Topic 3, Lecture 2

Question 1

What are the modifications that must occur before mRNA can leave the nucleus?

Answer 1

5’ Capping, RNA Splicing, and 3’ Polyadenylation

Question 2

What are the functions of the 5’ cap? Hint: There are six functions.

Answer 2

1. Protect the mRNA from enzymatic degradation
2. Promotes rapid elongation
3. Assists in exports to the cytoplasm
4. Helps initiate translation
5. Identifies it as an mRNA
6. Lets the protein synthesis machinery know that the 5
   end of the mRNA is present

Question 3

What are the major features of the 5’ cap?

Answer 3

There is a 7-methyguanosine, 5’ to 5’ triphosphate linkage, and a methylated 2’-OH group on the ribose of the first (and sometimes second) nucleotide.

Question 4

What are the functions of the 3’ Poly-A tail? Hint: There are five functions.

Answer 4

1. Protects the mRNA from degradation
2. Involved in translation
3. Signal termination of transcription
4. Identifies it as an mRNA
5. Lets the protein synthesis machinery know that the 5’ end of the mRNA is present

Question 5

How many adenylic acid residues are added to the end of the 3’ terminus?

Answer 5

200-250

Question 6

What enzyme adds the adenylic acid residues to the 3’ terminus?

Answer 6

Poly-A Polymerase

Question 7

Describe termination of transcription.

Answer 7

Termination is functionally connected with cleavage and polyadenylation of the nascent transcript’s 3′ end. In the two-step pre-mRNA 3′-processing reaction, transcription of the poly(A) signal triggers the endonucleolytic cleavage of the nascent transcript, generating an upstream cleavage product that is immediately polyadenylated. The remaining downstream cleavage product, with an uncapped phosphate at its 5′ end, is highly unstable and is rapidly degraded.

Question 8

What is the cleavage and polyadenylation complex?

Answer 8

Cleavage and polyadenylation specificity factor is involved in the cleavage of the 3’ signaling region from a newly synthesized pre-messenger RNA molecule in the process of gene transcription. It remains bound to the CTD until the sequence signaling cleave is transcribed by the polymerase.

Question 9

Describe polyadenylation.

Answer 9

Polyadenylation is the addition of a poly-a binding protein and then 12 adenylic acids over and over

Question 10

Can there be more than one polyadenylation site?

Answer 10

There can be weak and strong polyadenylation sites.

Question 11

Describe cleavage stimulatory factors.

Answer 11

CstF have a high affinity for polyadenylation sites. More CstF can allow for the binding to a weaker polyadenylation signal.

Question 12

What is an intron?

Answer 12

Any noncoding region of a gene that is removed because it disrupts the coding region of the mRNA.

Question 13

What is splicing (simple)?

Answer 13

The very precise removal of introns

Question 14

Describe consensus sequences for splice sites.

Answer 14

Introns are typically very long. There is always a 5’ splice site and a 3’ splice site on each intron.

Question 15

Describe the invariant sequences for splicing

Answer 15

Somewhere in the middle of an intron there will be a GU and an AG

Question 16

BBP

Answer 16

Branch point binding protein

Question 17

Spliceosome First step

Answer 17

The U1 snRNP forms base pairs with the 5’ splice junction. BBP recognizes the branch-point site and binds cooperatively with U2AF, which recognizes the polypyrimidine tract and the 3’ splice junction.

Question 18

snRNA

Answer 18

Small nuclear

Question 19

RNP

Answer 19

Ribonuclear protein

Question 20

Spliceosome Second Step

Answer 20

The U2 snRNP displaces BBP and U2AF and forms base pairs with the branch-point site consensus sequence.

Question 21

Spliceosome Third Step

Answer 21

The U4/U6+U5 “triple” snRNP enters the reaction. In this triple snRNP, the U4 and U6 snRNAs are held firmly together by base-pair interactions. Subsequent rearrangements break apart the U4/U6 base pairs, allowing U6 to displace U1 at the 5’ splice junction and ejecting the U4 snRNP and some of the proteins o the U6 snRNP.

Question 22

Spliceosome Forth Step

Answer 22

Addition of the NTC/NTR protein complex positions the snRNPs to form the active site of the spliceosome and brings the branch point in proximity to the 5’ splice site.

Question 23

Spliceosome Fifth Step

Answer 23

Lariat formed. Additional rearrangements bring the two exon segments together and place them in the active site.

Question 24

Spliceosome Sixth Step

Answer 24

Spliced RNA is released. Spliceosome components recycled using ATP hydrolysis to “reset” them.

Question 25

Where do exon junction proteins bind?

Answer 25

Exon junction complex

Question 26

U1

Answer 26

5’ splice site binding

Question 27

First transesterification reaction

Answer 27

The first step of splicing involves rearrangement of snRNPs to allow a nucleophilic attack by the 2′-hydroxyl group of an adenosine in the BPS on the guanine nucleotide at the 5′ss, which results in cleavage of the 5′ss and production of an intron lariat

Question 28

Second transesterification reaction

Answer 28

The hydroxyl group at the 3′ end of the upstream exon attacks the phosphodiester bond at the 3′ss. The adjoining exons are thereby covalently bound and the intron is released in the form of a lariat, which is degraded by an exonuclease

Question 29

How long are exons

Answer 29

Around 150 bp

Question 30

How long are introns

Answer 30

Around 3500 bp

Question 31

How does splicing occur at correct locations when intron are very long?

Answer 31

SR Proteins

Question 32

List the components of the exon recognition complex.

Answer 32

Branch point, 3’ splice site, ESE, SR, 5’ splice site

Question 33

SR Proteins

Answer 33

RNA binding proteins that interact with exonic splicing enhancers, directing the U1 proteins to 5’ splice site and directing U2 proteins to the branch point adenine

Question 34

ESE

Answer 34

Exonic splicing enhancers, only found on exons, binding sites for SR proteins

Question 35

What is the goal of SR proteins?

Answer 35

Increased affinity that the U1, U2, and snRNAs have for their corresponding sequences

Question 36

Why don’t we just use random GU sequences?

Answer 36

They must be attached to a series of SRs

Question 37

Isoforms

Answer 37

Same but different

Question 38

What percent of human genes undergo alternative splicing?

Answer 38

75%

Question 39

How many genes do humans have?

Answer 39

30,000

Question 40

Explain the Dscam gene

Answer 40

There are 24 exons that produce 38,016 different isoforms. We use all of them because dendrites

Question 41

“Cryptic” splice sites

Answer 41

Accidentally skip over exons

Question 42

Can we remove terminal exons by splicing?

Answer 42

No, we have to have a 5’ splice site and a 3’ splice site

Question 43

Can we remove terminal exons without splicing?

Answer 43

Yes, we could remove the last exon by using a different polyadenylation site, and the first exon by having a different +1 site

Question 44

Splicing silencers

Answer 44

Proteins that sit on mRNA at splice site and blocks the formation of U1 and U2 binding

Question 45

Splicing enhancers

Answer 45

Ensures appropriate binding due to weakness

Question 46

Constitutive Splicing

Answer 46

Splicing occurs because there is an intron sequence ambiguity: the standard spliceosome mechanism for removing intron sequence is unable to distinguish clearly between two or more alternative pairing of 5’ and 3’ splice sites so that different choices are made by chance on different transcripts

Question 47

Give an example of a big change in sequencing.

Answer 47

C-to-U editing for Apo B