mRNA Stability and Localization Flashcards
ORF
(open reading frame) protein coding region
AUG
Start codon, 1st amino acid
UAG, UGA, UAA
Stop codon, no amino acid
mRNP
messenger ribonucleoprotein
3’ poly-A, poly(A) polymerase (PAP)
enzyme adds stretch of polyadenylic acid to 3’ end of RNA
Hairpin/stem-loop
2ndary structure RNA of base-paired region (stem) + terminal loop of ssRNA
Why is mRNA unstable?
Ribonucleases
Endoribonuclease
Cleaves RNA at internal site(s)
Exoribonuclease
Removes terminal ribonucleotides from RNA
Enzyme remains associated with substrate while catalyzing removal of nucleotides
Processive nuclease
Enzyme that catalyzes removal of only one or a few nucleotides before dissociating from substrate
Distributive nuclease
Bacterial mRNA half-lives
Few minutes
Eukaryotic mRNA half-lives
10-20 minutes yeast, 2-3 hrs mammals
Importance of differential mRNA stability?
mRNA abundance and spectrum of proteins
Steady state
Concentration of population of molecules where rates of synthesis and degradation are constant
RBPs
(RNA-binding proteins) large number exist, uncharacterized
RNA regulons
Overlapping sets of regulatory proteins and mRNAs
Prokaryotic mRNA degradation
Before translation. Degradosome (RNAse E, PNPase, helicase & accessory enzyme)
mRNA that is simultaneously being translated by multiple ribosomes
Polyribosome
mRNA that codes for one polypeptide
Monocistronic mRNA
mRNA that codes for several polypeptides
Polycistronic mRNA
Protects mRNA from direct 3’ attack (prokaryotes)
Stem-loop structure
Most mRNA degradation occurs via…?
Two deadenylation-dependent pathways
PABP
Poly(A) binding protein, binds to 3’ stretch of poly(A) on eukaryotic mRNA
Deadenylation-dependent pathways
1) 5’ -> 3’ (Dcp + Xm1)
2) 3’ -> 5’ (exosome)
Initiated by deadenylation by poly(A) nucleases
Competes with translation intitiation complex for 5’ cap binding
Decapping enzyme
Exosome
Large protein complex, 9 core subunits, interacts with Ski complex
Translationally repressed mRNAs particles (aka mRNP granules)?
Maternal mRNA granules, neuronal granules, stress granules
Where degradation may occur in cytoplasm
Processing bodies (PBs)
Alternative degradation pathways that target specific mRNAS?
Deadenylation-independent decapping, histone degradation via addition of poly(U)-tail, endonucleotic cleavage, miRNA-mediated silencing
Cis-elements that affect rate of degradation in mRNAs
Stabilizing and destabilizing elements
Destabilizing element example
AU-rich elements (ARE)
Stabilizing element example
Iron-response element (IRE)
AREs
No strict sequence conservation, promote rapid deadenylation-dependent exosome degradation
Iron-response element (IRE)
Stability or translation of mRNAs regulated by cellular iron concentration, Fe low -> Transferrin mRNA stable and IRP in high-affinity binding state
Aberrant nuclear RNAs identified and destroyed by…?
RNA surveillance system
Yeast TRAMP Complex
Recruits exosome to aberrant RNAs and facilitates 3’ to 5’ exonuclease activity
Defective RNP examples
Misfolded RNA, missing poly(A) tail
CUTs
Cryptic unstable transcripts
Cryptic unstable transcripts
Non-protein coding RNAs, majority of RNA Pol II transcripts
Nonsense-mediated decay (NMD) targets….
mRNAs with premature stop codons, conserved Upf and SMG proteins
Recognition of premature termination codon in mammals?
Presence of downstream exon junction complexes (EJC)
Recognition of premature termination codon in most organisms?
Unusual 3/ UTR structure or length
Nonstop decay (NSD) targets…
mRNAs lacking an in-frame termination codon, requires conserved SKI proteins
No-go decay (NGD) targets…
mRNAs with stalled ribosomes in coding regions
Localization of mRNA mechanisms (3)?
Uniform distribution but degradation at all sites except translation, freely diffusible but trapped at translation sites, active transport to translation site
Zipcode (localization) signal
Any of mRNA cis-elements involved in directing cellular localization
Predominant active transport mechanism of mRNPs?
Cytoskeletal tracks
