Pre-RNA Processing (Bently)

Question 0

What are some major differences between pre-mRNA and mature mRNA?

Answer 0

Pre-mRNA has its introns, no 5’ cap and no poly A tail

Question 1

Name the 3 major ways in which all pre-mRNA’s are processed.

Answer 1

1. 5’ - 7-methylguanosine cap with a 5’-5’ linkage
2. Splicing
3. cleavage / polyadenylation

Question 2

Describe the addition of the 5’ cap to pre-mRNA

Answer 2

1. Since the 5’ end has a triphosphate, the gamma phosphate is cleaved
2. An “upside down” guanosine monophosphate (MGP) is added - 5’-5’ linkage
3. Methylate the guanosine at the C7 position

The result is a triphosphate in the middle of a 5’-5’ linkage of the methylated guanosine and the 5’ residue of the new RNA

Question 3

What are the conserved sequences of introns and where are they located in the intron?

Answer 3

1. GU (GT) at the 5’ end
2. A in the middle closer to the 3’ end
3. AG at the 3’ end

Question 4

What are the functions of the 5’ cap?

Answer 4

1. Stabilize 5’ end. Protect from exonuclease activity.
2. Recognition site for
   i. CBC in the nucleus -
   a. enhances splicing
   b. enhances 3’ processing
   c. enhances export from nucleus
   ii. EIF4F in the cytoplasm (initiates translation by binding the cap and bringing the mRNA to the ribosome)

Question 5

What is the conserved sequence at the 3’ end of the pre-mRNA and what is it used for?

Answer 5

AATAAA roughly 10-30 bp’s upstream from the cleavage point in the process of adding the poly A tail

Question 6

List some genetic disorders caused by splicing defects.

Answer 6

1. Marfans
2. SMA (spinal muscular atrophy)
3. CD44 - alt. splicing predicts metastasis in cancers

Question 7

What is CD44?

Answer 7

Codes for a cell surface protein that moderates cell migration. Alt. splicing can predict cancer metastasis.

Question 8

What is marfans syndrome?

Answer 8

splicing defect in the fibrolyn gene.

presents with tall, protruding chest, weak aorta

Question 9

SMA (spinal muscular atrophy)?

Answer 9

name is very descriptive…

splicing defects causing mtr neuron degeneration

Question 10

Name the factors that bind to the intron during splicing.

Answer 10

U1 snRNA
U2 snRNA
U2 associated factor protein

Question 11

Where does U1 snRNA bind?

Answer 11

At the 5’ end of the intron on the GT (GU) site.

Question 12

Where does the U2 snRNA bind?

Answer 12

at the A site in the “middle” of the intron

Question 13

Where does the U2 associated factor protein bind?

Answer 13

at the 3’ end of the intron at the AG site

Question 14

What is the A site in the intron also known as?

Answer 14

the branch point

Question 15

Describe how the U1, U2 snRNA’s and the U2 assoc. factor bind to the intron vs. the exon…

Answer 15

From the A site in the first intron then to the GU site in the second intron, bridging an exon in between.

They “bridge” an exon because exons are shorter (easier to identify) than introns.

Question 16

Describe the rxns in splicing once all the factors have bound.

Answer 16

1. 2’-OH of the A site nuc. attack on the 5’ end’s phosphate
2. 3’ -OH at the 5’ end of the intron nuc. attack on the phosphate at the 3’ end
3. The two exons are joined by the usual phosphodiester bond.

Question 17

Describe the two rxns that process the 3’ end of the pre-mRNA.

Answer 17

Add’n of poly A tail:

1. cleavage about 20 bp downstream from the AATAAA sequence in the non-coding region
2. Addn of 150 - 200 A’s

Question 18

What enzyme does the poly A addition at the 3’ end?

Answer 18

polyadenylate polymerase

Question 19

Describe two instances of alternative poly adenylation.

Answer 19

1. IgM heavy chain - stay on lymphocyte cell membrane vs. go into the serum
2. Thalassemia - alpha-globin production altered with alternative poly A tail site (AATAAA corrupted to AATAAG)

Question 20

What is the termination function of the 3’ end processing?

Answer 20

poly A tail signals termination of trxn

Question 21

What are two major functions of the poly A tail?

Answer 21

1. Stabilize the 3’ end. Protect from exonuclease activity.

2. Aid in translation later on in the ribosome.