L14 Eukaryotic Transcript Processing 2

Question 1

Self-splicing introns

Answer 1

See onenote diagram

• RNA catalysed reaction compared to spliceosomal-based
• e.g. protozoan rRNA genes and some mitochondrial genes

The only way introns could’ve genetically survive over time is if they were self-splicing; introns are slightly negatively deleterious as it is essential interrupting genes

Question 2

Two theories of intron evolution

Answer 2

1. introns evolved and proliferated early but lost in some genes
2. introns evolved late and are proliferating
   - to address this, comparison of intron positions in related genes in closely related species (orthologues)

Question 3

Intron evolution - orthologue comparison

Answer 3

See onenote diagram

- intron position can vary

Question 4

Intron splicing, gain, loss

Answer 4

See onenote diagram

1. spliceosomal intron splicing
2. intron gain by reverse splicing
3. intron loss

Question 5

Intron Sliding

Answer 5

See onenote diagram

Change in intron position

Intron gets inserted in a concerted reaction but in a different position that is close to the original site

Sequence of intron exactly the same BUT now in a different position in the gene

Question 6

Intron function

Answer 6

See onenote diagram

Introns may code for snoRNA

• small nucleolar RNA
• generally transcribed by RNA pol2
• modify bases in rRNA and tRNA
• introns can have genes in them, usually non-coding genes
• snoRNA sitting in introns
• intron lariat needs to be linearised and processed to give mature snoRNA

Alternative splicing

Question 7

snoRNA

Answer 7

Some snoRNA can exists as a single gene -monocistronic
- Need exonuclease activity to trim them

Back to back snoRNA - polycistronic

• Each snoRNA needs to be separated and trimmed
• Need endonuclease activity to separate them
• Need exonuclease activity to trim them

Specific snoRNA structure

• Can bind to their target sequence
• Complementary to target RNA
• As snoRNA is part of the snorp protein complex, the enzyme in the snorps modifies the rRNA and tRNA

Question 8

SV40

Answer 8

Differential intron splicing

- T gene has two different forms 
- Within the intron, there is an in-frame stop codon => makes truncated protein, small t-antigen 
- If intron spliced out, the two exons are spliced together and makes the large t-antigen 
- Large t-antigen causes cells that are infected by SV40 to undergo transformation (neoplasty transformation), cells become immortalised (like cancerous cells), large t-antigen drives proliferation of these cells 
- Usually the immune system recognises those cells or the cells recognise it themselves and they tend to die (apoptosis)
- Small t-antigen blocks apoptopic mechanism, cell cannot kill itself 
- From SV40 infection, cells become cancerous and makes lots of SV40 by using both small and large t-antigen

Question 9

Drosophila DSCAM

Answer 9

See onenote diagram

Orthologue of down syndrome cell adhesion molecule

24 alternatively spliced exons

38,016 combinations of mature mRNAs

Two roles:
1. neural patterning and antigen recognition

Neural patterning:

• self or non-self recognition
• neurite self-avoidance

These molecules are cell surface molecules, involved in identifying cells to other cells - cells have slightly different properties as they have different exons in them e.g. two neurons can recognise that they’re different as they have different molecules on their surface

Question 10

Transcript splicing - repression and activation

Answer 10

See OneNote diagram

If the stochiometry of the protein at the end gives you what you need, then no further regulation is required
BUT if you want a specific form in a specific cell type then it needs to be regulated

Repression
Protein binds to the intron as it recognises the sequence to the RNA and blocks the spliceosome complex from binding and cutting that intron

Activation

- Intron not spliced out efficiently
- Activator protein binds to intron and interacts with the spliceosome complex, stabilises it, intron can be spliced

Question 11

Transcript processing - RNA editing

Answer 11

see apo-B example in onenote

• post-transcriptional modification of mRNA sequence
• DNA sequence differs from mRNA sequence
• protein sequence differs from that predicted from DNA sequence

2 mechanisms:

1. nucleotide modification (site-specific deamination)
2. nucleotide insertion by guide RNAs

more common than originally thought but not as common as some would have us believe

Question 12

RNA editing - nucleotide modification by specific enzymes

Answer 12

Cytosine deamination by cytidine deaminase:
- cytosine to uracil

Adenine deamination by adenine deaminase (ADAR):

• adenine to inosine
• Inosine has altered base pair properties, will base pair will A,C,G => tRNA can recognises triplet of codons due to inosine

Question 13

RNA editing - insertion or deletion of U’s

Answer 13

See onenote diagram

• RNA in mitochondria of some protozoans
• Protein looks nothing like what you predicted if you looked at the coding DNA

Question 14

RNA editing - carried about by guide RNAs

Answer 14

See onenote diagram

• base pairing mediated (to maintain reading frame)

Question 15

Post-transcription - RNA localisation

Answer 15

See onenote diagram

• pre-mRNA is retained in the nucleus
• mature mRNA exported out of the nucleus
• proteins specifically bound to fully processed mature mRNA signal transport out of the nucleus

Will affect whether the protein will be made
Can maintain mature RNA in nucleus and block gene expression
Control when it is transported into the cytoplasm, controls when translation occurs