mRNA Surveillance & RNA interference Flashcards
What are the pathways to eliminate faulty mRNA?
- non-stop decay
- nonsense-mediated decay
What is the non-stop mRNA decay pathway?
- if mRNA is missing a stop codon, translation continutes through the poly(A) sequence
- this makes a poly lysine tail (AAA = lys)
- Ski7 protein recruited
- Ski dissociates ribosome and recruits exosome to degrade mRNA 3’-5’
- polypeptide degraded by a protease that recognizes poly(Lysine)
Nonsense-mediated mRNA decay (NMD) is a — mechanism that eliminates mRNAs containing premature translation-termination codons (PTCs…nonsense codons UAA, UAG or UGA).
translation-coupled
NMD was first discovered through studies on the genetic basis of
B-thalassemia
How does B-thalassemia disease vs no disease occur in the gene?
Disease: premature termination codon in last exon (mRNA not degraded)
No: PTC in other exons (mRNA degraded)
This is in the beta gene of hemoglobin
NMD requires a protein complex known as the
Where is this protein placed?
exon junction complex (EJC)
20-24 nt upstream of every single exon-exon junction after splicing
What is the exon junction complex made of?
many proteins: UPF1, 2, 3 (up-frameshift protein
What are the steps of the NMD pathway?
- EJCs placed on every junction
- ribosome displaces EJCs from each exon:exon junction if normal mRNA
- mRNA translated by ribosome
- if premature termination codon is more than **50-55 nts **away from the exon:exon junction, NMD occurs
- ribosome will stop at PTC
- UPF1 and UPF2 recruited by downstream EJCs
- UPF1 and UPF2 recruits protein that removes 5’ cap
- 5’ end susceptible to 5’-3’ exoribonuclease degradation
short RNAs are called — or —
miRNA (microRNAs)
siRNA (short interfering RNAs)
What are the two sources of RNA for interfering RNA? (RNAi)?
- extracellular - dsRNA viruses
- intracellular - miRNA genes
canonical miRNA biogenesis pathway?
- miRNA gene transcripbed with RNAPII
- pri-miRNA forms stem-loop structure (RNA-RNA bp)
- Drosha enzyme cleaves to make pre-miRNA
- Exportin-5 exports pre-miRNA to the cytoplasm from nucleus
- pre-mRNA processed by Dicer enzyme to remove loop and just have miRNA duplex
- AGO (slicer) protein associates with duplex which is the pre-RISC complex
- One strand (passenger strand) is lost
- RISC complex formed
pri-miRNA
What is the RISC complex?
What does it do and what does it include
RNA-Induced Silencing Complex ie guide RNA
- interferes with gene expression (mRNA degradation or translational suppression)
- AGO (slicer) protein + 1 mature, guide miRNA strand that complementary bps with the target
- complementarity of RNA to target determines functionality
What is the non-canonical pathway to generate miRNA?
introns
- pre-miRNA embedden in intron
- spliceosome releases lariat
- this is the pre microRNA
- continute to be exported
- will usually interfere with their own gene because they have the complementarity
intron generated
What are the sections of the miRNA when bound to its target?
- region at 5’ end of miRNA called the seed region that binds to target site
- bulge
- 3’ end has varied complementarity
more miRNAs bind in the — of target mRNA
3’ UTR (between ORF termination codon sequence and polyA tail)
If the seed region is perfectly complementary to the target …
Ago slices mRNA -> deadenylation of target and degradation
if the seed region is imperfecting boind to the target mRNA…
translational inhibition
The major difference between siRNAs and miRNAs is that
siRNA inhibits the expression of one specific
target mRNA
miRNA regulate the expression of multiple mRNAs
How does siRNA function?
siRNAs can not..
- dsRNA recognnized by Dicer
- makes siRNA duplex (perfectly bped)
- Ago2-RISC complex binds to target and degrades mRNA
repress translation (only induce cleavage)
sources of siRNA?
endogenous, virus (dsRNA)
