Translating into the new year. A new role for eIF4A and more methyl modifications

Translation, the conversion of the mRNA code into a polypeptide, is a very important process, one that by now you’d think we would have sussed. However, it is the way with science that with further experiments and improvements in techniques, our opinions on cellular mechanisms can change. The process of translation involves hundreds of factors, both protein and RNA, and hence a wealth of regulatory modifications can be loaded upon them as well. With recent advances in the characterisation of RNA nucleotide modifications, many of these are beginning to be linked to critical steps in the life of mRNA implicating its translational efficiency. m6Am has recently been linked to the stability of mRNA. However, are knowledge of the roles of the essential factors and their interplay are also expanding.

A quick introduction to translation initiation

During translation, triplets of mRNA nucleotides are ‘read’ by the ribosome and the corresponding amino acid that the trinucleotide (codon) encodes is added to the elongating polypeptide chain. A key step in translation is finding where to start. Translation initiation typically occurs at the first AUG codon downstream of the 5’end. This involves both the loading of the ribosome onto the mRNA followed by AUG recognition. The ribosomal complex achieves this in association with a host of initiation factors.

The process begins with the small subunit of the ribosome (40S) in combination with the initiation factors eIF1, eIF1A, eIF2-GTP-Met-tRNAi*, eIF3, and eIF5 to form the 43S preinitiation complex (PIC). The PIC is recruited onto the 5’end of the mRNA where it is joined by yet more factors eIF4E, eIF4G, eIF4A (collectively eIF4F) and eIF4B forming the 48S complex. The complex then scans along the mRNA until it encounters the AUG, recruits the large ribosome and begins protein synthesis (Figure 1).

 

trans 2.png
Figure 1: Simplified model of translation initiation. Note green represents growing polypeptide

 

This is by no means a simple task.

*eIF2-GTP-Met-tRNAi constitutes the ternary complex which contains the first amino acid, methionine, to be added to polypeptide. tRNAi is the special i=initiation tRNA.

eIF4A – eukaryotic initiation factor 4 A (for awesome.., probably)

The role of eIF4A (4A) is to aid in the scanning of the complex down mRNA. mRNA is not as bare nor as straight as indicated in Figure 1 and can conform to a variety of structures that hinder the scanning. 4A uses the energy from ATP hydrolysis to ‘strip-bare’ the mRNA. This is 4A’s well-known role as a helicase.

However, a paper published in PNAS by Sokabe’s group (1) have reported an additional role for 4A. You see, mRNA has to be accommodated into the 43S complex before scanning can start. eIF3j, a domain of eIF3, blocks part of the mRNA binding site in 40S providing a barrier to this step. Sokabe’s team used fluorescence anisotropy to demonstrate that eI3j has reduced affinity for the 43S-PIC during recruitment of mRNA. Reduced affinity was ATP-dependent but helicase-independent activity of eIF4A. To ignore the helicase activity the group used unstructured mRNA (CAA)42 and non-hydrolysable AMP-PNP (instead of ATP).

 

 

A simplified, proposed mechanism for what occurs can be seen in Figure 2. It seems possible that eIF4A-ATP enables full accommodation of mRNA into the entry channel by inducing conformational changes in the small ribosome subunit.

 

epa.png
Figure 2: Accommodation of mRNA into the PIC by eIF4A-ATP reduces the affinity of eI3j. Modified from (1)

 

Since, this role is independent to its function as a helicase and it requires ATP, it serves as another regulatory step in mRNA recruitment and may explain why 4A is required for translation of mRNAs regardless of their structure. Before these results can be taken as gospel, further structural details will be needed – these could come from cryoEM structures.

..But A is also for adenine

mRNA doesn’t hang around in a cell forever. Eventually it gets degraded. skull The rates of degradation provide an extra layer of regulation for protein production since mRNAs that hang around for longer have more time to synthesise protein.

Mauer’s group recently published a paper in Nature (2) that demonstrates how methylation of the first adenine (A) nucleotide in mRNA can alter its stability. Adenine is known to be methylated at at least two positions; 2’-OH and N6 (Figure 3), the former of which is normal for the majority of mRNA. It is the presence of the additional N6 methylation that Mauer’s team found with increased stability. They propose the additional methylation provides resistance against the decapping activity of DCP2, a step which initiates mRNA decay. Interestingly, it was found that the mark is reversible (m6Am can be converted back to Am) by the protein FTO. This additional mark may also help to explain the discrepancy in efficacy of miRNA-mediated decay.

 

meth a.JPG
Figure 3: Adenine modifications (taken from (2))

 

This is just one paper out of thousands that are now examining this new area of the epitranscriptome which is all very exciting! I suspect many more findings in 2018!

Further Reading

(1) (Really, really nice paper, if you’re interested) Sokabe, M., & Fraser, C. S. (2017). A helicase-independent activity of eIF4A in promoting mRNA recruitment to the human ribosome. Proceedings of the National Academy of Sciences, 114(24), 6304–6309. https://doi.org/10.1073/pnas.1620426114

(2) Mauer, J., Luo, X., Blanjoie, A., Jiao, X., Grozhik, A. V., Patil, D. P., … Jaffrey, S. R. (2017). Reversible methylation of m6Amin the 5′ cap controls mRNA stability. Nature, 541(7637), 371–375. https://doi.org/10.1038/nature21022

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