Dependent Start Codon Recognition
In this study strong evidence is provided that Rps5 T38 phosphorylation is important for efficient canonical translation initiation. The phospho-deficientrps5-T38Amutation causes a cap-dependent translation defect in vitro and a defect in de novo protein synthesis in vivo (Fig. 2.4). Specifically, the formation of 48S initiation complexes is decreased in
rps5-T38Amutant cells compared toRPS5wt cells (Fig. 2.9 and Fig. 2.10). Moreover, the
rps5-T38Amutation causes increased initiation from non-AUG start codons (Fig. 2.12). In addition, the discrimination against good and poor start codon context is eliminated in the
rps5-T38A mutant cells (Fig. 2.13), indicating that Rps5 T38 phosphorylation functions in context dependent start codon recognition.
During translation initiation first the MFC (eIF1, 3, 5 and the TC) and eIF1A bind to
the 40S subunit forming the 43S PIC. The pre-assembled 43S PIC is recruited to the 5’ cap of the mRNA and subsequently starts to scan the 5’ UTR for a start codon in
a suitable consensus context (see Section 1.4.1). The recognition of the AUG by perfect complementarity with the Met-tRNAi anticodon in the ribosomal P-site completes scanning
Figure 3.3: Localization of Rps5 T21 and T38 on the ribosome and protein sequence conservation of the N-terminus of Rps5. (A) Phosphorylated amino acids are depicted as red spheres, Rps5 protein in green and all other small ribosomal proteins in yellow and rRNA in gray. The 40S structure was taken form Ben-Shem et al. (2011). (B) Multiple sequence alignment of Rps5 protein sequence (UniProt, www.uniprot.org). Colors used are purple for S and pink for T; light - weakly, dark - highly conserved; boxed are the two sites T21 and T38.
(Huang et al., 1997; Algire et al., 2005). The recognition of the start codon and base pairing of the Met-tRNAi anticodon with the start codon is crucial for stable 48S PIC
formation (Maag et al., 2005). Thus, the inability of rps5-T38A mutant cells to efficiently recognize the start codon might also cause the decreased formation of 48S PICs observed
in vitro and in vivo.
Although it remains to be elucidated how the rps5-T38A mutation impedes start codon selection, initiation factors might be involved since the rps5-T38A mutant ribosomes are able to translate the CrPV IRES containing mRNA which does not need any initiation
factors. In addition, the release of several initiation factors is altered inrps5-T38Amutant ribosomes (Fig. 2.11). Possible candidates are eIF1, 1A, 2, 3 and 5 since mutations in these
confer an increased initiation from UUG codons as observed for the rps5-T38A mutant cells (Castilho-Valavicius et al., 1990; Fekete et al., 2007; Saini et al., 2010; Pestova
and Kolupaeva, 2002; Valasek et al., 2004; Huang et al., 1997). Moreover, the T38 phosphorylation site lies in the variable N-terminus of Rps5 and forms a part of the mRNA
exit channel (Fig. 3.3). The yeast Rps5 N-terminus is 21 aa longer than the human Rps5 N-terminus. Replacing yeast Rps5 with its human homologue altered translation fidelity
and affected the recruitment of specific mRNAs to the ribosome (Galkin et al., 2007). Using different truncated Rps5 variants Lumsden et al. (2009) showed that the first 24 aa
only play a minor role whereas deletion of aa 1-30 and 1-46 impairs translation initiation by affecting the association with eIF2 and eIF3. In addition, both Rps5 and eIF2α were crosslinked to -3 thioU upstream of the AUG start codon on mRNAs in mammalian 48S PICs (Pisarev et al., 2006, 2008), and also eIF3 locates in close proximity being crosslinked
to nucleotides -8 to -17 upstream of the AUG codon (Pisarev et al., 2008), supporting eIF2 and eIF3 as possible candidates.
In contrast, Rps5 might also be directly involved in start codon selection. First, it crosslinked specificlly to the -3 position which is crucial for optimal AUG sequence context
in yeast (Pisarev et al., 2006) explaining the loss of discrimination against poor AUG context in the rps5-T38A mutant. Additionally, -3 thioU also cross-linked specificlly to the prokaryotic homologue rpS7 in prokaryotic ribosomal initiation complexes indicating a conserved function of rps5/rps7 (La Teana et al., 1995). Second, Rps5 T38 phosphorylation
might influence the structure or conformation of initiation complexes. During start codon recognition the 43S PIC rearranges from an open, scanning permissive to a closed
conformation mediated by eIF1 and eIF1A (Passmore et al., 2007). It is tempting to speculate that Rps5 T38 phosphorylation might influence the transition from the open,
scanning competent conformation to the closed after start codon recognition either directly or via interaction of initiation factors with the mRNA and the 43S during translation
initiation.
The fact that the rps5-T38D mutation causes a ribosome biogenesis or stability defect suggests that the phosphorylation of Rsp5 T38 needs to be reversible. Provided that the phospho-mimicry D mutation indeed resembles the phosphorylation of Rps5 at
phospho-deficient rps5-T38A mutation.
Although the loss of Rps5 T38 phosphorylation in the rps5-T38A mutation does not lead to a general growth defect under specific conditions – starvation of amino acids (3-AT), inhibition of elongation (cycloheximide), low carbon or ethanol as carbon source – the
growth of rps5-T38A mutant cells is more severely affected than wild-type cells whereas under rapamycin, inhibiting TOR signaling, the mutant cells grew better (Fig. 2.15).
Therefore, the phosphorylation and dephosphorylation of Rps5 T38 is needed for survival under specific conditions.
Concluding, the phosphorylation of Rps5 on T38 is important for efficient 48S initiation complex formation and context dependent start codon selection. Since Rps5 belongs to
the conserved family of Rps5/Rps7 proteins and the T38 residue is conserved in higher eukaryotes (Fig. 3.3 B) also the function of this phosphorylation site might be conserved
in other organisms.