Differential contribution of the m7G-cap to the 5′ end-dependent translation initiation of mammalian mRNAs

Abstract

Many mammalian mRNAs possess long 5′ UTRs with numerous stem-loop structures. For some of them, the presence of Internal Ribosome Entry Sites (IRESes) was suggested to explain their significant activity, especially when cap-dependent translation is compromised. To test this hypothesis, we have compared the translation initiation efficiencies of some cellular 5′ UTRs reported to have IRES-activity with those lacking IRES-elements in RNA-transfected cells and cell-free systems. Unlike viral IRESes, the tested 5′ UTRs with so-called ‘cellular IRESes’ demonstrate only background activities when placed in the intercistronic position of dicistronic RNAs. In contrast, they are very active in the monocistronic context and the cap is indispensable for their activities. Surprisingly, in cultured cells or cytoplasmic extracts both the level of stimulation with the cap and the overall translation activity do not correlate with the cumulative energy of the secondary structure of the tested 5′ UTRs. The cap positive effect is still observed under profound inhibition of translation with eIF4E-BP1 but its magnitude varies for individual 5′ UTRs irrespective of the cumulative energy of their secondary structures. Thus, it is not mandatory to invoke the IRES hypothesis, at least for some mRNAs, to explain their preferential translation when eIF4E is partially inactivated.

The Current Status of Vertebrate Cellular mRNA IRESs

“…It is much more likely that there is differential affinity for different mRNAs, in which case those mRNAs with high affinity will continue to be translated moderately efficiently via the scanning mechanism even when eIF4F activity has been significantly reduced. A likely reflection of this can be found in the observation that addition of cap analog (m 7 GTP) or especially 4E-BP1 to a Krebs-2 in vitro system resulted in less inhibition of a reporter with the Apaf-1 (especially), c-myc or Hsp70 5 0 -UTRs than with the actin 5 0 -UTR (Andreev et al 2009). In addition, it should be appreciated that the relief of competition consequent on inhibition of bulk mRNA translation following partial eIF4F inactivation will increase the availability of other initiation factors and ribosomal subunits for those mRNAs with high functional affinity for the residual eIF4F.…”

Alternative Ways to Think about Cellular Internal Ribosome Entry

“…Most putative cellular IRESs are much less active than their viral counterparts when tested in assays that reliably measure translational activity (in vitro translation or in vivo translation of transfected in vitro transcribed mRNAs) (8,9,16). Although it is true that the presumed raison d'être of most cellular IRESs, to permit expression of key regulatory proteins, often of low abundance, under conditions of global inhibition of cap-dependent translation, does not require that cellular IRES-dependent initiation be nearly as efficient as viral IRES-dependent translation (which typically drives unregulated high-level expression of viral proteins), the very low level of activity of most putative cellular IRESs makes it far more difficult to rule out alternative explanations for activity in bicistronic reporter assays, mechanisms that do not involve translation at all, such as cryptic promoter activity.…”

“…A typical Northern blot exposure is simply not adequate to rule out the possibility that 1% of the total mRNA encoding the 3Ј-cistron is monocistronic. This is the appropriate level of detection to consider for most putative cellular IRESs, whose demonstrably IRES-dependent translation is only ϳ1% as efficient as cap-dependent translation of a control reporter mRNA (8,9,16). It is important to note that the popular Renilla/firefly luciferase reporter is not the only bicistronic reporter system vulnerable to cryptic promoter artifacts.…”

“…It was therefore quite surprising when the long GC-rich 5Ј-UTRs of several putative cellular IRES-dependent genes (including HIF-1␣, c-myc, and Apaf-1) were found to mediate cap-dependent translation nearly as efficiently as the 5Ј-UTR from ␤-globin or a short unstructured control 5Ј-UTR. Even somewhat less efficiently translated 5Ј-UTRs were translated almost exclusively by a cap-dependent mechanism (8,9). Clearly, our current understanding of what makes an mRNA amenable to cap-dependent translation is insufficient to allow accurate predictions.…”

“…One attractive hypothesis to explain this observation is that 5Ј-UTR sequences that are capable of recruiting eIF4G in the absence of eIF4E greatly enhance the efficiency of translation in the presence of eIF4E and an m 7 G cap. Several mammalian 5Ј-UTRs that are capable of (relatively inefficient) IRES-dependent initiation are translated with surprising efficiency in a cap-dependent context, given that these 5Ј-UTRs are quite long and GC-rich (8,9). The in vitro IRES activity of these 5Ј-UTRs is strongly dependent on the level of eIF4G (20).…”