mRNA and protein transport across nuclear membrane

Question 1

Describe the structure of the nuclear pore complex

Answer 1

Large complex with only roughly 30 nucleoporin proteins

FG repeat nucleoporins line the pore

Question 2

Discuss the difference between cytosolic and nuclear RAN

Answer 2

RAN is an essential cofactor in nuclear transport
In the cytoplasm, Ran is weakly associated with GDP
In the nucleus, Ran is tightly associate with GTP

Question 3

Discuss the mechanism of import karyopherins

Answer 3

Importin binds cargo protein via nuclear localization signal - transported in to the nucleus
the tight binding of Ran to GTP (Ran associated with importin) causes release of cargo protein in the nucleus

Question 4

Discuss the mechanism of export karyopherins

Answer 4

Bind cargo proteins by NES. Only bind cargo protein when complexed with RAN GTP in nucleus. Cargo protein is efficiently bound in nucleus.
When passed into the cytoplasm, cargo protein dissociates due to weak RAN-GDP interactions with exportin

Question 5

What is the purpose of NXF1 in mRNA export from the nucleus?

Answer 5

Binds to the mRNA and to FG repeats in nucleoporins.

Question 6

What is NXF1 binding to mRNA dependent on?

Answer 6

Binding of NXF1 to mRNA dependent on RAF, which is a component of the EJC deposited on mRNA during splicing

Question 7

How is the NXF1 mRNA complex disassembled?

Answer 7

Disassembled by helicase Dbp5 at mRNP remodelling

Question 8

What is the function of the HO gene in yeast?

Answer 8

HO gene transcribes an endonuclease inducing a mating type switch in mother cells

Question 9

What is the function of ASH1?

Answer 9

ASH1 mRNA localizes to daughter cell in division to prevent expression of HO gene.

Question 10

How is ASH1 directed in nuclear division?

Answer 10

ASH1 is localized to the daughter cell via a zip code protein.
Localised to distal tip of anaphase daughter cells by active transport along actin cables

Question 11

How does ASH1 induce repression?

Answer 11

ASH1 binds to URS and recruits Rdp3 HDAC which represses expression

Question 12

Discuss the complex formed on mRNA to initiate translation

Answer 12

eIF4F binds to m7g cap complex on mRNA
PABP binds to polyadenylated tail
Complexes interact, circularizing mRNA priming it for translation

Question 13

Discuss global translational control to stress in eukaryotes

Answer 13

Stress activated ribosomal protein kinases (eg PKR) phosphorylate eIF2-a (initiation factor and GTPase)
Phosporylated eIF2-a blocks dissociated from its GEF, irreversibly bound. Cannot initiate translation.
Some mRNAs which code for stress response proteins are more efficiently translates at low eIF2-a levels.

Question 14

How does MTOR affect translational control?

Answer 14

MTOR promotes cell signaling and proliferation - stimulates production of eIF4F.

Question 15

How does MTOR achieve its desired affects?

Answer 15

MTOR phosphorylates 4EBPs, and S6 kinases.

Prevents inhibition of eIF4F by EBPS and kinases stimulate translation.

Question 16

What is the function of IRP?

Answer 16

IRP= iron regulatory proteins. Detect levels of iron in the human metabolic system.

Question 17

How does IRP/FE2+ complex impact protein expression?

Answer 17

IRP bound to iron = high levels of iron in the system. IRPS will in this case not bind to mRNA and uninterrupted expression of mRNA leads to ferritin protein production.
Ferritin can bind excess iron.

Question 18

How does IRP interact with mRNA in low iron levels?

Answer 18

IRP binds to stem loop structure in 5’ leader sequence of ferritin mRNA in apo-state. Blocks 43s preinitiation complex interacting with cap binding complex and therefore blocks transcription