L13 Eukaryotic Transcript Processing

Question 1

Genes transcribed by RNA pol1

Answer 1

See OneNote

• core RNA pol complex and GTFs
• ribosomal RNA (rNRA) genes except 5S (RNA pol3)

RNA pol 1 has its own GTF but shares the TBP

Question 2

Genes transcribed by RNA pol3

Answer 2

See OneNote

• core RNA pol complex and GTFs: TF2A,B,C
• TF2B has TBP as a component
• small RNAs: 5S RNA, snRNAs, snoRNAs, gRNAs

RNA pol 3 has its own GTFs
Like RNA pol 1 and 2, has TBP as its core component

Question 3

Primary transcript processing

Answer 3

See OneNote diagram

• coordinately processed in the nucleus
• capping, splicing, polyadenylation and termination
• different functions depend on phosphorylation of CTD

Question 4

RNA pol2 recruits RNA processing enzymes via CTD

Answer 4

See OneNote diagram

CTD = repeats of 7 aa in C-terminus of large RNA pol2 subunit
- phosphorylated by GTFs

Phosphorylation at serine 5 drives capping reaction
Phosphorylation at serine 2 drives splicing process

Kinase adds phosphate

Specific enzymes target specific aa in the heptamer repeat

Phosphorylation allows for recruitment of different proteins during different states

Involves machinery that recognises modification of CTD

Question 5

Formation of 5’ mRNA cap

Answer 5

See onenote diagram

• 7 methyl guanine
• triphosphate bound
• 5’ hydroxyl group

5’ end of eukaryotic mRNA

• G added on backwards => ends up with a triphosphate bond and a hydroxyl group that sticks out, no 5’ phosphate to be degraded by endogenous nucleases
• Methylation of guanine
• Specific enzymes recruited to the complex by serine 5 phosphorylation

Question 6

Transcription termination

Answer 6

See onenote diagram

CTD no longer phosphorylated, CstF and CPSF initially binds to CTD
CstF and CPSF then binds to polyA sequence once it has been transcribed
CstF clips RNA

polyA pol - template independent pol that adds A’s to 3’ end
Poly A binding protein stabilises poly A tail
Length of tail can vary significantly
Longer poly A tail = longer half life

Question 7

Transcription termination - Rat1/hXrn2

Answer 7

See onenote diagram

For RNA pol2 dissociation:

Rat1/hXrn2 bound to RNA pol complex
Clipping leads to uncapped end (no serine 5 phosphorylation => no capping machinery)
Rat1/hXrn2 chews away the transcript and the RNA pol dissociates

Clipping itself can destabilise the complex and the RNA pol falls off

Question 8

Transcript splicing

Answer 8

See onenote diagram

Spliceosome catalyses intron splicing

• complex consists of: 150 proteins, 5 small nuclear RNAs (U1,U2,U4/U6,U5 snRNAs)
• snRNAs associate with proteins to form snRNPs (small nuclear ribonucleoproteins - Snurps)
• snRNPs bind in succession to pre-mRNA

5’ splice site consensus sequence = GU-purine-AGU
3’ splice site consensus sequence = CAG

branch site

Question 9

Transcript splicing mechanism

Answer 9

See onenote diagram

Question 10

The role of snRNPs - “Snurps”

Answer 10

1. recognise 5’ splice site and branch site: determine specificity of splicing
2. bring together 5’ splice site and the branch site
3. catalyse RNA cleavage and joining reactions

Question 11

snRNA

Answer 11

See onenote diagram

• snRNA molecules provide the specificity of spliceosomal intron splicing
• snRNA recognises specific sequences by complementary base-pairing

Question 12

Splicing errors

Answer 12

See onenote diagram
e.g. Thalassemia diseases

• proteins can interact with introns and exons to help define these and assist in accurate splicing
• particularly important for genes with very large introns, If introns are large, exons that need to be joined may be far apart
• exon skipping
• cryptic splice site selection

SR = splice regulators, bind to exonic material

Question 13

hnRNPs

Answer 13

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are complexes of RNA and protein present in the cell nucleus during gene transcription and subsequent post-transcriptional modification of the newly synthesized RNA (pre-mRNA). The presence of the proteins bound to a pre-mRNA molecule serves as a signal that the pre-mRNA is not yet fully processed and therefore not ready for export to the cytoplasm.

Question 14

3 types of RNA splicing

Answer 14

See onenote diagram

1. major
2. U12-type
3. trans

Question 15

trans-splicing

Answer 15

See onennote diagram

• generally rare but in Trypanosomes all mRNAs are trans-spliced
• allows a common region to be attached

If you want to put the same domain on many other proteins
Leader exon
- Proteins that you want to direct to a particular location in a cell e.g. traverse/tether itself to a membrane
- Only need one leader sequence instead of 5 individual leader sequence

Question 16

Trans-splicing in C.elegans

Answer 16

See onenote diagram

• mRNAs are trans-spliced to attach a 5’ leader sequence
• polycistronic pre-mRNAs: cleaved cis-spliced and trans-spliced