RNA surveillance and translation I

mRNA surveillance

• improperly processed pre-mRNAs are recognized in the nucleus and marked for degradation

• incompletely spliced pre-mRNAs that remain associated with spliceosome components are restricted from export

nonsense-mediated decay (NMD)

targets mRNAs with stop codons that occur before the last splice junction

sources of mRNAs with stop codons before last splice site

• errors in splicing that produce frameshifts

• mutation that produces a stop codon or a frameshift

dominant negative mutations

generated by frameshifts, loss of function

frameshift mutation

causes a drastic reduction in transcript levels

mechanism of nonsense-mediated decay

1. after splicing, exon-junction complex (EJC) is deposited at the exon-exon junction

2. Upf3 (EJC component) remains associated during export

3. ribosomes displace Upf3 during pioneer round

4. ribosomes can’t displace Upf3 if it is downstream from a premature stop codon

5. if Upf3 remains associated with the mRNA it triggers formation of a complex that targets the mRNA for degradation

6. mRNA targeted for degradation is sent to P-bodies (membraneless organelles)

non-decay

targets mRNAs that lack an in-frame stop codon due to premature polyadenylation or mutation

no-go decay

targets mRNAs that are stalled during translation because of damage or the presence of an unusually stable secondary structure

types of RNA involved in protein synthesis

• mRNA

• tRNA

• rRNA

bacterial ribosomes

• 23S + 5S rRNA + 31 proteins → 50S large subunit

• 16S rRNA + 21 proteins → 30S small subunit

• assembled: 70S

eukaryote (vertebrate) ribosomes

• 28S:5.8S + 5S rRNA + 50 proteins → 60S large subunit

• 18S + 33 proteins → 40S

• assembled: 80S

S

unit used to describe size of structure, based on gradient from ultracentrifuge (represents density in gradient)

rRNA structure

highly conserved stem-loop secondary structure

pre-tRNA processing

• 5’ end sequence is removed

• short segment is removed

• CCA is added on to the 3’ end (amino acid attachment site)

• extensive modification of internal bases

aminoacyl-tRNA synthetases

• recognize and bind to their cognate tRNAs

• can bind to more than one unique tRNAs (each tRNA will bind the same amino acid)

degenerate genetic code

• a single codon can be bound by more than one single tRNA (anticodon)

• only a few tRNAs can bind to a unique codon (most are redundant)

tRNA_i^Met

• exclusively used for initiation of a polypeptide chain

• can interact with P site of small subunit of ribosome

• methionine in bacterial tRNA_i is modified by addition of a formyl group

tRNA^Met

exclusively used for elongation of a polypeptide chain

tRNA_i^Met and tRNA^Met

charged by the same aminoacyl-tRNA synthetase

assembly of pre-initiation complex

1. eIF2 ternary complex formation: eIF2-GTP binds to tRNA_i^Met

2. 43S complex formation: small subunit of ribosome in non-translating form (40S) associates with eIF5

3. mRNA activation: eIF4 binds the 5’ cap of the mRNA

4. attachment to mRNA: eIF4G (subunit of eIF4) binds eIF3 in the 43S preinitiation complex and PABPC at 3’ end of mRNA

5. 5’ to 3’ scanning

regulated of protein synthesis

negatively regulated by phosphorylation of eIF2, inhibits its ability to bind GTP and blocks translation

cap-binding protein

required for efficient translational initiation

capping of mRNA transcripts

dimeric guanylyltransferase binds to the phosphorylated CTD of RNA pol II, so only mRNA is capped and translated efficiently

eIF4

• eIF4E binds 5’ cap of mRNA, activity is tightly regulated and overexpression is associated with tumour formation, may remain associated with scanning complex forming a loop at 5’ end of mRNA

• eIF4G binds eIF3 on 43S preinitiation complex and PABPC (poly(A) binding protein, cytoplasmic) at 3’ end of mRNA, forming a loop

• eIF4A is an ATP-dependent RNA helicase and removes secondary structures of mRNA

• eIF4B enhances eIF4A

scanning complex

43S preinitiation complex and eIF4

initiation of translation

1. when the scanning complex associates with the AUG start codon, eIF2-GTP is hydrolysed to eIF2-GDP, causing a conformational change and dissociation of factors except eIF1A and eIF5B

2. stable binding of the initiation complex with the start codon triggers subunit joining and formation of the 80S ribosome

3. once translation is initiated the ribosomal complex becomes irreversibly bound to the mRNA until translation is terminated