3 - mRNA Export - Wilson Flashcards
what does the image in q1 325 - 3 word show? describe how this image was obtained
- shows mRNA locations in HeLa cells. distributed throughout nucleus and cytoplasm (NOT nucleoli)
- fluorescent oligo(dT) probe which hybridises to the polyA tail of mRNA
- FISH used to visualise
what processing events need to take place for mRNA to be exported to the cytoplasm? why is this important?
- capping, splicing, CPA
- important becuase if export of unprocessed mRNA occurred could lead to incorrect protein formation, toxic fo the cell OR if contained an intron -> NMD in cytoplasm
give the first stage of mRNA export. what protein binds, what proteins aid this recruitment, where is this protein located on the mRNA?
- TREX binding stabilised the mRNA and aids in mRNA export
- initially Cap binding protein complex (CBP80 + 20) bind to the CAP structure. CBP80 recruits ALYREF, recruits TREX to the 5’ cap structure
- from here, TREX can jump to internal sites on the mRNA
- TREX interacts with the exon junction complex protein eIF4AIII. TREX can be found all along the mRNA
- TREX stabilised on mRNA by splicing
325 - 3 word
give the name of the major mRNA export factor. state its structure, function and how specifically it aids in mRNA export
- Nxf1
- exists as a heterodimer with p15
- multi domain protein with RNA binding domains (RBD) etc (cargo-binding domain) and a nuclear pore complex binding domain
- binds specifically to F/G repeats that line the nuclear pore and aid translocation
- function; acts at a late stage of mRNA export taking mRNP complexes to the nuclear pore and aiding its translocation to the cytoplasm
draw a diagram of Nxf1 associated with mRNA and state how this association occurs
- Nxf1 recruited to TREX so everywhere TREX bound, Nxf1 also present
325 - 3 word
how does Nxf1 transition to the open complex? what does this allow it to do? why is this stage important? DAG
- TREX binding to Nxf1 releases the RBD allowing it to bind to mRNA and aid in translocation
- specifically, ALYREF binds to the RBD allowing it to dissociate from NTF2 and bind RNA
- a separate TREX domain binds to NTF2
- important becuase this ensures that ONLY SPLICED mRNA is exported. TREX is present only @ EJC of spliced mRNA
325 - 3 word
how does the length of RNA determine the export route? explain the process of each export route
- hnRNPC proteins measure the Length of mRNA as it is being transcribed
- < 300 nt uses the snRNA pathway, > 300 nt uses the mrna export pathway
mRNA EXPORT; - hnRNPC (wraps around 230nt) can wrap around this mRNA
- preventing the recruitment of the PHAX binding protein and allowing ALYREF recruitment
- mRNA export factors recruited eg Nxf1
snRNA EXPORT;
- hnRNPC cannot wrap around the smaller mRNA. cannot prevent PHAX recruitment
- PHAX recruitment -> this recruitment pathway used
why is it necessary to recycle export factors ? give the names of these export factors in humans and yeast
- because we would get many export factors in the cytoplasm and none in the nucleus if we weren’t to recycle them
- humans; Nxf1, p15
- yeast; Mex67 (Nxf1 alternative) and Mtr2 (p15)
describe how we get recycling of the export factors
- activation of RNA helices on cytoplasmic side triggers release of Mtr2 and Mex67 from the mRNA
- ATP hydrolysis cycle and recycling of the factors
- proteins that play an important role in this = Gle1 and DBP5
why is it important for RNA to never be naked in the cell (when we are undergoing transcription)
formation of R loops;
- ssRNA can invade the transcription bubble and base pair to ssDNA
- RND:DNA interaction stronger than DNA:DNA interaction therefore hard to disrupt
- therefore leading to innapropriate or inhibition of protein synthesis -> cell death/cancer
give examples of the proteins that bind to mRNA and help to prevent R loop formation
- packaging mRNA for export = v important eg proteins hnRNP, PHAX, ALYREF, TREX, Nxf1
- other proteins; EJC - eIF4AIII, SR like proteins, PABP (poly A binding protein)
draw a diagram of both versions of SMN1 gene in the cell and explain what happens if we get a mutation of one of them
- SMN2 gene creates faulty (low SMN protein levels) because naturally occurring mutation in Exon 7 therefore this Exon cannot be included in the mRNA (skipped out)
- if we get mutation in SMN1 gene (which has naturally high SMN protein levels) leads to lower SMN protein levels leading to neuronal death
- leading to spinal muscular atrophy
325 - 3 word
how can we potentially treat spinal muscular atrophy?
force the correct splicing of SMN2 gene using antisense oligonucleotides
what are the functions of the SMN protein? state specifically how it does this and draw a diagram of this process. also state where the SMN proteins reside
- allows the biogenesis of snRNPs in the nucleus
- SMN located in Gems in the nucleus
- SMN promotes the formation of the Sm protein ring on the snRNP in the cytoplasm
- once sufficient modifications to the snRNP made in the cytoplasm (eg 3’ end trimming) the snRNP is bound by importins and taken back to the nucleus where it resides in Cajal bodies. this is important in creating active snRNPs that can then function in pre-mRNA splicing
325 - 3 word
what are the functions of gems? in which disease are they not present?
- where the SMN proteins reside
- function still largely unclear
- mutations of the FUS gene in motor neurone disease (MND) result in no gems
