Abstract
We report the initial characterization of an N‐terminal oligopeptide ‘2A‐like’ sequence that is able to function both as a signal sequence and as a translational recoding element. Owing to this translational recoding activity, two forms of nascent polypeptide are synthesized: (i) when 2A‐mediated translational recoding has not occurred: the nascent polypeptide is fused to the 2A‐like N‐terminal signal sequence and the fusion translation product is targeted to the exocytic pathway, and, (ii) a translation product where 2A‐mediated translational recoding has occurred: the 2A‐like signal sequence is synthesized as a separate translation product and, therefore, the nascent (downstream) polypeptide lacks the 2A‐like signal sequence and is localized to the cytoplasm. This type of dual‐functional signal sequence results, therefore, in the partitioning of the translation products between the two sub‐cellular sites and represents a newly described form of dual protein targeting.
Highlights
The function of the 2A oligopeptide sequence (18aa) was first characterized from the positive-stranded RNA picornavirus Foot-and-Mouth Disease Virus (FMDV)
Our data show that this N-terminal form of 2A possesses both translational recoding and signal sequence activities in mammalian and plant cells. We propose that these translational recoding signal sequences represent a newly characterized form of dual protein targeting: in the proportion of cases where a peptide bond is formed between the SpNLR2A signal sequence and the downstream protein, the fusion protein is targeted to the exocytic pathway
While we have not investigated the fate of the oligopeptide SpNLR2A signal sequence [interaction with signal recognition particle (SRP) or degradation?], we show that lacking an N-terminal signal sequence the downstream protein localizes to the cytoplasm – taken together, a novel form of dual protein targeting
Summary
The function of the 2A oligopeptide sequence (18aa) was first characterized from the positive-stranded RNA picornavirus Foot-and-Mouth Disease Virus (FMDV). In support of this model, eRF3 was recently shown to mediate termination of translation of yeast ribosomes stalled on polylysine tracts [17], other recent analyses using re-constituted translation systems did not support the involvement of eRFs 1 and 3 [18] Our model of this translational recoding event predicts that three alternative outcomes arise; having synthesized sequences upstream of 2A, ribosomes either (i) resume translation of the downstream sequences, (ii) translation is www.traffic.dk 923. Terminated at that point, or, (iii) that no translational recoding occurs: the glycyl-prolyl peptide bond is formed and the protein is synthesized in the normal manner The ratio of these translation products is dependent upon the specific 2A-like sequence in question – a model supported by the observations from many other laboratories that have used 2A for biomedical or biotechnological protein co-expression applications [19]. A motif (−D[V/I]ExNPGP−) is conserved at the C-terminus of all 2A/2A-like sequences: this motif must, be accompanied by an appropriate, but much
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