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Codon-specific ribosome stalling reshapes translational dynamics during branched-chain amino acid starvation

ABSTRACT: This study investigates the immediate effects of single, double, and triple branched-chain amino acid (BCAA) deprivation on translational dynamics in NIH3T3 cells. Using a multi-omics approach combining RNA-seq, ribosome profiling (Ribo-seq), quantitative proteomics, and tRNA charging assays, we systematically assessed global translational output and codon-specific ribosome dwell times. Our findings reveal that BCAA starvation induces diverse and non-additive translational control patterns, reflecting a complex interplay between mTORC1 and GCN2 pathway activation, tRNA isoacceptor availability, and transcript codon usage. Notably, we identified a positional effect where the enrichment of valine codons at the 5' end of transcripts creates an elongation bottleneck under valine and triple BCAA starvation, impacting downstream ribosome occupancy and overall protein output. This study uncovers a previously underappreciated layer of complexity in BCAA-specific translational regulation.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture, Fibroblast

SUBMITTER: Cédric Gobet

LAB HEAD: Felix Naef

PROVIDER: PXD067949 | Pride | 2025-09-13

REPOSITORIES: Pride

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			Action	DRS
	LW_230802_0730_Lumos_A.msf	Msf
	LW_230802_0730_Lumos_Fr01.raw	Raw
	LW_230802_0730_Lumos_Fr01_uncalibrated.mgf	Mgf
	LW_230802_0730_Lumos_Fr02.raw	Raw
	LW_230802_0730_Lumos_Fr02_uncalibrated.mgf	Mgf

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Project description:The branched-chain amino acid (BCAA) pathway and high levels of BCAA transaminase 1 (BCAT1) have recently been associated with aggressiveness in several cancer entities. However, the mechanistic role of BCAT1 in this process remains largely uncertain. Here, by performing high-resolution proteomic analysis of human acute myeloid leukaemia (AML) stem-cell and non-stem-cell populations, we find the BCAA pathway enriched and BCAT1 protein and transcripts overexpressed in leukaemia stem cells. We show that BCAT1, which transfers α-amino groups from BCAAs to α-ketoglutarate (αKG), is a critical regulator of intracellular αKG homeostasis. Further to its role in the tricarboxylic acid cycle, αKG is an essential cofactor for αKG-dependent dioxygenases such as Egl-9 family hypoxia inducible factor 1 (EGLN1) and the ten-eleven translocation (TET) family of DNA demethylases. Knockdown of BCAT1 in leukaemia cells caused accumulation of αKG, leading to EGLN1-mediated HIF1α protein degradation. This resulted in a growth and survival defect and abrogated leukaemia-initiating potential. By contrast, overexpression of BCAT1 in leukaemia cells decreased intracellular αKG levels and caused DNA hypermethylation through altered TET activity. AML with high levels of BCAT1 (BCAT1high) displayed a DNA hypermethylation phenotype similar to cases carrying a mutant isocitrate dehydrogenase (IDHmut), in which TET2 is inhibited by the oncometabolite 2-hydroxyglutarate11,12. High levels of BCAT1 strongly correlate with shorter overall survival in IDHWTTET2WT, but not IDHmut or TET2mutAML. Gene sets characteristic for IDHmut AML13 were enriched in samples from patients with an IDHWTTET2WTBCAT1high status. BCAT1highAML showed robust enrichment for leukaemia stem-cell signatures14,15, and paired sample analysis showed a significant increase in BCAT1 levels upon disease relapse. In summary, by limiting intracellular αKG, BCAT1 links BCAA catabolism to HIF1α stability and regulation of the epigenomic landscape, mimicking the effects of IDH mutations. Our results suggest the BCAA–BCAT1–αKG pathway as a therapeutic target to compromise leukaemia stem-cell function in patients with IDHWTTET2WT AML.

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Codon-specific ribosome stalling reshapes translational dynamics during branched-chain amino acid starvation

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