Lecture 12 - RNA splicing and polyadenylation

Question 1

what links transcription to splicing and polyadenylation and how

Answer 1

CTD tail of Pol II by recruiting splicing and poly(A) enzymes

Question 2

___ imaging of DNA and __________ transcript hybridization shows what

Answer 2

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

Question 3

Key residues in introns for splicing that are 100% conserved (4)

Answer 3

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

Question 4

adenosine vs adenine

Answer 4

adenine = nitrogeneous base. adenosine = adenine (the nitrogeneous base) + ribose

Question 5

Splicing occurs via ___ consecutive _________ reactions

Answer 5

2. transesterification

Question 6

1st transesterification reaction in splicing (3 things)

Answer 6

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

Question 7

2nd transesterification reaction in splicing

Answer 7

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

Question 8

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 ________.

Answer 8

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

Question 9

Important about the branch point : The adenosine is not __________

Answer 9

base-paired

Question 10

Essential for splicing : Base pairing of pre-mRNA with _______ and ________. Proven by _________ on pre-mRNA. (blocks splicing)

Answer 10

U1 snRNA and U2 snRNA. mutations

Question 11

Mutation in pre-mRNA 5’ splicing site consequence explanation + solution

Answer 11

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)

Question 12

U2 snRNA binds to a sequence that contains ___________ but it is not _________

Answer 12

the branch point adenosine, base-paired

Question 13

snRNPs form something called a ________ in which ___ different ______(name them) interact via base pairing with _________and with _________ and __________ to form the ___________.

Answer 13

spliceosome. 5 different snRNAs (U1,U2,U4,U5,U6), one another, pre-mRNA and proteins. spliceosome.

Question 14

What spliceosome does and what it does ultimately

Answer 14

1) catalyzes 2 transesterification reactions 2) join 2 exons and remove the intervening intron as an excised lariat structure which is degraded

Question 15

First interactions in spliceosome formation (4)

Answer 15

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

Question 16

2nd interactions in spliceosome formation

Answer 16

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

Question 17

3rd interactions in spliceosome-mediated splicing

Answer 17

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

Question 18

4th interactions in spliceosome-mediated splicing

Answer 18

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.

Question 19

_______ 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 _____________.

Answer 19

Group II. mitochondrial. chloroplast. predecessors. group II. 1 RNA self-splicing. spliceosome’s

Question 20

which RNAs are an exception in that they are not polyadenylated

Answer 20

Histone mRNAs

Question 21

3 signals/sites at 3’ end of mRNA that are associated with polyadenylation

Answer 21

In order from 5’ to 3’ : Poly(A) signal -> AAUAAA. Poly(A) site. Poly(A) signal -> GU or U rich sequence

Question 22

which factors bind to poly(A) signals and sites

Answer 22

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

Question 23

What links cleavage and polyadenylation and how it starts poly(A)

Answer 23

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)

Question 24

what happens after cleave (factors and 3’ portion)

Answer 24

CPSF, CStF and CFI/CFII are released. 3’ portion containing the GU or U rich sequence is degraded.

Question 25

After addition of 12 or so A residues, what happens

Answer 25

Binding of PABPII (Poly(A)-binding protein) via its RRM domain speeds up poly(A). Rapid addition of 200-250 A residues.

Question 26

How polyadenylation ends

Answer 26

PABPII signals signals PAP to stop adding residues after 200-250 where added. (unknown mechanism)