Understanding the purpose of Differential CDS/Leader read density Analysis of Ribosome Profiling
Ribosome Profiling: A Window into Translation Dynamics
Ribosome profiling provides a snapshot of active translation by sequencing ribosome-protected mRNA fragments1 allowing the identification of which mRNAs are being translated, thereby offering insights into the translational control of gene expression. Ribo-seq data are instrumental in revealing the intricacies of translation initiation, elongation, and termination, as well as uncovering hidden features such as alternative translation start sites and small open reading frames (sORFs).
Understanding the purpose of Differential CDS/Leader read density Analysis of Ribosome Profiling.
In eukaryotes, the leader (5′ untranslated region) plays a significant role in regulating the main coding sequence (CDS). This regulation is partly achieved through the translation of open reading frames (ORFs) located within the leader. Translation in the leader is pervasive in humans, occurring in roughly half of all genes2,3. In most cases, the translation of upstream ORFs (uORFs) is thought to inhibit the CDS region. This is because the ribosome initiating translation of the uORF reduces the availability of ribosomes to initiate translation of the CDS.
Currently, only a few dozen well-characterized regulatory uORFs are known. These include genes that are activated through the integrated stress response, such as ATF4, ATF5, SLC35A4, and IFRD1, genes involved in polyamine synthesis regulation such as AMD1, AZIN1, and EIF5 which is sensitive to changes in ribosomal start codon recognition. Although all these genes are regulated by uORF translation, the specific regulatory mechanisms vary.
It appears that much of the translation in leader ORFs does not serve a strong regulatory function but occurs for other reasons. Some peptides produced from this translation may have a role in immune responses by acting as antigens4. In other cases, the translation of certain ORFs may be non-functional, occurring simply due to insufficient selective pressure or it may aid in the robustness of the translation processes to various stresses.
The Differential CDS/Leader analysis on EIRNA Bio Connect platform attempts to identify genes that display differences in translation between two conditions. This can reveal genes that are differentially translationally regulated between two conditions.
Figure 1 Differential CDS/Leader plot on the Connect platform of sodium arsenite treated samples compared to control samples. Genes are stratified on the x-axis based on expression (i.e. the minimum in the two conditions). The fold change in Ribo-seq count of the ratio of CDS/Leader reads between the two conditions is indicated y-axis. Those found to be regulated are indicated in orange. These are easily discernible using the dynamic interactive Connect platform. Gene names can be dynamically highlighted and further explored on the platform with some of the regulated genes highlighted here.
Figure 2 Comparison profile on EIRNA Bio Connect of ATF4, showing the ribosome profiles of both the control (green) and sodium arsenite treated samples. The lower panels show the open reading frame organisation plot with ATG indicated with white vertical bars while stop codons are indicated with black vertical bars. Notice the higher ratio of CDS/Leader reads in that arsenite-treated samples compared to the control samples. This gene is a very well characterized case of translational regulation achieved by translation in its leader.
The EIRNA Bio Connect Approach
The EIRNA Bio Connect browser-based platform contains a tool for the identification of differential CDS/Leader counts between two groups using Ribo-seq counts. The user selects the samples of interest to belong to each group and chooses whether or not to include ambiguously mapped reads in the analysis.
Applications and Implications
To illustrate the use of differential CDS/Leader analysis we conduct this analysis in one of the most well-characterized translational programs: the integrated stress response (ISR). The ISR is a response converging from various cellular stresses, leading to a general inhibition of translation and activation of a transcriptional program as the cell works to alleviate stress5.
Figure 1 presents results comparing sodium arsenite-treated samples (which induces the ISR) to control samples. Genes showing significant changes in the CDS/Leader ribosome profiling count ratios are identified. Some of the previously well-characterized stress-responsive genes6 that were found to be regulated are also highlighted.
Figure 2 displays the ribosome profile of ATF4 under both control and stress conditions. As shown in Figure 1, the relative density of ribosome footprints in the leader region compared to the CDS is much higher in the control group than in the arsenite-treated group.
Conclusion
The Differential CDS/Leader analysis tool available on EIRNA Bio Connect offers a simple means to identify cases of potential regulation owing to differential translational in the 5’leader. As this analysis is conducted using only Ribo-seq counts the tool may on occasion identify cases owing to the occurrence of alternative splicing as well as by genuine regulation at the translation level. Using the intersection of the group of genes found to be significant using this analysis with the group of genes found to be regulated at the TE level using differential expression analysis will result in identification of the high confidence candidates.
Stay tuned for how EIRNA Bio Connect can provide actionable insights for your own data.
Over the course of the coming months, we will highlight additional EIRNA Bio Connect functionality to illustrate how our interactive platform can help advance your own research questions.
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- Kochetov AV, Sarai A, Rogozin IB, Shumny VK, Kolchanov NA. The role of alternative translation start sites in the generation of human protein diversity. Molecular Genetics and Genomics. 2005 Jul;273:491-6.
- McGillivray P, Ault R, Pawashe M, Kitchen R, Balasubramanian S, Gerstein M. A comprehensive catalog of predicted functional upstream open reading frames in humans. Nucleic acids research. 2018 Apr 20;46(7):3326-38.
- Jürgens L, Wethmar K. The emerging role of uORF-encoded uPeptides and HLA uLigands in cellular and tumor biology. Cancers. 2022 Dec 7;14(24):6031.
- Pakos‐Zebrucka K, Koryga I, Mnich K, Ljujic M, Samali A, Gorman AM. The integrated stress response. EMBO reports. 2016 Oct;17(10):1374-95.
- Andreev DE, O’Connor PB, Fahey C, Kenny EM, Terenin IM, Dmitriev SE, Cormican P, Morris DW, Shatsky IN, Baranov PV. Translation of 5′ leaders is pervasive in genes resistant to eIF2 repression. elife. 2015 Jan 26;4:e03971.
