A Leaderless mRNA Can Bind to Mammalian 80S Ribosomes and Direct Polypeptide Synthesis in the Absence of Translation Initiation Factors

Abstract

Translation initiation in eukaryotic cells is known to be a complex multistep process which involves numerous protein factors. Here we demonstrate that leaderless mRNAs with initiator Met-tRNA can bind directly to 80S mammalian ribosomes in the absence of initiation factors and that the complexes thus formed are fully competent for the subsequent steps of polypeptide synthesis. We show that the canonical 48S pathway of eukaryotic translation initiation has no obvious advantage over the 80S pathway of translation initiation on leaderless mRNAs and suggest that, in the presence of competing mRNAs containing a leader, the latter mechanism will be preferred. The direct binding of the leaderless mRNA to the 80S ribosome was precluded when such an mRNA was supplied with a 5 leader, irrespective of whether it was in a totally single-stranded conformation or was prone to base pairing. The striking similarity between the mechanisms of binding of leaderless mRNAs with mammalian 80S or bacterial 70S ribosomes gives support to the idea that the alternative mode of translation initiation used by leaderless mRNAs represents a relic from early steps in the evolution of the translation apparatus.More than a decade ago, we showed using the toeprint assay that the prokaryotic leaderless mRNA coding for the cI repressor of lambda phage assisted by initiator Met-tRNA was able to bind directly to undissociated 70S ribosomes in the absence of initiation factor 1 (IF1), IF2, and IF3 (1). Under the same conditions, no initiation complex was observed with the 30S ribosomal subunit. In the presence of initiation factors, the 30S subunit invariably bound to internal sites on the mRNA immediately downstream of the closest Shine-Dalgarno (SD) sequence, whereas undissociated 70S ribosome strongly preferred the initiation triplet as close as possible to the 5Ј end of the mRNA. These studies were substantially extended and confirmed in other laboratories both in vitro and in vivo (12,13,15,28) and resulted in similar conclusions. Natural leaderless mRNAs have been identified in all kingdoms of life (see references 10 and 11). This class of mRNAs is well represented in archaea and mammalian mitochondria. Although they have not yet been found among cellular mammalian mRNAs, leaderless monocistronic N and dicistronic N-P RNAs of measles virus were found to be associated with polysomes in infected mammalian cells (4). In addition, it is unlikely that a systematic search for such mRNAs in mammalian cells has ever been undertaken. There is one well-documented example of a translatable leaderless mRNA in unicellular eukaryotes. It encodes a variant specific surface protein of Giardia lamblia and is certainly translated in these cells (14). Leaderless mRNAs are efficiently translated in mammalian cell-free systems (see reference 12). However, it is not known whether they use a similar (80S) pathway for translation initiation in eukaryotes, given the remarkable quantitative and qualitative differences in the organization of eukaryotic and prokaryotic t...

Dynamic evolution of translation initiation mechanisms in prokaryotes

“…Therefore, we believe that the SD interaction has been considered the universal mechanism for effective initiation of translation in prokaryotes because the organisms used for most of the experiments have high SD presences. According to our results and those of preceding studies, the origin of the mechanisms seems to use both the SD sequence and a leaderless mRNA, considering the evolutionary conservation of the anti-SD sequence and broad usage of leaderless mRNAs including all three domains, respectively (25,38). It is known that the origin of eukaryotes is a hybrid of bacteria and archaebacteria, and translation-related proteins are shared by eukaryotes and archaebacteria (39).…”

The bacterial toxin RelE induces specific mRNA cleavage in the A site of the eukaryote ribosome

“…Such factor binding to the A site would, in analogy with previous results obtained for the Escherichia coli system (Pedersen et al 2003;Zavialov et al 2005a,b), have inhibited the hydrolytic action of RelE. To sort out these issues, we turned to our eukaryote in vitro system for assembly of translation initiation complexes from purified components (Pestova et al 1996(Pestova et al , 1998aDmitriev et al 2003;Andreev et al 2006); as will be described next.…”

“…In order to identify a minimal set of factors that allows for mRNA-induced cleavage by RelE on the eukaryote ribosome, we took advantage of the prokaryote-like hepatitis C virus (HCV) IRES-mediated and the c1 leaderless mRNA eukaryote translation systems (see Pestova et al 1998b;Andreev et al 2006). The HCV IRES element forms a stable initiation intermediate with the 40S ribosomal subunit (40S-HCV IRES binary complex henceforth), which in the presence of Met-tRNA i , eIF2, and eIF3 generates a functional 48S initiation complex (Pestova et al 1998b).…”

“…Moreover, RelE induces cleavage of a stop codon positioned in the A site of a pretermination complex assembled from purified components (Andreev et al 2006). The hydrolytic action of RelE requires a vacant A site and appears to require correct accommodation of the mRNA in the mRNA-binding channel of the ribosome.…”

Ribosomes bind leaderless mRNA in Escherichia coli through recognition of their 5′-terminal AUG

“…The expression of leaderless mRNAs in Bacteria, Archaea, and Eukarya suggests that the ability to bind and translate these mRNAs represents a common, possibly conserved, function of all ribosomes (Janssen 1993;Moll et al 2002;Andreev et al 2006;Brenneis et al 2007). Evidence that 59-AUG recognition is a conserved function of perhaps all ribosomes suggests that this capability may have persisted since early evolution of the translational apparatus.…”