eIF3j facilitates loading of release factors into the ribosome

Abstract

eIF3j is one of the eukaryotic translation factors originally reported as the labile subunit of the eukaryotic translation initiation factor eIF3. The yeast homolog of this protein, Hcr1, has been implicated in stringent AUG recognition as well as in controlling translation termination and stop codon readthrough. Using a reconstituted mammalian in vitro translation system, we showed that the human protein eIF3j is also important for translation termination. We showed that eIF3j stimulates peptidyl-tRNA hydrolysis induced by a complex of eukaryotic release factors, eRF1-eRF3. Moreover, in combination with the initiation factor eIF3, which also stimulates peptide release, eIF3j activity in translation termination increases. We found that eIF3j interacts with the pre-termination ribosomal complex, and eRF3 destabilises this interaction. In the solution, these proteins bind to each other and to other participants of translation termination, eRF1 and PABP, in the presence of GTP. Using a toe-printing assay, we determined the stage at which eIF3j functions – binding of release factors to the A-site of the ribosome before GTP hydrolysis. Based on these data, we assumed that human eIF3j is involved in the regulation of translation termination by loading release factors into the ribosome.

Recognition of 3′ nucleotide context and stop codon readthrough are determined during mRNA translation elongation

“…4B). Western blotting showed that under low-salt (LS) conditions, significant amounts of eIF3 remained in the preTC(LS), which was also shown in our previous study (45). After high-salt (HS) centrifugation, we observed a significant decrease of eIF3 in the preTC(HS).…”

“…The 3ʹ context UGU GUG was chosen as the Standard. This context has been used in all our previous studies in the reconstituted mammalian translation system (3,39,(41)(42)(43)(44)(45)(46)(47). The frequencies of triplets from this sequence in the human genome calculated earlier (1-1.8%) were close to that for random triplet NNN 1/64 = 1.56% (39).…”

“…It is noteworthy that the local concentrations of eRFs in various regions of the translated mRNA are different. At the stop codon of the main reading frame, eRFs are recruited and activated by the poly(A)-binding protein PABP, eIF3j, and other factors (42,(45)(46)(47). The stop codons of the upstream ORFs are recognized by eRFs recruited by eIF3j and eIF3 (45).…”

“…At the stop codon of the main reading frame, eRFs are recruited and activated by the poly(A)-binding protein PABP, eIF3j, and other factors (42,(45)(46)(47). The stop codons of the upstream ORFs are recognized by eRFs recruited by eIF3j and eIF3 (45). However, the local concentration of eRFs near the PTC is low because it lacks determinants (poly(A) tail and 5 0 end of mRNA) for the binding of protein loading and activating eRFs.…”

“…We recently showed that several eIFs, including eIF3 and eIF3j, stimulate translation termination (45). As we performed previous experiments on readthrough and termination in the reconstituted in vitro system in the presence of all translation factors, the influence of eIFs on these processes should be excluded.…”