Multiple Mechanisms of Reinitiation on Bicistronic Calicivirus mRNAs

Abstract

Reinitiation is a strategy used by viruses to express several cistrons from one mRNA. Although extremely weak after translation of long ORFs on cellular mRNAs, reinitiation occurs efficiently on subgenomic bicistronic calicivirus mRNAs, enabling synthesis of minor capsid proteins. The process is governed by a short element upstream of the restart AUG designated “termination upstream ribosomal binding site” (TURBS). It contains the conserved Motif 1 complementary to h26 of 18S rRNA, displayed in the loop of a hairpin formed by species-specific Motifs 2/2*. To determine the advantages conferred on reinitiation by TURBS, we reconstituted this process in vitro on two model bicistronic calicivirus mRNAs. We found that post-termination ribosomal tethering of mRNA by TURBS allows reinitiation by post-termination 80S ribosomes, and diminishes dependence on eIF3 of reinitiation by recycled 40S subunits, which can be mediated either by eIFs 2/1/1A or by Ligatin following ABCE1-dependent or -independent splitting of post-termination complexes.

Toward the mechanism of eIF4F-mediated ribosomal attachment to mammalian capped mRNAs

“…Thus, in the absence of eIF3, eIF1 was not able to discriminate against AUGs located too close to the 5 ′ end of mRNA. We previously noted that, in the absence of eIF3, eIF1 was also unable to discriminate efficiently against formation of 48S complexes at near-cognate codons during reinitiation on calicivirus mRNAs (Zinoviev et al 2015). Taken together, this indicates that eIF3 additionally has a role in maintaining the fidelity of initiation codon selection and, with eIF1A, cooperates with eIF1 in this process.…”

De Novo Mutations in DENR Disrupt Neuronal Development and Link Congenital Neurological Disorders to Faulty mRNA Translation Re-initiation

“…These capped mRNAs are recognized by eIF4F, which functions to recruit the ribosomal 43S complex containing eIF2a to facilitate translation. However, the ribosomal 43S complex can also bind uncapped mRNAs and internal ribosomal entry sites (IRES) of viral transcripts in conjunction with DENR (Skabkin et al, 2010(Skabkin et al, , 2013Zinoviev et al, 2015). DENR facilitates the efficient translation re-initiation of transcripts bearing an stuORF either by release of deacylated tRNA or recruitment of aminoacylated tRNA (Schleich et al, 2014;Skabkin et al, 2010Skabkin et al, , 2013.…”

“…DENR facilitates the efficient translation re-initiation of transcripts bearing an stuORF either by release of deacylated tRNA or recruitment of aminoacylated tRNA (Schleich et al, 2014;Skabkin et al, 2010Skabkin et al, , 2013. Another translation factor, ligatin/eIF2D, contains a SWIB/MDM2 domain homologous to DENR in its N-terminal half, as well as an eIF1-like/SUI domain homologous to MCTS1 in its C-terminal half, and has functions in translation that overlap with those of DENR and MCTS1 (Dmitriev et al, 2010;Skabkin et al, 2010Skabkin et al, , 2013Zinoviev et al, 2015). Whereas it has recently been demonstrated that loss of DENR during Drosophila embryo development leads to impaired tissue growth and larval formation (Schleich et al, 2014), reductions in ligatin gene dosage were found to exacerbate the defective development of DENRknockout flies (Schleich et al, 2014), suggesting overlapping functions for DENR and LIGATIN in embryo development.…”

“…The importance for canonical mRNA translation in human neuronal development is reflected by the finding that genetic mutations in translation factors, such as eIF2g and eIF2B, cause X-linked intellectual disability and leukoencephalopathy (a neurological disorder with vanishing white matter), respectively (Borck et al, 2012;Bugiani et al, 2010;Li et al, 2004). However, there are several non-canonical mechanisms for translation initiation of viral mRNAs in eukaryotic cells that do not bear a 5 0 cap or possess neither a 5 0 cap nor a 3 0 poly(A) tail (Firth and Brierley, 2012;Skabkin et al, 2010;Zinoviev et al, 2015). The translation of such mRNA types involves the functions of several eukaryotic proteins, such as the density regulated protein, known as DENR, and its obligate binding partner MCTS1 (Skabkin et al, 2010;Zinoviev et al, 2015).…”

“…However, there are several non-canonical mechanisms for translation initiation of viral mRNAs in eukaryotic cells that do not bear a 5 0 cap or possess neither a 5 0 cap nor a 3 0 poly(A) tail (Firth and Brierley, 2012;Skabkin et al, 2010;Zinoviev et al, 2015). The translation of such mRNA types involves the functions of several eukaryotic proteins, such as the density regulated protein, known as DENR, and its obligate binding partner MCTS1 (Skabkin et al, 2010;Zinoviev et al, 2015). DENR is known to play important roles during mammalian translation, in the processes of ribosome recycling, in initiation on some viral mRNAs (Skabkin et al, 2010), and in termination-dependent re-initiation after long open reading frames (ORFs) on calicivirus mRNAs (Zinoviev et al, 2015).…”