Lec 49: RNA Metabolism

Question 1

What type of RXN takes place to start intron excision?

Answer 1

A transesterificaiton rxn breaks the phosphodiester bond between the 5’ end of the intron and the 3’ end of the exon.

Question 2

What party is responsible for starting the attack on the 3’ end of the exon in the transesterification rxn in the first step?

Answer 2

The OH group on an Adenylate molecule.

Question 3

Which are taken out of pre-mRNA introns or exons?

Answer 3

Introns. Introns are IN the way and so have to be taken OUT.

Question 4

What structure forms when the intron is excised?

Answer 4

A lariat (loop)

Question 5

What is the mechanism for the second step of intron excision that allows the release of the lariat?

Answer 5

The OH group from the phosphodiester bond of the 3’ end of the exon (that just lost its connection to the 5’ end of the intron) attacks the 3’ end of the intron and cuts it out - in the process, reconnecting the first exon with its following exon.

Question 6

What recognizes the “splicing enhancers” for binding?

Answer 6

SR proteins.

Question 7

SR proteins are made up of two regions, what are they?

Answer 7

An RNA binding domain that connects to the ESE sequence. And an “activation domain” that helps recruit parts of the splicesome and is rich in Arg/Ser.

Question 8

How many proteins are there in the SR family?

Answer 8

10. And they all have the RNA binding domain and the Arg/Ser rich activation domain.

Question 9

What group of proteins helps to inhibit the SR protein action?

Answer 9

HNRNP proteins will work with the “splicing inhibitors” (ESS/ISS) to assist them in modulation of the splicing process.

Question 10

ESE/ISE and ESS/ISS stand for what?

Answer 10

Exon splicing enhancer/Intron splicing enhancer and Exon splicing silencer and Intron splicing silencer (respectively).

Question 11

Apart from Spinal Muscular Atrophy, another disease (involving the globin gene is mentioned as part of lecture as an example of splicing mutations causing disease, what was it?

Answer 11

Beta-Thalassemia

Question 12

The splicesome consists of 5 parts, what are they?

Answer 12

U proteins numbered 1, 2, 4, 5, and 6.

Question 13

What is the function of U1?

Answer 13

It binds the 5’ splice site.

Question 14

What two U proteins catalyze the splicing?

Answer 14

U2/U6

Question 15

What does U4 do?

Answer 15

It masks the catalytic activity of U6.

Question 16

What does U5 do?

Answer 16

It binds the 5’ splice site and then the 3’ splice site.

Question 17

In what order are the U proteins assembled?

Answer 17

U1/U2 first, (U1 binds the 5’ site and U2 binds at the branch site) the U4/5/6 bind. U1/U4 are eventually kicked out.

Question 18

What category of protein are the U proteins that make up the splicesome?

Answer 18

snRNPs (small ribonucleoprotein particles)

Question 19

The “Lariat intermediate” has an odd bond that holds it together, tell me about it?

Answer 19

It’s a bond that involves the adenylate in a 2’ to 5’ fashion and holds the ends of the intron together.

Question 20

Spinal muscular atrophy involves a specific gene being dysfunctional and its counterpart being unable to keep up with production, what are these genes?

Answer 20

SMN1/SMN2 (SMN1 is the primary gene)

Question 21

Why can’t SMN2 keep up with demand if SMN1 is knocked out of commission?

Answer 21

Only about 20% of its proteins get folded correctly due to only 20% splicing correctly.

Question 22

How is the 5’ end of an intron recognized?

Answer 22

A consensus sequence containing GU is recognized U1 (exact recognition).

Question 23

How is the 3’ end of an intron recognized?

Answer 23

It has a long pyrimidine chain that precedes an AG ending. This is recognized by U2. (Well, mostly).

Question 24

Why is U2 only “mostly” responsible for spotting the 3’ end of the intron?

Answer 24

U2 needs a hand from a pair of auxiliary proteins U2AF65 and U2AF35 (U2AF stands for U2 Auxiliary Factor).

Question 25

U2AF65 and U2AF35 attach to what?

Answer 25

U2AF65 recognizes the pyrimidine chain at the 3’ end of an intron to bind while U2AF35 recognizes the AG ending.

Question 26

What is SF1?

Answer 26

It binds to the U2AFs and then gets kicked out when U2 shows up at the branch site.

Question 27

How does the machinery make sure that Exon one connects to Exon 2 and so on until the strand is ready for translation?

Answer 27

RNA polymerase II carries SR proteins with it and drops them where they’re supposed to be. (This isn’t the whole picture but it’s an important part of it).

Question 28

What should be remembered about the role of splicing when it comes to diversity or protein creation?

Answer 28

Splicing is a major contributor of diversity by alternate splicing locations and quantities that leads to a major change of proteins structure, and thereby, function.

Question 29

What does SMN in the SMN gene stand for?

Answer 29

Survival of Motor Neurons

Question 30

What is the difference between the SMN1 and SMN2 genes in terms of gene contents?

Answer 30

There’s a C to T substitution in section 7 (of 8) in the SMN2 gene.