Structural Biochemistry/Nonsense-Mediated mRNA Decay

NMD Introduction
NMD is a short-handed term for “Nonsense-mediated mRNA decay”. NMD is a mechanism of mRNA surveillance which functioned as detecting nonsense mutations and preventing the expression of reduction or incorrect proteins. During RNA translation, the mRNAs can produce abridged proteins carry dominant negative activities. After transcription, mRNA performs a convergence of ribonucleoprotein components first, and then undergoes the regulation of pre-mRNA. As average intro sizes in eukaryotic cells are relatively large, the probability of aberrant mRNA splicing will highly increases. This results in the production of nonsense/stopping codon such as UAA, UAG, and UGA to increase. NMD is then triggered by exon junction complexes, also known as “EJCs”, which are stored during the pre-mRA processing. The presence of EJCs can further promote ribosomal recruitment before the replacement by translation. The EJCs existed in the downstream of a nonsense codon are not performed since the ribosome is reduced from the transcript without reaching it. The remaining EJCs sign of recruitment of UPF1 after the mRNA’s transport away from the nucleus and intro the cytosol, the RNA degrading center. Overall, NMD is considered both a process of degrading truncated mRNA and also a method to regulate normal transcripts’ expression.

NMD Factors
Essential proteins for NMD are: UPF1, UPF2 and UPF3 with the core NMD machinery. For UPF1, proteins including SMG-1, SMG-5, SMG-6 and SMG-7 can intercede the both cycles of phosphorylation and dephosphorylation.

UPF and SMG proteins
The UPF and SMG proteins are the core machineries for NMD mechanism.

Here are some of the proteins and their functions:
 * 1) UPF1: located mainly in cytoplasmic reticulum and some nuclear.  Its functions are promoting translation; histone mRNA decay; ATPase; helicase; phosphoprotein substrate for SMG-1 and requited by eRFs to stop codons; undergoes a cycle of phosphorylation and dephosphorylation.
 * 2) UPF2: located in cytoplasmic but has nuclear localization signals. It s functions are to promoting translation, EJC adapter protein that binds to both UPF1 and UPF3; binds to RNA in vitro.
 * 3) UPF3: located mainly in nuclear, some in cytoplasmic. Its functions are promoting translation; EJC protein with short and long isoforms that differentially distribute into distinct cytoplasmic protein complexes with UPF1
 * 4) SMG-1: located in cytoplasmic. Its functions are phosphoionsitide 3-kinase-related kinase family member; phosphorylates UPF1
 * 5) SMG-5: located mainly in cytoplasmic and some nuclear. Its functions are interacting with PP2A and promoting UPF1 dephosphorylation.
 * 6) SMG-6: located mianly in cytoplasmic, and some nuclear. Its functions are interacting with PP2A and promoting UPF1 dephosphorylation.
 * 7) SMG-7: located mianly in cytoplasmic, and some nuclear. Its functions are interacting with PP2A and promoting UPF1 dephosphorylation; when over expresses, it recruits UPF1 to P-bodies.

Exon-Junction Complex (EJC)
The Exon Junction Complex (EJC) contains four major proteins: eIF4AIII, MAGOH, MLN51, and Y14. All of the four proteins form the core functions in NMD mechanism such as cross-linking, coimmuno-precipitation, mutation analysis, and RNase H footprinting techniques. The purpose of EJC is to serve as station to attach the transient EJC components, and therefore associate more transiently with the mRNA during its journey from the nucleus to the cytoplasm.

NMD during a pioneer round of translation
NMD during a pioneer round of translation can produce large amount of truncated proteins with deleterious activities. In order to undergo this process, mRNAs are scanned for PTCs and degraded during the early rounds of translation. This method further suggests that mRNAs are scanned either in the nucleus or after entering the cytoplasm when the mRNAs are associated with the nucleus.

During the pioneer round of translation, NMD occurs when UPF1 interacting with UPF2, which is bound to UPF3. EJC core, attaching with UPF2 and UPF3, is recruited to exon-exon junctions during mRNA splicing. In an aberrant transcript, at least one EJC deposited downstream of the premature stop codon. It then can interact with UPF1 recruited by CBC and the eukaryotic release factors eRF1 and eRF3. The interaction between UPF1 and UPF2 is strengthened by CBC when undergoing the pioneer round of translation. In the final step, mRNAs eventually decay. Comparing to a PTC-containing mRNA, a normal mRNA transcript prohibits NMD because the presence of EJC stops codon and further displaced by the ribosome before UPF1 is recruited. After this step, normal transcripts exchange theirs proteins at both 5’ and 3’ ends and proceed to enlarge translation continuously.

Molecular Interactions that Define Nonsense Codons
The second signal, such as EJC, is required to define a stop codon as being premature and thus trigger another NMD. Before the mechanism, mRNA is first degraded by NMD. mRNA then undergoes the pioneer round of translation, which leads to the recognition of the stop codon by the eukaryotic release factors eRF1 and eRF3. UPF1 recruits the protein kinase SMG-1, which together with the eRFs forms a transient complex called SURF in third step. For the next step, the SURF complex interacts with an EJC as a requirement for SMG-1 to phosphorylate UPF1. The interaction further triggers the degradation of mRNA, which eventually reduces the release factors and the 40S and 60S ribosomal subunits.

UPF1 Dephosphorylation, P-Body Recruitment, and mRNA Decay
After UPF1 is phosphorylated by SMG-1 in the previous step, it further recruits the dephosphorylation. Late molecular activity in NMD degrades the mRNA. The phosphorylation of UPF1 recruits the SMG-5/SMG-7 heterodimers and the phosphatase PP2A indicated in the first two steps. P-body recruitment then trigger PP2A to dephosphorylate UPF1. The dephosphotylate UPF1 then releases PP2A and SMG-5/SMG-7 from the mRNP and also decapping, which the mRNA body rapidly decays by 5’ to 3’ exonucleas.

Recent Study
Recent researches suggest that mammalian NMD has other uses other than being a linear pathway. All the discoveries of NMD involves the study of EJCs and other independent EJCs.

Reference

 * 1) NMD
 * 2) Yao-Fu Chang, J. Saadi Imam, and Miles F.Wilkinson The Nonsense-Mediated Decay RNA Surveillance Pathway  Link: