Validated previous discoveries.

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8
Q

What are the snRNA/Ps?

A

U1, U2, U4, U5 and U6.

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9
Q

What are properties of snRNAs?

A
  • Highly structured (hairpin loops)
  • Interact with proteins to form snRNPs
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9
Q

What structures do the Sm and Lsm proteins form when binding snRNAs?

A

Heptameric rings.

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10
Q

Which snRNAs bind Sm proteins?

A

U1, U2, U4 and U5.

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11
Q

Which snRNA binds Lsm proteins?

A

U6.

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12
Q

Do snRNAs also bind proteins unique to each od them?

A

Yes; there are >20 snRNA specific proteins.

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13
Q

Which 2 snRNAs are base-paired?

A

U4 and U6.

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14
Q

What is the function of Sm and Lsm proteins?

A
  • Maturation of snRNAs.
  • Nuclear localisation of snRNAs.
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15
Q

Which snRNPs are in the minor spliceosome?

A

U11, U12, U4atac, U5, U6atac.

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16
Q

What proportion of splicing is done by the minor spliceosome?

A

0.5-1%

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17
Q

What is the purpose of the minor spliceosome?

A

We don’t really know.

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18
Q

How are the major and minor spliceosomes different?

A

They have slightly different sequences (cis-acting motifs).
- The minor spliceosome can also use AU as a 5’ SS and AC as a 3’ SS (where the atac name comes from).
- The minor spliceosome BP sequence is more constrained.
- The minor spliceosome introns are clustered in specific gene families.

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19
Q

What are minor spliceosome introns hypothesised to be involved in?

A

Alternative splicing decisions.

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20
Q

Are the catalytic steps (nucleophilic attacks) of the splicing reaction ATP dependent?

A

No.

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21
Q

How does splicing use ATP?

A

Various steps of spliceosome assembly, disassembly and function require ATP.

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22
Q

How is the complexity of the spliceosome advantageous?

A

It allows it to work on many substrates (different introns).

23
Q

Why is spliceosome ATP dependence advantageous?

A

It allows it to work with high accuracy – rejects sub optimal substrates.

24
Q

What is the first step in spliceosome assembly?

A

Assembly of the A complex.

25
Q

What is the A complex?

A

pre-mRNA with U1 snRNP base paired at the 5’ SS (ATP independent) and U2 snRNP base paired at the BP (ATP dependent).

26
Q

What makes a splice site ‘strong’ or ‘weak’?

A

The base pairing of the pre-mRNA with the snRNA sequences.

27
Q

How does U2 binding at BP facilitate nuclear attack by the adenosine?

A

It causes the BP A to bulge from the RNA duplex.

28
Q

What catalyses U2 assembly at the BP?

A

Prp5 - an RNA-dependent ATPase.

29
Q

What is the second step in spliceosome assembly?

A

Assembly of the B complex (catalytic core scaffold).

30
Q

What is the function of the Prp8 protein?

A

Anchors the U2/5/6 snRNAs and intron in place for reaction.

31
Q

What catalyses the splicing reactions?

A

The RNA catalytic core (not any proteins!). There are 2 Mg2+ ions that are coordinated by phosphate residues in the B complex snRNAs. These ions facilitate the reactions by interacting with more phosphate groups in the pre-mRNA (breaks the bonds). One ion catalyses step 1, and the other ion catalyses step 2.

32
Q

What is the B complex?

A

U2, U5 and U6 snRNPs with various stabilising proteins. Forms a complex tertiary structure.

33
Q

How does the B complex form?

A

U4/U5/U6 trisnRNP complex is recruited.
U4 and U6 are initially base paired, but this is unwound by the Brr2 helicase (ATP dependent) so U6 can base pair with U2. U2 and U6 also base pair with the BP and 5’ SS respectively (U6 displaces U1), so hold the pre-mRNA in a specific conformation.
U5 loop 1 binds both exons via weak base pairing and protein interactions, stabilising the exons next to each other in the intermediate.

34
Q

Which organelle can splice its own RNA?

A

The ribosome.

35
Q

Which organelle is thought to be an evolutionary ancestor of the spliceosome?

A

The ribosome.

36
Q

What is the kinetic proof reading model for splicing?

A

As the splicing reaction progresses, it must ‘pass’ all of the ATP-dependent steps - if the substrate is not perfect then it is rejected.
This ensures high accuracy.

37
Q

Why does splicing need to be highly accurate?

A

The splicing motifs are only short, so there are lots of similar sequences in the genome that should not be spliced!

38
Q

What promotes splicing step 2?

A

FAST ATP-dependent Prp16 removal of Cwc25 (protein involved in step 1) from the spliceosome. Occurs when substrates have the right consensus sequences.

39
Q

What inhibits splicing step 2?

A

SLOW ATP-dependent Prp16 removal of Cwc25 (protein involved in step 1) from the spliceosome. Occurs when substrates have mispairings in consensus sequences. In this case, the spliceosome disassembles from the pre-mRNA.

40
Q

What happens at the end of the splicing cycle?

A

The components are recycled for the next splicing reaction - why it can be viewed as a cycle!

41
Q

How many proteins are involved in splicing in yeast?

A

≈150

42
Q

How many proteins are involved in splicing in humans?

A

> 300

43
Q

What type of reactions are those in step 1 and 2?

A

Transesterifications. Attack on the RNA phosphodiester bonds.

44
Q

What are the cis-acting sequence elements required for splicing?

A

5’ SS, 3’ SS, BP.

45
Q

What are the trans acting elements required for splicing?

A

snRNPs, auxiliary proteins (assist snRNPs).

46
Q

How is budding yeast and human genome architecture different?

A
  • Yeast have much fewer genes with introns.
  • Most yeast genes with introns only have 1 intron (human median is 11 introns)
  • Humans have longer introns (about 1000 times longer).
47
Q

Why does splicing use more energy than capping or polyadenylation (in humans)?

A

It must happen multiple times per transcript, compared to only once.

48
Q

How does the commitment complex form?

A

ATP independent folding of the pre-mRNA, mediated by SR and SR-like proteins that form cross bridges to join the 5’ SS and 3’ SS and therefore bring the 2 exons into close proximity.

49
Q

What are SR proteins?

A

Proteins that promote splice site recognition. They contain serine and arginine repeats to facilitate protein-protein interactions. They have RNA binding domains to facilitate bridging of the pre-mRNA. They recruit U2 snRNP (ATP dependent).

50
Q

How are SR proteins regulated?

A

Phosphorylation / dephosphorylation can change their activity or cellular localisation.

51
Q

What are hnRNPs?

A

Ribonucleoproteins that inhibit splice site recognition via binding to the splice sites and blocking their recognition by other spliceosome factors.

52
Q

What is the intron definition model?

A

The 5’ SS, 3’SS and BP within the intron are recognised by the splicing factors.

53
Q

What is the exon definition model?

A

(When introns are much longer than exons) The splicing machinery recognises the exons. SR proteins recognise exon splicing enhancers (ESEs) (purine rich) and recruit splicing factors to either side of the exon.

54
Q

Is the intron or exon definition model correct?

A

There is evidence for both occurring, so both occur. Which is used depends on the pre-mRNA architecture.

55
Q

What is evidence for the intron definition model?

A

Increasing the length of small introns flanking a large exon (i.e. lower eukaryote genome architecture) causes exon skipping. Since the exon is unchanged, it implies increased intron length has prevented spliceosome assembly.

56
Q

What is evidence for the exon definition model?

A

A single exon construct (no introns, just flanking SS sequences) was able to recruit known splicing machinery in the same way as a construct with a full intron and flanking exons.

57
Q

Why does initial exon definition need to switch to an intron definition process to complete splicing?

A

The 3’ SS (precedes exon) needs to interact with the 5’ SS preceding it instead of the 5’ SS after the exon so that the intron can be removed.

58
Q

Is the spliceosome able to work on different genome architectures?

A

Yes, it is malleable and can work in yeast and human genomes.

59
Q

What did cryo-EM recently reveal about the splicing process steps?

A

Splicing machinery is initially recruited to either side of an exon (exon definition) and forms the A complex. When the trisnRNP is recruited there is remodelling to an intron definition B complex structure (spans intron).

60
Q

In which organisms does intron definition dominate?

A

Budding yeast.

61
Q

In which organisms does exon definition dominate?

A

Mammals.

Gene Expression (9 decks)

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