Eukaryotic RNA Processing

Question 1

Actinomycin D

Answer 1

Inhibitor of Transcription

• slips into the major groove between the DNA strands (intercalates), preventing the unwinding of the DNA template strands
• inhibits initiation and elongation
• effective in both prokaryotes and eukaryotes

Question 2

In bacterial mRNA, the 5’ end would have the structure:

Answer 2

5’ PPP

b/c mRNA (bacterial) has no processing and it means the 1st base is a triphosphate and it won’t be cleaves

Question 3

What is hnRNA?

Answer 3

heterogenous nuclear RNA –> precursor of mRNA in eukaryotic cells

Question 4

Post-transcriptional modification in mRNA (eukaryotes)

Answer 4

5’ cap
3’ poly A tail
Splicing to remove introns

Question 5

Where does post-transcriptional modification happen in eukaryotes?

Answer 5

Nucleus, to allow for transport into cytoplasm

Question 6

mRNA cap

Answer 6

GTP added backwards to 5’ end to form 5’-5’ triphosphate linkage thru Guanylyl Transferase

Question 7

How many phosphates released in making the mRNA cap?

Answer 7

Release of gamma phosphate from RNA and release of PPi from GTP

Question 8

Function of Poly-A tail-3’ end

Answer 8

related to transcription termination
Protects 3’ end from degradation
Stabilization of mRNA

Question 9

Function of AAUAAA in poly A tail formation

Answer 9

Functions as a signal for dephosphylation and endonuclease to cleave upstream 3’ –> poly A polymerase using ATP adds around 200 A’s –> poly A binding proteins bind for protection of 3’ end and stabilizes mRNA

Question 10

What does splicing require?

Answer 10

Involves snRNA + proteins= snRNPs
U1,2,4,5,6

2’-5’ phosphodiester bonds at branch point

Question 11

Consensus Sequences on Exon/Intron borders

Answer 11

All introns begin with GU and end with AG

Question 12

What is the branch point

Answer 12

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

Question 13

Role of snRPs

Answer 13

Involved in mRNA precursor splicing

contains one small snRNA and several proteins

Question 14

U1

Answer 14

Binds to 5’ splice site (there can be mismatched sequences in this bindings, but there is a conservative sequence at which U1 binds to)

Question 15

U2

Answer 15

Binds the branch site and forms part of the catalytic center

Question 16

U5

Answer 16

Binds the 3’ splice site, loops over to the 5’ site

Question 17

U4

Answer 17

Masks the catalytic activity of U6, until ready to splice

Question 18

U6

Answer 18

Catalyzes splicing- it is a ribozyme

Question 19

Why does U1 snRNP bind the 5’ splice site?

Answer 19

Bc it is complimentary to the sequence on the 5’ end

Question 20

Pathway for Spliceosome Assembly

Answer 20

1) U1 binds to 5’ end of intron
2) U2 binds to branch site
3) U4,5,6 complex comes in and brings exon1/intron (U1 area) junction closer to the branch site (A) thru looping –> creation of spliceosome
- -> structured so that A is close to the 3’ end of exon 1
4) U1/U4 leave

Question 21

Actual splicing pathway

Answer 21

1) U2 and U6 have complementary sequences and can base pair- they form the catalytic site

U6 catalyzes:

1) attack of 2’ OH on A to the 5’ splice site
2) Newly formed 3’ OH on exon 1 attacks the 3’ splice site on the other exon still attached to the intron
3) Exons are joined
4) Intron is released in the form of a lariat

Question 22

Key Points in Splicing

Answer 22

Highly regulated process- introns removed in order unique to each mRNA

Alternative splicing

Mutations in splice sites are involved in many diseases processes

Question 23

RNA binding proteins

Answer 23

Facilitate alternative splicing
Also important as many genes are alternatively spliced in malignant cells

Blocks snRPs from binding so those exons are excluded from specific tissues

Question 24

SR

Answer 24

Serine/arginine protein (positive-favors splicing)- binds on exons

Question 25

hnRNPS

Answer 25

heterogeneous nuclear ribonucleoprotein (negative- inhibits splicing)
binds both on exons and introns or both

Question 26

ESE, ISE

Answer 26

exon splicing enhancer- binds SR

intron splicing enhancer- binds SR

Question 27

ESS, ISS

Answer 27

exon splicing silencer- binds hnRNP

intron splicing silencer- binds hnRNP

Question 28

beta-thalassemia major

Answer 28

one base mutation in the beta globin gene (G to A)
causes low beta globin protein and severe anemia

Single point mutation of G –> A in an intron of the beta-globin gene results in mis-splicing..creates a new 3’ splice site –> protein cut short and not functional

Question 29

Variant exons

Answer 29

V5 forms in many metastatic cancers–> facilitates movement of cancer cells

Question 30

Inclusion of variant exon

Answer 30

SAM binds ESS site –> hnRNP cannot bind –> SR binds –> U2 binds –> splicing occurs –> exon will be included in that variant protein

Question 31

Exclusion of variant exon

Answer 31

hnRNP + silencer –> prevents binding of SR proteins and blocks E2 from binding –> prevents splicing of variant exon –> variant exon expressed in final product