June 16th, 2024

Recent Publications Harnessing the Power of Translatomics.

Every week we provide a digest of a small number of recent interesting papers in the field of translatomics.

In this week’s Sunday papers, David, F. et al investigated ribosome composition changes in human cardiomyocytes under ER stress induced by tunicamycin and Shepelev N.M. et al investigated the translation and impact of a uORF within PRPF19 on cell viability using reporter constructs in human cells. Lastly, Samuels, T.J. et al focused on the intricate process of stem cell differentiation on Drosophila female germline stem cells.

Mitochondrial Ribosomal Protein MRPS15 Is a Component of Cytosolic Ribosomes and Regulates Translation in Stressed Cardiomyocytes

International Journal of Molecular Sciences, 2024

David F, Roussel E, Froment C, Draia-Nicolau T, Pujol F, Burlet-Schiltz O, Henras AK, Lacazette E, Morfoisse F, Tatin F, Diaz JJ, Catez F, Garmy-Susini B and Prats AC.

Stress inhibits global translation but activates internal ribosome entry site (IRES)-dependent translation. Traditionally seen as simple translation machines, ribosomes are now understood to exhibit heterogeneity, crucial for translational control. Variations in ribosomal RNA modifications and protein composition enable selective mRNA translation, especially under stress. Endoplasmic reticulum (ER) stress triggers the unfolded protein response (UPR), increasing GRP78 expression and activating stress pathways, which is vital for cardiomyocyte survival and angiogenesis in ischemic heart conditions. This study investigates ribosome composition changes in human cardiomyocytes under ER stress induced by tunicamycin.

In this study, AC16 cardiomyocyte cells were treated with tunicamycin to induce ER stress, with 4 hours selected for detailed analysis due to significant GRP78 upregulation and eIF2-α phosphorylation, indicating inhibited global translation. Cells were lysed, and ribosomes were separated by ultracentrifugation through a 10–50% sucrose gradient, distinguishing monosomes from polysomes. The polysome/monosome ratio, which decreased from 2.49 to 1.71 after tunicamycin treatment, confirmed the impact of ER stress on translation. Mass spectrometry revealed no significant changes in overall ribosomal protein composition in stressed cardiomyocytes, but mitochondrial ribosomal protein MRPS15 increased in cytosolic polysomes, confirmed as a ribosomal component by PLA and immunoprecipitation, and shown to activate IRES-dependent translation through knock-down and overexpression experiments.

These findings offer insights into translational remodelling and have implications for developmental brain disorders characterized by aberrant translation.

PRPF19 mRNA Encodes a Small Open Reading Frame That Is Important for Viability of Human Cells

Doklady Biochemistry and Biophysics2024

Shepelev NM, Kurochkina AO, Dontsova OA and Rubtsova MP

Ribosome profiling has unveiled translation in previously considered non-coding RNA regions, notably small open reading frames (small ORFs) in mRNA 5′ untranslated regions (UTRs), termed upstream ORFs (uORFs). These uORFs regulate gene expression by impacting downstream main ORF translation and can encode functional microproteins. Examples include ATF4, crucial for stress response, and ASNSD1, involved in asparagine biosynthesis. Genome-wide CRISPR-Cas9 screenings identified PRPF19 as essential for cell viability. PRPF19 encodes a highly conserved splicing factor within the Prp19/NTC complex, pivotal in RNA processing and DNA damage response. This study investigates the translation and impact of a uORF within PRPF19 on cell viability using reporter constructs.

Ribosome profiling was utilized to identify translating ribosomes in the mRNA of the PRPF19 gene, particularly focusing on its 5′ UTR using the Trips-Viz transcriptome browser. This analysis revealed ribosome association with an upstream ORF within the 5′ UTR, indicating its potential translation despite lacking a canonical start codon. By examining ribosome initiation profiles, it was confirmed that the upstream ORF could begin translation from non-canonical start codons. Subsequent experiments, including 5′ RACE analysis and reporter constructs, validated the translation of this upstream ORF in human cells. Furthermore, sequence conservation analysis suggested the functional significance of the encoded small protein. Finally, CRISPR-Cas9-mediated mutagenesis demonstrated that disrupting the upstream ORF led to decreased cell viability, highlighting its importance in cellular function.

Ribosome profiling facilitated the identification and characterization of this previously overlooked translation event, providing insights into the functional significance of upstream ORFs in gene regulation and cellular physiology.

Two distinct waves of transcriptome and translatome changes drive Drosophila germline stem cell differentiation

The EMBO Journal, 2024

Samuels TJ, Gui J, Gebert D and Karam Teixeira F.


Precisely managing fate transitions during stem cell differentiation is crucial for ensuring proper tissue development and upkeep. Yet, the difficulties in systematically studying adult stem cells, which are often sparsely distributed, have impeded our understanding of how the complex regulation of gene expression programs drives stem cell differentiation in living organisms. This study delves into the intricate process of stem cell differentiation by focusing on Drosophila female germline stem cells (GSCs). The researchers synchronized the differentiation of these stem cells in vivo, allowing for a detailed examination of gene expression regulation at various stages. Through comprehensive transcriptome and translatome analyses, they observed dynamic alterations in mRNA levels, promoter usage, exon inclusion, and translation efficiency.

Ribo-seq was used to directly measure translation activity during Drosophila female germline stem cell (GSC) differentiation. Of 6444 genes from RNA-seq, 5922 were translated, with 43 showing heat shock effects. Validation confirmed translation dynamics of key genes like bam and osk. Notably, osk mRNA remained translationally repressed, while nos translation decreased early despite constant transcription, indicating post-transcriptional regulation. A key discovery was the identification of two distinct phases in the differentiation process. Initially, the master regulator gene Bam triggers significant changes in gene expression, particularly impacting the cell cycle program. However, as Bam activity diminishes, differentiating cells surprisingly revert to a transcription and translation profile reminiscent of stem cells, albeit with crucial modifications that set the stage for terminal differentiation into oocytes.

Ribo-seq reveals dynamic translation patterns in GSC differentiation, vital for understanding stem cell fate transitions. The study challenges linear progression, emphasizing orchestrated waves of gene expression. Temporally regulated changes drive stem cells to specialized types, illuminating tissue development’s complexity.

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