Composition and arrangement of genes define the strength of IRES-driven translation in bicistronic mRNAs

Abstract

In addition to the cap-dependent mechanism, eukaryotic initiation of translation can occur by a cap-independent mechanism which directs ribosomes to defined start codons enabled by internal ribosome entry site (IRES) elements. IRES elements from poliovirus and encephalomyocarditis virus are often used to construct bi- or oligocistronic expression vectors to co-express various genes from one mRNA. We found that while cap-dependent translation initiation from bicistronic mRNAs remains comparable to monocistronic expression, internal initiation mediated by these viral IRESs is often very inefficient. Expression of bicistronic expression vectors containing the hepatitis B virus core antigen (HBcAg) together with various cytokines in the second cistron of bicistronic mRNAs gave rise to very low levels of the tested cytokines. On the other hand, the HBcAg was well expressed when positioned in the second cistron. This suggests that the arrangement of cistrons in a bicistronic setting is crucial for IRES-dependent translation of the second cistron. A systematic examination of expression of reporter cistrons from bicistronic mRNAs with respect to position was carried out. Using the dual luciferase assay system we show that the composition of reading frames on a bicistronic mRNA and the order in which they are arranged define the strength of IRES-dependent translation. Although the cellular environment and the nature of the IRES element influence translation strength the dominant determinant is the nature and the arrangement of cistrons on the mRNA.

Translational Regulation of the JunD Messenger RNA

“…10) (29). Expression from the EMCV IRES control was consistent with previous studies, being up-regulated 10-fold when compared with the no insert construct (24,49). Insertion of a synthetic hairpin sequence into a dicistronic vector was shown previously to inhibit expression of the 5Ј-ORF while simultaneously increasing expression of the 3Ј-ORF in the presence of an IRES (21).…”

Improved co-expression of multiple genes in vectors containing internal ribosome entry sites (IRESes) from human genes

“…It remains to be determined if the observations made here are applicable in different gene therapy vector systems including viral (eg retroviral) and non-viral systems. 25 As it has been reported that the nature of the upstream gene influences the translation efficiency of the viral IRES, 7 it would also be important to determine if our observations are also applicable when different therapeutic genes are introduced upstream and downstream of these cellular IRESes. The applicability of other cellular as well as artificial IRESes 26 for the co-expression of multiple genes for gene therapy can also be explored.…”

“…[4][5][6] Furthermore, it has been reported that the order of arrangement of the genes on the transcript greatly influences the efficiency of viral IRES-dependent translation. 7 IRES sequences that have been identified in mammalian genes include those of the immunoglobulin heavy chain binding protein (BIP), 8 eukaryotic initiation factor 4G (EIF4G), 9 c-myc proto-oncogene (MYC), 10 and vascular endothelial growth factor (VEGF). 11,12 Thus far, they have not been utilized in gene therapy vectors for the coexpression of multiple genes.…”

F2A sequence linking MGMTP140K and MDR1 in a bicistronic lentiviral vector enables efficient chemoprotection of haematopoietic stem cells

“…8 The transgene itself might influence the IRES-dependent translation owing to its structure or sequence. 56 In addition, an influence of the cell type or of the available cellular components might be possible. 57 The observation that a significant position dependence for MGMT P140K expression under the control of both IRES elements could be found only in K562 but not in HL-60 cells argues for a cell typespecific effect maybe in combination with a ternary structure of the MGMT mRNA.…”