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BIS477 World of RNA > Mechanisms for RNA localisation in eukaryotes > Flashcards

Mechanisms for RNA localisation in eukaryotes Flashcards

1
Q

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

A
  • Make a piece of DNA made of Ts with a fluorescent probe
  • Binds to polyA tails on mRNAs
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2
Q

What are nuclear speckles?

A

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

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3
Q

What needs to happen before mRNA export? (2)

A
  • 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
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4
Q

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

A
  • 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
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5
Q

Which way does mRNA exit the nucleus?

A

Goes through nuclear pore 5’ end first

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6
Q

What is the nuclear cap binding complex?

A

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

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7
Q

What is U2AF35?

A

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

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8
Q

What is UAP56? (2)

A
  • RNA helicase
  • Component of the TREX complex
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9
Q

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

A
  • 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
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10
Q

What is the exon junction complex (EJC)?

A

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

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11
Q

What is EIF4A3? (2)

A
  • RNA helicase
  • Component of the EJC
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12
Q

What is polyA binding protein (PABP)?

A

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

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13
Q

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

A
  • 5’ cap via CBP80
  • Internal EJCs via EIF4A3
  • PolyA tail via PABP
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14
Q

What is the TREX complex?

A

Intermediate complex in mRNA export

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15
Q

What is Nxf1 (Tap) protein? (3)

A
  • Nuclear RNA export factor 1
  • The major mRNA export receptor
  • Binds to RNA and the nuclear pore
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16
Q

What is the structure of Nfx1? (2)

A
  • 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
17
Q

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

A
  • Nxf1 heterodimerises with p15 (Nxt1) via NTF2 scaffold
  • NTF2 and UBA-like domain recognise phenylalanine-glycine (FG) repeats in nucleoporins
18
Q

What are nucleoporins?

A

Proteins which line the nuclear pore

19
Q

How is mRNA licenced for export? (3)

A
  • 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
20
Q

How is Nxf1 linked to TREX? (2)

A
  • 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
21
Q

How does RNA length determine export route? (5)

A
  • 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
22
Q

What is hnRNPC?

A

Molecular ruler

23
Q

How are mRNA export factors recycled? (5)

A
  • 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
24
Q

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

A
  • Protects the mRNA
  • Proteins packaging the RNA prevent it from re-annealing to the template DNA strand and prevent formation of R loops
25
Q

How are R loops formed? (3)

A
  • 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
26
Q

What happens if TREX is depleted?

A

Lots of R loops and lots of DNA damage

27
Q

How does SMN1 cause spinal muscular atrophy? (5)

A
  • 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
28
Q

What is spinal muscular atrophy?

A

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

29
Q

What is the life cycle of snRNAs? (6)

A
  • 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
30
Q

What genes tend to be mutated in motor neuron diseases?

A

RNA binding proteins e.g. FUS

31
Q

How does FUS mutation cause disease? (5)

A
  • 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

BIS477 World of RNA (14 decks)

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