Reducing ribosome biogenesis upregulates interferon-stimulated gene translation

The interferon response is a signaling pathway unique to vertebrates that links the innate and adaptive immune responses. Interferons signal through a cascade of factors, including the JAK-STAT pathway, to induce the transcription of hundreds of interferon-stimulated genes (ISGs). Although the main interferon signal transduction pathways and ISGs have been elucidated, translational regulation of ISG transcripts has not been fully investigated. Prior work demonstrated that ribosomal protein RPL28 negatively regulates a subset of ISGs. Recent findings suggest that ribosome biogenesis may impact the translational regulation of ISGs. The aim of this project is to examine the relationship between ribosome perturbations and ISG translation under interferon stimulation.

To determine how ribosome biogenesis and the interferon response are associated, we knocked down one of the key ribosome biogenesis factors, BOP1, in A549 cells, with and without interferon treatment. The resulting samples were analyzed by mass spectrometry and RNA-seq. Differential expression analysis revealed a subset of ISGs which had higher protein abundance in the BOP1-depleted condition, further supporting that ISGs are uniquely regulated. To validate that this was regulation at the translational level, the RNA-seq data revealed no significant differential expression in the candidate ISGs in the knockdown condition compared to control with interferon stimulation. The protein- and gene-level differential expression results of interferon-stimulated BOP1 knockdown were aligned for further analysis. Specifically, the codon adaptation index (CAI) was calculated to investigate the codon optimality of genes with protein differential expression greater than the transcript differential expression. In both the overall dataset and an ISG dataset, genes that had higher protein differential expression had a lower CAI, indicating codon non-optimality.

Canonically, codon non-optimality results in mRNA instability due to ribosome stalling and subsequent degradation; however, we propose that in instances of ribosome perturbation, such as ribosome biogenesis knockdown, the reduction in ribosome abundance prevents ribosome queuing and therefore increases mRNA stability and ultimately protein production (Figure 1). In times of stress, such as virus infection, ISGs and other immune-related proteins still need to be rapidly translated despite known reductions to host translation. It is possible that the reduced codon optimality of critical immune pathway transcripts is a method of ensuring robust translation of the immune response. Future work will clarify the role of codon optimality in the upregulation of both ISG and non-ISG proteins to further elucidate the proposed mechanism of translational regulation.

Figure 1: A proposed model for the increased protein levels of ISGs during BOP1 knockdown and IFN stimulation (created with Biorender).