5.17. Eukaryotic Translational Control
Eukaryotic mRNAs has longer life span than prokaryotic ones, so there is more chance for translational control. Initiation is rate-limiting factor in translation, thus most control apply at this stage. Generally the phosphorylation of initiation factors involve in control mechanism of initiation and this phosphorylation either stimulatory or inhibitory. For example, a protein binds directly to the 5’ untranslated region of an mRNA and inhibits its translation; exclusion of this protein activates translation.
5.17.1. Phosphorylation of Initiation Factor eIF2α
It is an example of inhibitory phosphorylation take place in reticulocytes, which make one protein haemoglobin. When reticulocytes are starved for heme, then formation of α-globins and β-globins seems wasteful. Deficiency of heme unmasks the activity of heme controlled repressor (HCR) which is a protein kinase and phosphorylate eIF2α, a subunit of trimeric eIF2. The phosphorylated eIF2 binds more strongly than normal to eIF2B. The elF-2B is GEF and also an initiation factor and block eIF2B to perform its function. thus other eIF2 remains in the inactive GDP-bound form and cannot attach Met-tRNAi Met to 40S ribosomes. Thus, responsible for translation initiation halt.
Another example comes from Interferons which is an antiviral protein. At the time of viral infection, due to the presence of interferon and doublestranded RNA a double-stranded RNA-activated inhibitor (DAI) a eIF2α kinase activated mechanism same like HCR to block the translation of virus in virus infected cell. Thus eIF2α phosphorylation condition unfavorable for cell growth, which shown in both above mention cases.
Phosphorylation of an eIF4E- cap binding protein which stimulates translation initiation. Phosphorylated eIF4E binds the cap with about four times more affinity then unphosphorylated eIF4E, thus cause stimulation of translation and favorable for cell growth. Cell division stimulation with insulin or mitogens cause increase in eIF4E phosphorylation and platelet derived growth factor (PDGF) also stimulate translation in mammals by an another pathway that occupy eIF4E.
Insulin binds to insulin receptor, present on cell surface. Insulin receptor is a receptor tyrosine kinase which activates a protein called mTOR. A protein called PHAS-I is a target of mTOR. PHAS-1 inhibit eIF4E activity.
PHAS-1 binds with elF4E. But once mTOR phosphorylate PHAS-I (2) It no longer can bind with elF-4E. Thus elF-4E is free and can participate in translation.
5.18. mRNA surveillance
mRNA surveillance mechanisms are pathways utilized by organisms to ensure fidelity and quality of messenger RNA (mRNA) molecules. There are a number of surveillance mechanisms present within cells.
mRNA surveillance is an enigmatic process because it requires a cellular machinery that can discriminate normal from aberrant mRNAs. mRNA surveillance has been documented in bacteria and yeast. In eukaryotes, these mechanisms are known to function in both the nucleus and cytoplasm.
Biology of mRNA turnover
The steady-state level of a given mRNA depends on the balance between its rates of synthesis and degradation. Importantly, the decay rate of mRNA can be changed to control the amount of polypeptide the cell produces.
The regular mRNA is decay either by decapping followed by 5’-3’ exonuclease or/ and by deadenylation followed by 3’-5’ exonuclease. There is one more mechanism exist to decay the mRNA that is degradation of mRNA by endonulcease followed by 5’-3’ exonuclease and 3’-5’ exonuclease.
Three surveillance mechanisms are currently known to function within cells:
- the nonsense-mediated mRNA decay pathway (NMD);
- the Nonstop Mediated mRNA decay pathways (NSD);
- the No-go Mediated mRNA decay pathway (NGD).
Deadenylation is the most common route of mRNA degradation followed by decapping and 5'® 3' exonucleolytic decay.
5.18.1. Nonsense-mediated mRNA decay (NMD)
The NMD pathway acts via deadenylation-independent decapping, followed by 5'® 3' exonucleolytic decay whereas nonstop decay appears to proceed via deadenylation-independent 3' ® 5' exonucleolytic decay. In bypassing the rate-limiting step of deadenylation, the mRNA surveillance pathways allow the rapid removal of irregular mRNAs from Nonsense-mediated decay (NMD). Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that exists in all eukaryotes.
NMD reduce errors in gene expression by eliminating mRNA transcripts that contain premature stop codons. Three interacting trans-acting factors, Upf1p, Upf2p, and Upf3p, are required for NMD but play no role in nonstop decay. Following splicing in the nucleus, the exon junction complex (EJC), which contains UPF3 (a core protein of the NMD pathway), is associated with the transcript, and the resulting messenger ribonucleoprotein is exported out of nucleus to the cytoplasm.
All three of these factors are trans-acting elements called up-frameshift (UPF) proteins. In mammals, UPF2 and UPF3 are part of the Exon-exon Junction complex (EJC). exon-exon junction is formed after splicing. UPF2 and UPF3 bound to mRNA other proteins, eIF4AIII, MLN51, and the Y14/MAGOH heterodimer, which also function in NMD. UPF1 phosphorylation is controlled by the proteins SMG.
In NMD eRF1, eRF3, Upf1, Upf2 and Upf3 make the surveillance complex. These protein scans the mRNA for premature stop codons. The assembly of this complex is triggered by premature translation termination. Premature termination codon can arise at the DNA level by mutations or at the level of RNA by transcription errors or alternative pre-mRNA splicing. If a premature stop codon is detected then the mRNA transcript is signaled for degradation – the coupling of detection with degradation occurs. Normally the premature stop codon are found 50-55 nucleotide upstream of exon exon junction.
In normal mRNA the Exon-exon Junction Complex (EJC) is upstream to stop codon. The Exon Junction complex get dissociated by the ribosome during the first round of translation. However the premature translation termination found upstream of exon exon junction. This implies that the Exon Junction complex protein remain bound to the mRNA even after this first round of translation, as the ribosome get dissociated before the exon exon junction. This activates the NMD. The premature termination of translation leads to the assembly of a complex composed of UPF1, SMG1 and the release factors, eRF1 and eRF2, on the mRNA.
If an exon Junction complex is left on the mRNA because the transcript contains a premature stop codon, then UPF1 comes into contact with UPF2 and UPF3, triggering the phosphorylation of UPF1. Phosphorylation of UPF1 by SMG1 leads to dissociation of eRF1 and eRF3 and binding of the SMG7 adaptor protein. If the premature translation termination within about 50 nucleotides of the final exon-junction complex then the transcript is translated normally. However, if the termination codon is further than about 50 nucleotides upstream of any exon-junction complexes, then the transcript is down regulated by NMD.
The phosphorylated UPF1 then interacts with SMG-5, SMG-6 and SMG-7, which promote the dephosphorylation of UPF1. SMG-7 is the most important protein for NMD thus called as terminating effector in NMD. SMG-7 also accumulates in P-bodies, which are cytoplasmic sites for mRNA decay. In both yeast and human cells, the major pathway for mRNA decay is initiated by the removal of the 5’ cap followed by degradation by XRN1, an exoribonuclease enzyme. The other pathway by which mRNA is degraded is by deadenylation from 3’-5'.
In the cytoplasm, a second NMD core protein, UPF2, binds to UPF3. Ribosomes associate and translate the mRNA, but are stalled on encountering a premature termination codon (PTC). This results in binding of the SURF complex (comprising SMG1, UPF1 and the peptide-release factors eRF1 and eRF3) to the ribosome. UPF1 also binds UPF2, thereby linking the EJC to the PTC. Subsequent steps that are still being elucidated lead to mRNA decay by various pathways.
The second method for degradation of mRNA is Non Stop decay.
5.18.2. Non-stop decay.
Non stop mRNA lacks stop codon i.e the mutation in DNA create a condition in which the stop codon of mRNA is converted into a sense codon and allow translation to continue. Translation of a mRNA which lacks a stop codon results in ribosomes traversing the poly(A) tail, displacing poly(A)-binding protein (PABP) and stalling at the 3' end of the mRNA. To release the ribosome form mRNA releasing factor binds with stop codon and later ribosome recycling factor dissociates the ribosome form mRNA.
In yeast and mammalian cells, Ski7 play a role in non stop decay. Ski7 is an adaptor protein that functions as a molecular mimic of tRNA, binds to the A site on the stalled ribosome to release the transcript, and then recruits the exosome. The exosome degrades the poly(A) tail and later the complete mRNA .
In another pathway described in Saccharomyces cerevisiae, in the absence of Ski7, the displacement of PABP by the translating ribosome renders the mRNA susceptible to decapping and 5' 3' decay by the 5' 3' exoribonuclease Xrn1.
5.18.3. An another mechanism for decay of mRNA
Several time because of the strong secondary RNA structure formation within the open reading frame (ORF) the ribosomes stall on the mRNA. That means the ribosome is not able to move on the mRNA thus called as No-go decay.
The Dom34 and Hbs1 proteins bind the transcript near the stalled ribosome and initiate an endonucleolytic cleavage event near the stall site. This releases the ribosome and generates two mRNA fragments, each with a free end exposed for exonucleolytic decay by the exosome and Xrn1, respectively.