Termination-reinitiation occurs in the translation of mammalian cell mRNAs.

Abstract

Many examples of internal translation initiation in eucaryotes have accumulated in recent years. In many cases terminators of upstream reading frames precede the internal initiation site, suggesting that translational reinitiation may be a mechanism for initiation at internal AUGs. To test this idea, a series of recombinants was constructed in the mammalian expression vector pSV2. Each contained a dicistronic transcription unit comprising the coding sequence for mouse dihydrofolate reductase (DHFR) followed by the gene for xanthine-guanine phosphoribosyl transferase (XGPRT) from Escherichia coli. Various versions of this pSV2dhfr-gpt recombinant plasmid altered the location at which the DHFR reading frame was terminated relative to the XGPRT initiation codon and demonstrated that this is a critical factor for the expression of XGPRT activity in transfected Cos-1 cells. Thus, when the DHFR frame terminated upstream or a very short distance downstream of the XGPRT initiator AUG, substantial levels of XGPRT activity were observed. When the DHFR frame terminated 50 nucleotides beyond the XGPRT initiator, activity was reduced about twofold. However, when the DHFR and XGPRT sequences were fused in-frame so that ribosomes which initiated at the DHFR AUG did not terminate until they encountered the XGPRT terminator, production of XGPRT activity was abolished. This dependence of internal translation initiation on the position of terminators of the upstream reading frame is consistent with the hypothesis that mammalian ribosomes are capable of translational reinitiation.

Human purine nucleoside phosphorylase and adenosine deaminase: gene transfer into cultured cells and murine hematopoietic stem cells by using recombinant amphotropic retroviruses.

“…3). Although coding sequences are usually translated from monocistronic messages in eucaryotes (21), translation of multicistronic message has been observed in several natural and recombinant systems (34). LHM,PL-infected cells appeared to contain more human PNP than LHM,PL-infected cells (Fig.…”

Detection of the ORF3 polypeptide of feline calicivirus in infected cells and evidence for its expression from a single, functionally bicistronic, subgenomic mRNA

“…This close coupling of termination and initiation codons suggests that the two events may be linked and that expression of ORF3 may occur via a termination-reinitiation mechanism. Reinitiation of translation at a downstream AUG codon has been found to take place in both naturally occurring and artificially constructed bicistronic mRNAs (Peabody & Berg, 1986;Kozak, 1987;Horvath et al, 1990).…”

The P2 and P3 Regions of the Poliovirus Genome are Preferentially Translated at Alkaline pH in Infected HeLa Cells

“…Moreover, internal initiation has been demonstrated in mRNAs constructed artificially by recombinant DNA methods (Lomedico & McAndrew, 1982;Subramani et al, 1982;Peabody & Berg, 1986 A large body of evidence indicates that the translation of picornavirus RN A in infected cells is modulated by the ionic concentrations present in the external medium Castrillo et al, 1987). These conditions influence the initiation rates but the site of initiation is not altered and thus the proportion of translation of each region of the genome remains unaltered.…”