Recap transcription and splicing Flashcards
What are the chemical differences between DNA and RNA
1) Nucleotides in RNA are ribonucleotides, rather than deoxyribose in DNA
2) Uracil replaces thymine in RNA
How does Uracil differ to thymine
It’s missing a methyl group
in what direction is RNA synthesis made?
5’ - 3’
In what direction is DNA read from the template or non-coding strand
3’ - 5’
True or false - Only the 3’ - 5’ strand can encode a complementary RNA sequence.
False - both the 5’ - 3’ and the 3’ - 5’
What is a flaw in traditional methods of measuring RNA expression (northern blot, RNA-seq, RT-qPCR)
They take the average level of expression from a bulk pf cells, so they are not representative of a heterogenous populations.
What does single cell data show about mRNA expression characteristics?
It occurs in discontinuous bursts. Gene expression is dependent on the frequency and size of these bursts
How is RNA transcription regulated? (2 types)
CREs - cis acting regulatory elements
TREs - Trans acting regulatory elements
What are CREs - give examples
Non protein coding DNA regions which regulate transcription of genes on the same strand of DNA
Promoter, Enhancers, Silencers, Insulators
What are TREs - give examples
Coding DNA regions which regulate gene transcription through their own genetic product.
Chromatin modifying enzymes, Transcription factors (general TFs which bind to promoters/RNA pol or gene regulatory proteins which bind to CREs)
What are the 4 essential components of transcription
DNA helicase, Promoter, RNA pol, General TFs
What is the role of DNA helicase
Unwinds DNA to create a transcription bubble at the promoter
Describe the promoter
CRE element which determins the initiation site of transcription. Within the promoter complex there is a TATA box and the INR region which is where transcription actually begins.
What is important about the INR
Its where transcription actually begins.
Which RNA pol is required in transcription in eukaryotes?
RNA pol II -
How does RNA pol II differ from RNA pol I/III
It has a long C terminal domain. Phosphorylation of of the CTD is vital for initiation, elongation and termination
Which serine residues are phosphorylated on the CTD of RNA pol II
Ser 2 and Ser 5
What general TFs are required for transcription?
TFIID, TFIIB, TFIIF, TFIIE, TFIIH
What is the role of the general TFs?
They associate with the promoter to form the pre-initiation complex
Which general TF associates with the TATA box to initiate transcription and how does it achieve this>
TFIID recognises the TATA box via its TATA binding protein (TBP), Distortion of the TATA due to histone acetylation by TFIID marks the location as an active promoter.
What step follows after TFIID association?
Recruitment of TFIIB to the active promoter via a BRE element
What is the role of associated TFIIB
It recruits RNA pol II to the INR
What is the role of associated TFIIF
It stabilises RNA pol II on the DNA
What is the role of associated TFIIH
It has helicase activity and unwinds DNA at the 5’ end
When TFIIB,D,F and H are associated, RNA pol can perform productive transcription? True or false
False, it can only perform abortive transcription as it is not very stable.
What does TFIIH do to RNA pol II to enable initiation ?
Phosphorylates RNA pol II CTD serines 2/5 causing a conformational change which allows for initiation.
Why is DNA looping required for transcription to occur?
It brings distal CRE/TRE elements into close proximal contact with the promoter/ RNA pol II/ Mediator complex
What is the first modification made to mRNA during elongation
5’ capping, facilitated by elongation factors associated with RNA pol II. phosphorylated Ser on RNA pol II CTD recruits capping enzymes
What are the three stages of 5’ capping
1 - Triphosphatase removes phosphate from 5’ nucleotide
2- Guanylyltransferase adds a guanine residue (GMP) in its reverse orientation
3- Methyltransferase adds a methyl group to the guanosine, creating a methyl guanine cap at the start of the mRNA transcript
What is the polyadenylation signal required for termination cleavage
AAUAA
What recognises the AAUAA sequence?
CPSF - recruited by phosphorylated serine on RNA pol II CTD
What makes up the cleavage complex
CstF, CFI and CFII
Where does exonucleic cleavage of mRNA occur
about 30 nucleotides downstream of the AAUAA site
How is RNA pol II released from DNA
A phosphatase removes the phsophate from serine 5 residues on the CTD compromising the stability of the RNA on DNA, causing it to fall off.
How does polyadenylation occur
RNA cleavage complex remains on the 3’ end and recruits poly-A polymerase (PAP). PAP sequentially adds adenosine to the 3’ end. Poly A binding proteins (PABs) bind along the A tail to stabilise PAP until around 200 - 250 adenosines have been added. At which point PAP falls off.
What is the necessity of having the 5’ cap on mRNA?
Association of cap binding complex enables export from the nucleus.
It prevents 5’ exonuclease degredation
Recognition signal for ribosomes and positions mRNA for translation
Why is the 3’ polyadenylation required in mRNA
It protects it from cytosolic enzymatic degredation
Why is splicing required in the formation of mature mRNA
Removes non-coding regions to produce a final coding sequence
Enables a wide range of isoform proteins to be produced from a single gene.
What are the 5 small nuclear ribonuclear proteins associated with splicing (snRNPs)
U1,U2,U4,U5,U6
Which snRNPs recognise the 5’ exon junction sequence?
U1 and U6
Which snRNPs recognise the branch junction sequence?
BBP and U2
Describe the process of splicing?
BBP recognises Adenosine at the branch point and recruits U2AF, U1 recognises the 5’ end junction sequence.
U2 is recruited to the branch point sequence, displacing BBP and U2AF.
The U4/U6-U5 triple snRNP binds to U1, an ATP mediated conformational change brings the 5’ end of the intron into close proximity to its 3’ end.
An OH group on adenosine attacks the phosphate group at the 5’ splice site, cutting the sugar phosphate backbone. The cut 5’ end of the intron becomes covalently linked to adenosine creating a loop in the RNA molecule.
U1 and U4 dissociate. The OH group at the 3’ end of the exon reacts with the start of the next exon sequence, joining the two exons together and releasing the intron in the shape of a lariet, which gets broken down into individual nucleotides to be recycled. U2, U5 and U6 dissociate.
What is the importance of alternative splicing?
Alternative splicing (excision of exons/ retention of introns) creates diverse mRNAs that are translated into functionally distinct protein isoforms.
How can alternative splicing induce cancerous phenotypes?
Mutations in intron excision consensus sequences, mutations in core spliceosome recognition sequences or aberrant splicing regulation can lead to an alternative splice. This alternative splice could produce a non-functional protein that would normally inhibit growth factor signalling etc; leading to cellular proliferation.
How do mutations in RBM10 induce lung cancer cell development?
RBM10 is an alternative splice regulator. When it is mutated it is unable to facilitate the skipping of exon 9 of the gene NUMB. This alternative splice forms a Numb protein that no longer inhibits notch signalling so cell proliferation is induced.
How can RBM10 deletions in lung cancer be targeted therapeutically and what is the result.
- Repair the splicing mechanism - alternative splicing which removes exon 9 prevents notch signalling by producing a functional Numb protein.
- Seletively target alternatively spliced oncogene/tumour suppressor
