eIF1A residues implicated in cancer stabilize translation preinitiation complexes and favor suboptimal initiation sites in yeast

The translation pre-initiation complex (PIC) scans the mRNA for an AUG codon in favorable context, and AUG recognition stabilizes a closed PIC conformation. The unstructured N-terminal tail (NTT) of yeast eIF1A deploys five basic residues to contact tRNAi, mRNA, or 18S rRNA exclusively in the closed state. Interestingly, EIF1AX mutations altering the human eIF1A NTT are associated with uveal melanoma (UM). We found that substituting all five basic residues, and seven UM-associated substitutions, in yeast eIF1A suppresses initiation at near-cognate UUG codons and AUGs in poor context. Ribosome profiling of NTT substitution R13P reveals heightened discrimination against unfavorable AUG context genome-wide. Both R13P and K16D substitutions destabilize the closed complex at UUG codons in reconstituted PICs. Thus, electrostatic interactions involving the eIF1A NTT stabilize the closed conformation and promote utilization of suboptimal start codons. We predict UM-associated mutations alter human gene expression by increasing discrimination against poor initiation sites. We examined the effect of tif11-R13P on global translational efficiencies (TEs) by ribosome footprint profiling of isogenic WT and tif11-R13P strains. The study includes 8 samples, comprised of 4 mRNA-Seq samples and 4 ribosome footprint profiling samples, derived from 2 biological replicates of tif11? mutant strains harboring plasmid-borne tif11-R13P or the WT TIF11 allele. Additional Ribosome profiling data were used for yeast uORF identification.

翻译预起始复合物（translation pre-initiation complex，PIC）会在信使RNA（mRNA）上扫描处于有利上下文环境中的AUG密码子，而AUG的识别可稳定该复合物的闭合构象。酵母eIF1A（eukaryotic translation initiation factor 1A，eIF1A）的无结构化N端尾（unstructured N-terminal tail，NTT）仅在复合物处于闭合状态时，通过5个碱性残基与起始tRNA（tRNAi）、mRNA或18S核糖体RNA（18S rRNA）发生相互作用。值得注意的是，改变人类eIF1A NTT的EIF1AX突变与葡萄膜黑色素瘤（uveal melanoma，UM）密切相关。我们的研究发现，在酵母eIF1A中对全部5个碱性残基进行替换，以及引入7种与葡萄膜黑色素瘤相关的氨基酸替换突变，均会抑制翻译起始过程——尤其是在近同源UUG密码子以及处于不利上下文环境中的AUG密码子处的起始效率。对NTT替换突变R13P进行核糖体足迹谱分析（ribosome profiling）后发现，该突变可在全基因组范围内增强对不利上下文环境AUG密码子的甄别能力。在重组构建的翻译预起始复合物中，R13P与K16D这两种替换突变均会降低UUG密码子处闭合复合物的稳定性。由此可见，涉及eIF1A NTT的静电相互作用能够稳定翻译预起始复合物的闭合构象，并促进细胞对亚最优起始密码子的利用。我们推测，与葡萄膜黑色素瘤相关的eIF1A突变，可通过增强对不利起始位点的甄别能力，改变人类基因的表达模式。我们通过对同基因背景的野生型（wild type，WT）与tif11-R13P突变菌株进行核糖体足迹谱分析，检测了tif11-R13P对全局翻译效率（translational efficiencies，TEs）的影响。本研究共包含8个样本，均来自携带质粒表达的tif11-R13P或野生型TIF11等位基因的tif11缺失突变菌株的2个生物学重复，其中包括4个mRNA测序（mRNA-Seq）样本与4个核糖体足迹谱分析样本。额外的核糖体谱分析数据被用于酵母上游开放阅读框（upstream open reading frame，uORF）的鉴定。