Lecture 12 - RNA splicing and polyadenylation Flashcards
what links transcription to splicing and polyadenylation and how
CTD tail of Pol II by recruiting splicing and poly(A) enzymes
___ imaging of DNA and __________ transcript hybridization shows what
EM . final mRNA transcript. Only part of the mRNA is hybridizing with the DNA and part of the DNA is left out so those are introns. Evidence for RNA splicing
Key residues in introns for splicing that are 100% conserved (4)
1) 5’ end of intron : GU (G (1st) and U (2nd)). 2) 3’ end of intron : AG (A (before last) and G (last)).3) 20-50 bp away from AG : Branch point -> adenosine. 4) Between branch point and AG : pyrimidine rich region of 15 bp
adenosine vs adenine
adenine = nitrogeneous base. adenosine = adenine (the nitrogeneous base) + ribose
Splicing occurs via ___ consecutive _________ reactions
- transesterification
1st transesterification reaction in splicing (3 things)
1) Adenosine (branch point) attacks phosphate at 5’ end of intron with its OH group 2) Exon at 5’ end of intron leaves 3) Lariat structure formation
2nd transesterification reaction in splicing
1) Exon that left (from 5’ end of intron) attacks phosphate at the 3’ end of intron 2) Phosphate leaves 3’ end of intron 3) Product : Excised lariat intron + Spliced (means connected) exons
Splicing is catalyzed by ____________. These include ____________, which are important for _________, and _________ associated _________. The whole complex is as big as a _________ so the process would be as difficult as ________.
small nuclear ribonucleoprotein particles (snRNPs). snRNA important for base-pairing with the pre-mRNA (with the conserved repeats at 5’ and 3’ end of intron). 170 associated proteins. ribosome. translation
Important about the branch point : The adenosine is not __________
base-paired
Essential for splicing : Base pairing of pre-mRNA with _______ and ________. Proven by _________ on pre-mRNA. (blocks splicing)
U1 snRNA and U2 snRNA. mutations
Mutation in pre-mRNA 5’ splicing site consequence explanation + solution
Mutation interferes with base-pairing at 5’ end of splicing site. (this blocks splicing). Solution : Compensatory mutation in U1 snRNA to restore base-pairing (base-pairing with the mutated nucleotide)
U2 snRNA binds to a sequence that contains ___________ but it is not _________
the branch point adenosine, base-paired
snRNPs form something called a ________ in which ___ different ______(name them) interact via base pairing with _________and with _________ and __________ to form the ___________.
spliceosome. 5 different snRNAs (U1,U2,U4,U5,U6), one another, pre-mRNA and proteins. spliceosome.
What spliceosome does and what it does ultimately
1) catalyzes 2 transesterification reactions 2) join 2 exons and remove the intervening intron as an excised lariat structure which is degraded
First interactions in spliceosome formation (4)
1)U1 snRNP binds to consensus 5’ splice site 2)SF1 (splicing factor 1) binds to branch point 3)U2AF (U2 snRNP associated factor) binds pyrimidine-rich region and U2 snRNP with one subunit and binds with AG region at 3’ end with another subunit 4) U2 snRNP associates with 3’ consensus splice site which contains branch point and this displaces SF1
2nd interactions in spliceosome formation
Trimeric snRNP complex of U4, U5 and U6 joins the initial complex to form the spliceosome. Spliceosome composed of approx. 70 proteins, some associated with snRNPs, others not
3rd interactions in spliceosome-mediated splicing
1) Release of U1 and U4 makes the spliceosome catalytically active
2) U6 and U2 catalyze the first transesterification reaction. Intermediate contains a 2’-5’ phosphodiester bond
4th interactions in spliceosome-mediated splicing
1) Second transesterification reaction joins 5’ and 3’ exons by a standard 3’ - 5’ phosphodiester bond
2) U2,U5,U6 are released. Excised lariat intron is converted into a linear RNA by a debranching enzyme and is then rapidly degraded.
_______ introns are only found in _________ and ________ genes and may be the evolutionnary ___________ of other introns. A ______ intron is __ (number) RNA that ___________. Their structure ressembles the _____________.
Group II. mitochondrial. chloroplast. predecessors. group II. 1 RNA self-splicing. spliceosome’s
which RNAs are an exception in that they are not polyadenylated
Histone mRNAs
3 signals/sites at 3’ end of mRNA that are associated with polyadenylation
In order from 5’ to 3’ : Poly(A) signal -> AAUAAA. Poly(A) site. Poly(A) signal -> GU or U rich sequence
which factors bind to poly(A) signals and sites
1) CPSF (Cleavage and polyadenylation specificity factor) binds to AAUAAA signal. 2) CStF (cleavage stimulatory factor) binds to GU or U rich sequence and CPSF so RNA bends 3) CFI and CFII (cleavage factors 1 and 2) binding stabilizes the complex
What links cleavage and polyadenylation and how it starts poly(A)
1) Poly(A) polymerase (PAP) binds to cleavage site (15-30 nt distance from the 5’ Poly(A) signal)
2) Stimulates cleavage at cleavage site and starts adding 12 A residues at SLOW RATE to the 3’-hydroxyl group generated by cleavage (ATP -> PPi - inorganic diphosphate)
what happens after cleave (factors and 3’ portion)
CPSF, CStF and CFI/CFII are released. 3’ portion containing the GU or U rich sequence is degraded.
After addition of 12 or so A residues, what happens
Binding of PABPII (Poly(A)-binding protein) via its RRM domain speeds up poly(A). Rapid addition of 200-250 A residues.
How polyadenylation ends
PABPII signals signals PAP to stop adding residues after 200-250 where added. (unknown mechanism)
