Mechanisms for RNA localisation in eukaryotes

Question 1

How can you see where mRNAs are located within cells? (2)

Answer 1

• Make a piece of DNA made of Ts with a fluorescent probe
• Binds to polyA tails on mRNAs

Question 2

What are nuclear speckles?

Answer 2

Structures containing RNAs and proteins in the nucleus, located between the chromatin

Question 3

What needs to happen before mRNA export? (2)

Answer 3

• Pre-mRNA must be fully processed before export (5’ cap, splicing, 3’ cleavage and polyA)
• RNA polymerase II acts as a tether as it is bound to DNA as RNA is coming out of it, therefore 3’ cleavage and polyA is essential

Question 4

How is the TREX complex recruited to the 5’ end of mRNA? (2)

Answer 4

• TREX complex is conserved from yeast to man and promotes mRNA export
• Key component ALYREF binds the CBP80 subunit of the nuclear cap binding complex which anchors TREX complex on the 5’ end of mRNA

Question 5

Which way does mRNA exit the nucleus?

Answer 5

Goes through nuclear pore 5’ end first

Question 6

What is the nuclear cap binding complex?

Answer 6

Heterodimer made of CBP80 and CBP20 which binds to the 5’ cap of RNA transcripts

Question 7

What is U2AF35?

Answer 7

Splicing factor which recognises the branch point sequence in intron of pre-mRNA (not spliced)

Question 8

What is UAP56? (2)

Answer 8

• RNA helicase
• Component of the TREX complex

Question 9

Why is TREX specifically bound to spliced mRNA? (4)

Answer 9

• TREX is first recruited to the 5’ cap of mRNA then jumps to internal sites next to the exon junction complex (EJC)
• EIF4A3 in EJC binds to ALYREF in TREX
• mRNA ends up with multiple TREX complexes bound to 5’ cap and internally next to EJC
• Therefore TREX is specifically bound to spliced mRNA because it is dependent on the EJC which is only present after splicing

Question 10

What is the exon junction complex (EJC)?

Answer 10

Protein complex deposited 20-24 nucleotides upstream from the exon-exon boundary in spliced mRNA

Question 11

What is EIF4A3? (2)

Answer 11

• RNA helicase
• Component of the EJC

Question 12

What is polyA binding protein (PABP)?

Answer 12

Binds to the polyA tail of mRNA in the nucleus and encourages recruitment of TREX to the 3’ end

Question 13

What are the 3 ways in which TREX is recruited to mRNA?

Answer 13

• 5’ cap via CBP80
• Internal EJCs via EIF4A3
• PolyA tail via PABP

Question 14

What is the TREX complex?

Answer 14

Intermediate complex in mRNA export

Question 15

What is Nxf1 (Tap) protein? (3)

Answer 15

• Nuclear RNA export factor 1
• The major mRNA export receptor
• Binds to RNA and the nuclear pore

Question 16

What is the structure of Nfx1? (2)

Answer 16

• N-terminal cargo-binding domain which contains RNA binding domain (RBD) and leucine-rich repeat (LRR)
• C-terminal nuclear pore complex (NPC) binding domain which contains NTF2 scaffold and UBA-like domain

Question 17

What is the function of the NPC-binding domain of Nxf1? (2)

Answer 17

• Nxf1 heterodimerises with p15 (Nxt1) via NTF2 scaffold
• NTF2 and UBA-like domain recognise phenylalanine-glycine (FG) repeats in nucleoporins

Question 18

What are nucleoporins?

Answer 18

Proteins which line the nuclear pore

Question 19

How is mRNA licenced for export? (3)

Answer 19

• Free Nxf1 doesn’t bind RNA in the nucleus because it sequesters its own RNA binding domain
• ALYREF in TREX binds to RBD in Nxf1 and THOC5 or Chtop in TREX binds to NTF2 in Nxf1 = causes Nxf1 to flip open and now will bind RNA with high affinity
• Nxf1 binds to the nuclear pore = export

Question 20

How is Nxf1 linked to TREX? (2)

Answer 20

• TREX drives Nxf1 into a conformation which allows it to bind RNA with high affinity
• Only get TREX on mRNA after processing and only get Nxf1 after TREX

Question 21

How does RNA length determine export route? (5)

Answer 21

• Some RNAs need to be kept in the nucleus
• Short RNAs (less than 200-300nt) e.g. snRNAs (splicing - nucleus) aren’t exported via mRNA export pathway (TREX and Nxf1)
• hnRNPC can wrap ~230-240nt of RNA around it
• If RNA is too short for hnRNPC, instead recruits Phax, Crm1 and Ran-GTP and this complex exports the mRNA
• If RNA is long enough to wrap around hnRNPC, recruitment of Phax/Crm1/Ran-GTP is blocked, binds ALYREF (TREX) and Nxf1 and is exported normally

Question 22

What is hnRNPC?

Answer 22

Molecular ruler

Question 23

How are mRNA export factors recycled? (5)

Answer 23

• Dbp5 RNA helicase forms a complex with ATP and binds to RNA in the nucleus with Met67 (Nxf1) and Mtr2 (p15)
• Complex goes through the nuclear pore, Gle1 joins, ATP hydrolysis triggers mRNA release into the cytosol leaving ADP bound
• Gle1 drives release of ADP and Dbp5 which triggers release of Met67 and Mtr2
• Nup159 joins and drives recycling of Dbp5
• Export factors have nuclear localisation signals so are imported via normal pathway without mRNA bound

Question 24

Why is packaging of mRNA for export important? (2)

Answer 24

• Protects the mRNA
• Proteins packaging the RNA prevent it from re-annealing to the template DNA strand and prevent formation of R loops

Question 25

How are R loops formed? (3)

Answer 25

- RNA polymerase II transcribes RNA - The RNA being produced re-invades the transcription bubble forming a DNA:RNA duplex - Dangerous because the affected DNA strand can't base pair with its other strand that is being transcribed so is vulnerable to cleavage/mutagenesis so causes genome instability

Question 26

What happens if TREX is depleted?

Answer 26

Lots of R loops and lots of DNA damage

Question 27

How does SMN1 cause spinal muscular atrophy? (5)

Answer 27

- SMN1 mRNA includes exon 6, 7, 8 and the resulting protein forms oligomers - SMN2 gene has C>T mutation in exon 7 causing skipping of exon 7 and the resulting protein can't form oligomers but if you have WT SMN1 its fine - SMN1 mutations cause spinal muscular atrophy, no functional SMN1 protein - Cure is antisense oligos which binds SMN2 pre-mRNA and forces splicing to include exon 7 so makes functional SMN protein - SMN complex is required for maturation of snRNP complex for splicing, motor neurons are susceptible to loss of spliceosome

Question 28

What is spinal muscular atrophy?

Answer 28

Genetic condition caused by SMN1 gene mutations which causes death of motor neurons

Question 29

What is the life cycle of snRNAs? (6)

Answer 29

- Transcription of snRNA in the nucleus - 150-200nt = too short for hnRNPC so Phax complex recruited and is exported to the cytoplasm for maturation - Has Gemin5 bound in the cytoplasm and interacts with SMN complex (mutated in spinal muscular atrophy) - SMN complex forms a ring (importance of oligomerisation) and recruits Sm proteins that also form a ring on the snRNA (don't get ring of Sm proteins on snRNA in spinal muscular atrophy, can't form snRNP complexes, can't form spliceosomes, can't do splicing properly) - Cap gets hypermethylated (tri-methyl) - snRNA recruited back into the nucleus into the Cajal body, drives final maturation of snRNP complex which can then be used in splicing

Question 30

What genes tend to be mutated in motor neuron diseases?

Answer 30

RNA binding proteins e.g. FUS

Question 31

How does FUS mutation cause disease? (5)

Answer 31

- Loss of function causes disregulation of splicing, motor neurons are particularly susceptible to loss of spliceosome - FUS binds to U1 snRNP (5' splice site), TDP43 and SMN (splicing factors) - FUS mutants mislocalise in the cytoplasm instead of the nucleus so bound U1 snRNA is in the cytoplasm rather than nucleus - RNA polymerase II binds to FUS which stabilises RNApolII interaction with U1 snRNP so loss of FUS means loss of coupling splicing and transcription - Uncoupled splicing is less efficient