Lecture 6. Post-Transcriptional Regulation of Gene Expression 1 Flashcards
What are the eukaryotic Pol II post-transcriptional events?
Possible attenuation (RNA transcript aborts)
5’ capping
Splicing
3’ end processing
Possible RNA editing
Nuclear export
What do all eukaryotic Pol II post-transcriptional events require?
Factors that bind the phosphorylated C-terminal domain (CTD) of RNA PolII
The CTD of RNA PolII comprises multiple repeats of a 7 amino acid sequence YSPTSPS Phosphorylation of seryls 2 and 5 marks the transition from transcriptional initiation to
elongation
Why is the 5’ cap put on backwards?
The 5; end of the transcript now masquerades as the 3’ end with a 3’ hydroxyl (no longer at risk of degreadtion)
How is the 5’ cap added?
Nascent RNA transcript has phosphate removed from 5’ end by RNA triphosphatase
Guanylyl transferase cleaves diphosphate from GTP and adds the G and P to the RNA transcript
Methylate (add CH₃) G to generate 7-methylguanosine and first ribose also methylated
What is the importance of the 5’ cap?
Distinguishes Pol II transcripts from other RNA molecules
Stabilises the RNA - there is no 5’ phosphate, so it is resistant to 5’ exonucleases
Aids in further processing (exon definition hypothesis) and export to the cytosol
And is required for efficient translation of mRNAs
What is the key principle of the 5’ cap?
5’ capping is co-ordinated with transcription
How does decapping of the 5’ cap take place?
A multi-subunit cytosolic ATP-dependent decapping enzyme complex removes the 5’ cap
It restores a 5’ phosphate on the mRNA. The mRNA can now no longer be bound by ribosomes and so can no longer be translated
The mRNA is now degraded by a 5’-3’ RNAse
What is ivs (intervening sequence)?
Intron found in the 26R rRNA gene
The intron is excised from the primary transcript and the RNA can do it by itself
How could the ivs intron be removed from the Tetrahymena nuclei?
α-amanitin fungal toxin (Pol II inhibitor) so no mRNAs could be made but rRNA genes were still transcribed
A nuclease inhibitor stabilises the RNA
And with ribonucleotides ATP, GTP, CTP and radioactive ³²P-UTP
How did Cech discover the ribozyme?
Deproteinised the rRNA by phenol (denatures proteins) extraction and/or boiled the rRNA for five minutes. The intron was still excised and still accumulated
Cloned the rDNA and made rRNA in vitro using bacterial RNA polymerase. The intron was still excised and still accumulated
Cech defined the RNA itself was catalytic, with the new principle that RNA could catalyse functions that had previously been thought of as enzyme functions (ivs = ribozyme)
How does the mechanism of introns function in the Tetrahymena?
- The intron folds. A co-factor s held in a pocket: guanosine, GMP, GDP, GTP. The 3’-OH of the co-factor is a nucleophile that attacks the phosphate at the 5’ splice site
- The 3’-OH of the upstream exon attacks the phosphate at the 3’ splice site
- The exons are fused, and the intron is ultimately degraded
Two sequential transesterifications fuse the exons and release the intron
How does the mechanism of introns function in humans?
- The intron folds and the 2’-OH of the ‘branch site’ adenosine attacks the phosphate at the 5’ splice site
- This adenosine now has three phosphodiester bonds: one is an unusual 2’, 5’ phosphodiester bond. The 3’-OH of the upstream exon attacks the phosphate at the 3’ splice site
- The exons are fused and the intron is released as a lariat
Two sequential transesterifications fuse the exons and release the intron
What controls the intron mechanism?
The spliceosome: an RNA and protein complex
What is the spliceosome and what makes up the spliceosome?
The spliceosome: an RNA and protein complex
5 small uridine-rich snRNPs (small nuclear ribonucleoproteins) U1, U2, U4, U5, U6
Each snRNP is a small nuclear RNA (snRNA) complexed with at least seven protein subunits. There are extra proteins ~200 in total
What allows the spliceosome to splice precisely?
A combination of RNA base-pairing (specificity) and protein binding (stabilisation) allows precise splicing because the splice sites and the branch site are brought close together
Where does the spliceosome recognise specific sequences at?
The 5’ splice site
The branch site and
The poly pyrimidine tract (poly Y)
How does the spliceosome function?
- U1 snRNP binds the 5’ splice site: base pairing provides specificity
- Branch binding protein (BBP) and the protein U2 auxiliary factor (U2AF) bind the branch site and associated pyrimidine tract (see later) respectively
- U2 is recruited and displaces BBP
- A pre-formed trimer of U4, U5 and U6 associates
- A molecular rearrangement (loss of U1 and U4) activates the complex
- The 2’-OH of the branch adenine attacks the 5’ splice site phosphate
- The lariat is formed and U6 is activated, guiding the 3’-OH of the upstream exon that attacks the 3’ splice site phosphate
- The exons fuse, and the intron is excised as a lariat. The snRNPs are recycled and the intron is degraded.
What are the key principles of how the spliceosome works?
In eukaryotic cells spliceosomes assemble at specific RNA sequences on the capped RNA by making specific base-pair bonds. Proteins stabilise the spliceosome. Molecular rearrangement activates the spliceosome. Two spatially and temporally regulated transesterifications fuse the exons and release the intron as a lariat.
How does U1 find the splice site?
- U1 is also recruited by the phosphorylated CTD of the large subunit of Pol II. U1 scans the growing mRNA for the 5’ splice site and binds it.
- The nascent chain keeps growing, forming a loop, while BBP/U2AF scan for a branch site/pyrimidine tract
- The spliceosome assembles and is released
What regulates the assembly of a spliceosome?
The process depends on the strength of binding of spliceosome components, so some introns are removed co-transcriptionally, others post transcriptionally
Spliceosome assembly is regulated by RNA Pol II and by the strength of the target binding sequences
What are the splicing signals of the spliceosome?
Consensus sequences: individual motifs have different affinities for spliceosome components – and so some are more efficient at stimulating splicing than others
What makes U1, U2, U4 and U5 snRNPs?
RNA Pol II
What happens in the biogenesis of U1, U2, U4 and U5 snRNPs?
Transcription and capping in the nucleus - shipped to cytosol
Cytosolic loading of a ring of seven Sm proteins loaded as a ring onto the snRNA by SMN (survival motor neuron) protein.
Cap hypermethylated signal to transport and return to nucleus. Maturation into U1 snRNP by association with other proteins
What are the functions of the protein components of snRNPs?
The snRNA provides specificity by base pairing with target: the proteins provide stability
Splicing associated proteins ‘define’ the exons
