Single polysome analysis of mRNP.

Eukaryotic translation is a complex process that involves the interplay of various translation factors to convert genetic information into a specific amino acid chain. According to an elegant model of eukaryotic translation initiation, the 3′ poly(A) tail of an mRNA, which is occupied by poly(A)-binding proteins (PABPs), communicates with the 5′-cap bound by eIF4E to enhance translation. Although the circularization of mRNA resulting from the communication is widely understood, it has yet to be directly observed. To explore mRNA circularization in translation, we analyzed the level of colocalization of eIF4E, eIF4G, and PABP on individual mRNAs in polysomal and subpolysomal fractions using single polysome analysis. Our results show that the three tested proteins barely coexist in mRNA in either polysomal or subpolysomal fractions, implying that the closed-loop structure generated by the communication between eIF4E, eIF4G, and PAPB may be transient during translation. •Individual mRNPs were analyzed at the single-molecule level. •The number of PABP bound to translating mRNA has a wide distribution. •Simultaneous appearance of eIF4E, eIF4G, and PABP on translating mRNA in polysomes was rare. •The closed-loop by the eIF4E-eIF4G-PABP complex is not a standard feature of active translation. [ABSTRACT FROM AUTHOR]