Decoding the Heterogeneity and Specialized Function of Translation Machinery Through Ribosome Profiling in Yeast Mutants of Initiation Factors

Abstract The nuanced heterogeneity and specialized functions of translation machinery are increasingly recognized as crucial for precise translational regulation. Here, high‐throughput ribosomal profiling (ribo‐seq) is used to analyze the specialized roles of eukaryotic initiation factors (eIFs) in the budding yeast. By examining changes in ribosomal distribution across the genome resulting from knockouts of eIF4A, eIF4B, eIF4G1, CAF20, or EAP1, or knockdowns of eIF1, eIF1A, eIF4E, or PAB1, two distinct initiation‐factor groups, the “looping” and “scanning” groups are discerned, based on similarities in the ribosomal landscapes their perturbation induced. The study delves into the cis‐regulatory sequence features of genes influenced predominantly by each group, revealing that genes more dependent on the looping‐group factors generally have shorter transcripts and poly(A) tails. In contrast, genes more dependent on the scanning‐group factors often possess upstream open reading frames and exhibit a higher GC content in their 5′ untranslated regions. From the ribosomal RNA fragments identified in the ribo‐seq data, ribosomal heterogeneity associated with perturbation of specific initiation factors is further identified, suggesting their potential roles in regulating ribosomal components. Collectively, the study illuminates the complexity of translational regulation driven by heterogeneity and specialized functions of translation machinery, presenting potential approaches for targeted gene translation manipulation.