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Results
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To investigate the functional implications of the Rpl10 loop mutation, we performed selective ribosome footprinting on the mutant ribosomes. Metagene analysis revealed that both Rpl10 mutant-bound and bulk ribosome footprints displayed a three-nucleotide periodicity, characteristic of active translation (Figure 4a, b). However, Rpl10 mutant ribosomes showed enrichment at the start site and within the first few codons compared to bulk ribosomes. Gene-level analysis using RiboGraph identified high peaks in a few genes, suggesting potential stall sites. To confirm that the observed enrichment at the 5' end of transcripts was not an artifact, we removed the top 50 genes and still observed the enrichment at the 5' end, supporting its validity.
Figure 4: Metagene analysis of Ribo-seq data. (a) Metagene plot showing ribosome footprint density around the start codons of transcripts. (b) Metagene plot illustrating ribosome occupancy near the stop codons of transcripts.
We found that most reads clustered around the start codon and the second amino acid, suggesting an elongation defect in the Rpl10 mutant ribosomes due to the absence of the P-site loop (Figure 5a, b). To assess whether these ribosomes translated further into the transcript, we analyzed region-specific read counts. In the bulk ribosome samples, >98% of the footprints mapped to coding regions, while in the Rpl10 mutant ribosomes, this value dropped to >92%, indicating that the mutant ribosomes translated deeper into the CDS (Figure 6a).
Figure 5: Rpl10 mutant ribosomes exhibit enrichment at the 5' end of transcripts. (a) Metagene plot showing ribosome footprint density across the first 14 codons of transcripts. (b) Normalized read counts corresponding to the first 20 amino acids, highlighting increased ribosome occupancy in this region.
Figure 6: Rpl10 mutant ribosomes are actively engaged in translation. (a) Distribution of ribosome profiling reads across the 5′UTR, coding sequence (CDS), and 3′UTR in immunoprecipitated (IP) and total (bulk) samples. (b) Read density at the 5′UTR of robustly expressed transcripts in control samples, comparing IP and bulk conditions.
Further analysis of IP samples showed that the 5' UTR junction was enriched with reads compared to total or bulk ribosomes, while the CDS reads were decreased, suggesting that the mutant ribosomes translate through the CDS but stall or slow down at the 5' UTR (Figure 6b). To identify specific stall sites and amino acid enrichment, we analyzed the composition of amino acids at genes with >50 reads at the 5' UTR junction. This analysis revealed that methionine was the most abundant at the stall sites (P-site), with cysteine in the second position (Figure 7c), indicating a potential issue with translation elongation. Additionally, alanine and serine were enriched at the A-site, suggesting problems in peptide chain elongation (Figure 7e).
Figure 7: Rpl10 mutant ribosomes show reduced efficiency in incorporating amino acids with small side chains. (a) Schematic representation of the ribosome highlighting the A and P site tRNAs. (b) Amino acid composition of transcripts with >50 reads at the 5′UTR-CDS junction, focusing on the first 14 amino acids. (c–d) Percentage of amino acids occupying the P site of the ribosome in transcripts with median read coverage >50 at the 5′UTR-CDS junction, shown for IP (c) and Total (d) samples. (e–f) Percentage of amino acids at the A site under the same conditions, shown for IP (e) and Total (f) samples.
We also examined the reading frame of these ribosomes and observed that, in IP samples, translation was skewed toward the third frame, further indicating a defect in ribosome function (Figure 8a). Ribosome footprint length distributions showed a peak at 28 nucleotides in the CDS for all samples, but at the 5' UTR junction, we observed two peaks at 28 and 30 nucleotides (Figure 8c), suggesting an abnormal population of ribosomes, likely due to disrupted ribosome rotation caused by the loop mutation.
Figure 8: Rpl10 mutant ribosomes exhibit defects in translation. (a–b) Analysis of ribosome reading frames in IP (a) and Total (b) samples. (c–d) Distribution of specific read lengths among total mapped reads from selective and total ribosome footprints at the 5′UTR-CDS junction (c) and within the CDS (d).
Gene-level footprint counts showed high reproducibility, with Spearman correlations of 0.974 and 0.979 between replicates of selective and conventional profiling, respectively, confirming data quality. Additionally, differential translation analysis revealed that genes involved in translation and translational control were particularly affected by the Rpl10 mutation (Figure 9a, b).
Figure 9: Rpl10 mutant ribosomes impact the synthesis of translation-related proteins. (a) Differential expression analysis (DESeq) comparing transcripts enriched in IP versus Total samples. (b) Gene Ontology (GO) analysis of significantly affected transcripts, highlighting enrichment in translation-associated processes.
In summary, our findings demonstrate that Rpl10 mutant ribosomes exhibit 5' end enrichment, altered reading frames, disrupted ribosome rotation, and slow translation, likely due to mispositioning of tRNAs and defects in elongation.
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