Evidence for Involvement of trans-Acting Factors in Selection of the AUG Start Codon during Eukaryotic Translational Initiation

Abstract

The molecular mechanism with which an appropriate AUG codon is selected as the start site for translational initiation by eukaryotic ribosomes is not known. By using a cell-free translation system, small RNA molecules containing single AUG codons, surrounded by various nucleotide sequences, were tested for their abilities to interfere with the translation of a reporter mRNA. RNAs containing the AUG in an ACCAUGG context (Kozak consensus sequence) were able to inhibit translation of the reporter mRNA. In contrast, RNAs containing the AUG in a less favorable context for start site selection (for example, CAGAUGC) had no effect on the translation of the reporter mRNA. The effect mediated by the ACCAUGG-containing RNAs was not due to sequestration of ribosomal subunits or to particular structural features in these RNAs. To identify potential trans-acting factors that might be preferentially bound by ACCAUGG-containing RNAs, ACCAUGG-and CAGAUGC-containing RNAs with a single 4-thiouridine residue at the AUG were incubated with partially fractionated extracts, and AUG-binding proteins were identified after irradiation of the complexes with UV light and subsequent analysis by gel electrophoresis. The analysis of such complexes in competition experiments revealed that proteins, approximately 50 and 100 kDa in size, were found to bind directly at the AUG codon embedded in the ACCAUGG motif. One of these proteins has been identified as the La autoantigen. These findings indicate that trans-acting factors may play a role in AUG start site selection during translational initiation.

Human La Protein: a Stabilizer of Histone mRNA

“…La was easily detected in the S130, which was the starting material from which it was purified. Some La was also detected in polysomes, which is consistent with the binding of La to 18S rRNA (50) and to cell mRNAs (38). La in the S130 appeared to migrate slightly faster than purified La.…”

“…We do not know if La is actually bound to histone mRNA or acts indirectly, although other in vitro studies suggest that La can bind to strong translation initiation regions of mRNAs (38). However, La does not appear to affect the half-lives of FIG.…”

“…It binds to the TAR stem-loop of human immunodeficiency virus type 1 RNA and facilitates the translation of downstream open reading frames (10,69). It might even play a role in translation initiation in uninfected cells, since it interacts in vitro with the translation start site (38). (ii) It facilitates Sindbis virus RNA replication (48).…”

Functional importance of RNA interactions in selection of translation initiation codons

“…By using a mammalian cell-free translation system, several protein factors, including the La autoantigen, were found, in mRNA competition experiments, to bind specifically to the initiation codon of small RNAs only if embedded in a favourable ACCAUGG context (McBratney and Sarnow, 1996). Svitkin et al (1996) recently showed that several eukaryotic RNA-binding proteins (including La, pyrimidine-tract-binding protein and others) can bind to various regions within mRNAs in vitro and perhaps block potential 'false' internal start sites of capped eukaryotic transcripts.…”

“…The efficient IRES evolved by picornaviruses and some cellular mRNAs indicates that eukaryotic ribosomes can also bind internally to mRNAs, perhaps requiring additional trans-acting proteins and prokaryotic-like interactions with 18S rRNA (Pilipenko et al, 1992;Jackson, 1996). Similar trans-acting proteins may also help ribosomes to recognize the correct translation initiation site or discriminate against 'false' start codons in 'capped' eukaryotic mRNAs (McBratney and Sarnow, 1996;Svitkin et al, 1996).…”