RNA Processing in Eu genes Flashcards
What sequence is at the end of introns?
GUAGs
Why is RNA capped at the 5’ end?
- Chem modified by addition of cap
- needs to be capped in order to mature
- provides barrier to 5’ exonuclease activity + stabilises transcript
- prevents degradation of fragile RNA e.g. by nucleotides on fingertips
What is added during 5’ capping?
A 7-methyl guanosine (M7G) residue is added to the 5’ end of the new transcript.
So, GMP nucleotide added to new transcript in reverse orientation compared with normal 3’-5’ bridge
= now a 5’-5’ triphosphate bridge
Which other RNA processing reactions does 5’ capping play a role in?
- splicing
- nuclear transport
- translation
What does polyadenylation result in?
- stabilisation of RNA
- log polyA tail results in longer half life
- helps in nuclear export
- involved in translation
Which proteins are involved in polyadenylation?
CPSFs - similar to TFs
Cleavage and poly-adenylation specificity factor
Binds to polyA site
What is the poly(A) signal and what is the poly (A) site?
Site:
Where A’s get added
Signal:
AAUAAA
What do CPSFs do?
Proteins which bind to polyA site on RNA
- have a high affinity for 3’ end/motifs
- binding causes a bend
What happens after CPSFs bind to the polyA site?
1) at least 3 additional proteins bind (CstfF, cleavage stimulators factor, and cleavage factors 1 and II) = stays bent
2) PAP poly A polymerase binds to the complex and causes enzymatic cleavage - 10-35 nt upstream of the polyA signal where the bend is
3) the free 3’ end generated is polyadenylated (slowly) using ATP
4) cleavage factors are released, as is the downstream cleavage product
5) multiple copies of PABII (polyA binding protein) bind to the short A tail
- binding of PABII accelerates the rate of A addition by PAP (rapid polyadenylation)
- after 200-250 residues have been added, PABII signals PAP to stop polymerisation
What is splicing?
Eu genes contain introns which are transcribed into hnRNA and need to be removed
What corresponds to the start and end of biological info for a protein-encoding gene?
Start = initiation codon
End = termination codon
What are the 2 ways RNA processing is regulated?
- differential splicing
- alternative splicing
= give rise to a series of mRNAs containing different exons and each specifying a different protein
What is the main mechanism for regulating mRNA processing?
Alternative splicing
Common in human genome (>60% human genes are alternatively spliced)
What are the general steps for processing eukaryotic genes?
1) transcription, 5’ capping
2) cleavage at poly(A) site
3) polyadenylation
4) RNA splicing
What is the spliceosome?
Large, protein-RNA complex that consists of five small nuclear RNAs (U1, U2, U4, U5, and U6) and over 150 proteins
What binds to specific sequences on the pre-mRNA substrate?
U1 and U2 snRNP complexes associate with pre-mRNA via base-pairing
- formed from snRNAs and their associated proteins
How is a spliceosome made?
1) U1 and U2 associate with different exons on the pre-mRNA substrate
2) More snRNPs (U4, U5, U6) come and join the complex = spliceosome
What are the steps in splicing?
1) All snRNPs join and form spliceosome
2) BP re-arranged between snRNAs = converts spliceosome into catalytically active configuration.
U1 and U4 are destabilised and released.
3) U2 and U6 (the catalytic core) catalysed the 1st TE reaction = forms intermediate containing 2’-5’ phosphodiester bond (proteins act like molecular scissors)
4) Further re-arrangements between snRNPs, the 2nd TE takes place:
Join 2 exons by a standard 3’,5’-phosphodiester bond + releases the intron as a lariat structure, and the snRNPs
5) The excised lariat intron is converted into a linear RNA by a debranching enzyme
PROTEINS MADE IN THR CYTOPLASM, COME BACK INTO NUCLEUS, BIND TO PRE-MRNA + HELP SPLICE EXONS OUT
What is differential splicing?
25k genes in humans
Many mRNAs undergo alternative or differential splicing
= give rise to a series of mRNAs containing different exons and each specifying a different protein
What are promoters, enhancers/silencers and insulators?
DNA sequences that regulate gene transcription
What is alternative/differential splicing?
A regulated process during GE which results in a single gene coding for multiple proteins.
Within this process, particular exons of a gene may be included within or excluded from the final, processed mRNA, produced from that gene.
What are splice junctions used for?
Differential splicing.
GUAG introns comprise >98% of splice junctions in the human genome.
Specific exons many be included or excluded in the RNA product by using or failing to use a pair of splicing junctions.
Selectively remove or include certain exons = change protein
What are 2 examples of RNA processing?
1) differential splicing to produce different proteins: sex determination in drosophila
2) how polyadenylation is regulated: by blocking the polyadenylation step to reduce the amount of protein made e.g. U1A protein
