Extended Data Fig. 4: LARP4 selectively targets hypertranslated mRNAs, particularly nuclear-encoded OXPHOS mRNAs, in exhausted CD8 + T cells.
From: LARP4-mediated hypertranslation drives T cell dysfunction in tumors
(a) Schematic diagram of workflow for searching the potential regulatory RBPs for hypertranslated mRNAs in d8 TEX cells. (b-d) Immunoblotting of LARP4 in CD8+ TN cells, TEFF cells, and TEX cells under in vitro chronic or acute antigenic stimulation with indicated days. (e) Box plots showing the Larp4 expression in Tumor TEX cells and TdLNs TPEX cells (n = 2). (f) Dot plots showing the Larp4 expression in three subsets of tumor-infiltrating OT-I T cells in the B16-OVA mice model (GSE218372). (g) Dot plots showing the LARP4 expression in six subsets of tumor-infiltrating CD8+ T cells in patients with melanoma (GSE159251). (h) IGV tracks showing signal of translation factors without significant signal enrichment at Larp4 loci and the nearby enhancer region, including NFAT (GSE64407), IRF4 (GSE54191), BATF (GSE149796), and BCL-6 (GSE182034). The promoter and enhancer regions are highlighted with a colored background. (i) Boxplot showing the Larp4 expression in WT and Tox-KO tumor-infiltrating CD8+ T cells (n = 3) by RNA-seq (GSE126973). P-values were calculated using two-sided unpaired Student’s t-tests. (j) Violin plot showing the Larp4 expression in WT and Nr4a1/2-KO tumor-infiltrating PD-1+ TIM-3+ CD8+ T cells by scRNA-seq (GSE247641). Gene expression was normalized by sctransform algorithm and ten neighboring cells were merged into metacells. P-values were calculated using two-sided unpaired Student’s t-tests. (k) IGV tracks showing the LACE-seq signal intensity for LARP4-IP and IgG control across mitochondrial-encoded RNA regions. (l) Bar plot showing the LACE-seq signal intensity of LARP4-IP and IgG control at reads-accumulated sites in mitochondrial-encoded RNA regions (n = 39). The reads-accumulated sites were identified by peak calling using LARP4-IP data alone. The diffBind was used to quantify signal intensity in LARP4-IP and IgG control at these sites. P value is calculated by two-sided unpaired Student’s t-tests. (m) Profile plot showing the LACE-seq signal intensity for LARP4-IP and IgG control at reads-accumulated sites in mitochondrial-encoded mRNA regions. The deeptools was used to quantify coverage differences between LARP4-IP and IgG control at these sites. (n) Profile plot showing the LACE-seq signal intensity for LARP4-IP and IgG control at LARP4-targeted sites in nuclear-encoded mRNA regions. (o and p) Top 5 motifs identified by HOMER across total LARP4-targeted sites (o) and on LARP4-targeted sites within OXPHOS mRNAs (p) in TEX cells after 8 days of in vitro antigenic stimulation. The asterisk (*) indicates the non-significant motif as labeled by HOMER. (q) Venn diagram showing the overlap between LARP4-targeted mRNAs and mitochondria-proximal mRNAs identified by APEX-seq (Fazal et al). P-value is calculated by one-sided Fisher’s exact test. The data represent three independent experiments (b-d).
