RNA Processing

Question 1

How does actinomycin D work?

Answer 1

Slips into the major groove between DNA strands (intercalates) preventing DNA unwinding (and thus initiation and elongation)

Question 2

Does prokaryotic mRNA get processed?

Answer 2

NO!

Question 3

What implication does the lack of mRNA processing in prokaryotes have for the 5’ end of mRNA?

Answer 3

The 5’ will still have a triphosphate attached (no cleavage or processing!)

Question 4

What modifications are made to eukaryotic mRNA?

Answer 4

5’ cap
3’ poly A tail
SPLICING!

Question 5

What does guanylyl transferase do to mRNA?

Answer 5

Adds a GTP “backwards” to the 5’ of the first base to create a 5’-5’ triphosphate linkage

Question 6

What are the 3 steps of placement of the mRNA cap? And where do they occur?

Answer 6

1. Guanylyl transferase
2. Methylation of N7 (nucleus)
3. 1 or more 2’ hydroxyls gets methylated (cytoplasm)

Question 7

What are the functions of the 5’ cap?

Answer 7

1. Protects from nuclease digestion
2. Forms scaffold for protein binding
3. Needed for efficient translation

Question 8

What is the significance of the AAUAAA sequence?

Answer 8

A polyadenylation cleavage signal that the polymerase transcribes signaling dephosphorylation and cleavage by endonucleases

Question 9

What does polyA polymerase do?

Answer 9

Add 180-200 A residues (polyA tail)

Question 10

What is the function of the polyA tail?

Answer 10

1. Protection of 3’ end

2. Stabilization of mRNA

Question 11

What are the snRNPs?

Answer 11

snRNA + proteins:

U1, U2, U4, U5, U6

Question 12

What unique bond exists at splice sites?

Answer 12

2’-5’ phosphodiester bond

Question 13

ALL introns begin with ____ and end with ____.

Answer 13

GU start

AG end

Question 14

What is the branch point?

Answer 14

An A located in a pyrimidine-rich sequence approx 50 bases from the 3’ end of the intron. Indicates splice site

Question 15

What does each snRNP do?

Answer 15

U1 binds to 5’ splice site
U2 binds the branch site (catalytic center)
U5 binds the 3’ splice site, loops over to the 5’ site
U4 Masks the catalytic activity of U6
U6 Catalyzes splicing

Question 16

What is the sequence of U1 binding

Answer 16

U1 -> U2 -> U4/5/6 complex -> U1 and U4 leave -> U2 and U6 remain to make catalytic site ]

Question 17

What type of snRNP is U6?

Answer 17

A ribozyme (a catalytic RNA) that carries out mRNA splicing

Question 18

Describe the reaction of of intron splicing.

Answer 18

2 transesterifications:

1. 2’ OH of intron attacks 5’ splice site (intron/exon junction)
2. 3’ OH of Exon attacks 3’ splice site (intron/exon junction at the other end)

Question 19

Introns are removed in _____ regulated order _____ to each mRNA

Answer 19

Highly regulated

Unique

Question 20

What proteins facilitate alternative splicing?

Answer 20

RNA binding proteins

Question 21

What are the 2 types of RNA binding proteins?

Answer 21

SR - Serine/arginine protein

and

hnRNP - heterogeneous nuclear ribonucleoprotein

Question 22

Which RNA binding protein favors splicing?

Answer 22

SR

Question 23

Which RNA binding protein favors no splicing?

Answer 23

hnRNP

Question 24

What sequences are present in exons to further regulate splicing?

Answer 24

ESE - Exon splicing enhancer binds SR

ESS - Exon splicing silencer binds hnRNP

Question 25

What sequences are present in introns to further regulate splicing?

Answer 25

ISE - Intron splicing enhancer binds SR

ISS - Intron splicing silencer binds hnRNP

Question 26

What mutation causes beta thalassemia?

Answer 26

A single point mutation (G to A) in an intron of the beta globin gene results in mis-splicing:

creates a new 3’ splice site -> final coding region/protein is truncated

Question 27

What mistake contributes to metastatic cancer?

Answer 27

Mis-splicing of V5 (exclusion of variant exon 5)

hnRNP binds and prevents binding of SR and U2 to the proper branch point -> prevents splicing of variant exon 5