The P-site loop of the ribosomal protein Rpl10 (also known as uL16 in eukaryotes) is essential for efficient protein synthesis. This loop is crucial in stabilizing the positioning of tRNA, a key step in ensuring the accuracy and efficiency of peptide bond formation during translation. By ensuring proper alignment of tRNA and the ribosomal machinery, the P-site loop enables smooth elongation of the nascent peptide chain. Mutations or disruptions near this loop have been linked to several diseases, including T-cell acute lymphoblastic leukemia, where they drive IRES-dependent translation of BCL-2, contributing to disease progression. This highlights the critical function of the loop in regulating cellular processes and maintaining cellular homeostasis. In this study, our goal is to investigate the translation status of ribosomes that lack the P-site loop of Rpl10. This involves assessing how the absence of this structural component affects ribosome activity, efficiency, and the overall translation process. To address this, we performed ribosome profiling on mutant ribosomes that have a deletion in the P-site loop. Ribosome profiling allows us to capture and analyze the positions of ribosomes on mRNA during translation, providing insight into how the mutation impacts translation dynamics.

Our primary goal is to analyze the ribosome profiling data to assess the translation activity of mutant ribosomes. We will begin by applying the RiboFlow workflow, which involves several critical steps to process the raw sequencing data. First, we will perform adaptor trimming and filter out rRNA reads to focus on protein-coding regions. Next, we will align the remaining reads to the transcriptome and remove unique molecular identifiers (UMIs) to eliminate any redundancy resulting in the creation of a Ribofile. This Ribofile will be used for visualization and further analysis in RiboR, where we will generate metagene plots to examine ribosome density along coding sequences. Additionally, we will create quality control (QC) plots, including average read length and CDS (coding sequence) coverage, to assess the quality of the data. Finally, we will investigate which genes are preferentially translated by ribosomes carrying the mutant Rpl10 P-site loop, providing insight into potential translation biases caused by the mutation.