Journal: Cancer Discovery

Article Title: LINE-1 Locus Transcription Nucleates Oncogenic Chromatin Architecture

doi: 10.1158/2159-8290.CD-25-1085

Figure Lengend Snippet: LINE-1 RNAs are primarily chromatin-associated nascent transcripts. A, Subcellular localization of LINE-1 RNAs in cancer cells. B, Immunoblot of LINE-1 ORF1p expression across 10 human cancer cell lines. GAPDH was used as a loading control. C, Relative abundance of RNAs in cytosolic, nuclear soluble, or chromatin fractions as assessed by qRT-PCR. The relative target sites of four LINE-1–specific qPCR primers (A, B, C, and D) are schematized along the L1HS consensus sequence. 45S rRNA is analyzed as a control for chromatin RNA. Exonic GAPDH is a control for cytosolic RNA. Results are shown as mean ± SD ( N ≥ 3 replicates). P values were calculated using an unpaired two-sided Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. D, Representative fluorescent micrographs of four human cancer cell lines probed with smRNA-FISH oligos conjugated with Cy5 (pink pseudo-colored, L1HS 5′UTR) or Cy3 (yellow pseudo-colored, β-actin). DAPI staining of nuclei is shown as blue pseudo-color. Quantification of the relative number of cytosolic versus nuclear foci per transcript per cell. N = 30 cells were quantified per cell line. P values were calculated using an unpaired two-sided Student t test. E, ChIRP-seq to assess genomic binding sites of LINE-1 5′UTR-containing transcripts. Scatter plot shows correlation of read counts from two ChIRP-seq biological replicates. Proportion of HOMER annotations overlapping ChIRP-seq 5′UTR peaks are shown as a pie chart. F, Top: binding sites for Northern blotting oligo probes A and B are depicted along the L1HS 5′UTR consensus sequence. Bottom: representative Northern radiograms with L1HS 5′UTR A or B vs. β-actin probes. Relative positions for 28S (5.8 kb) and 18S (1.8 kb) rRNAs were marked based on ethidium bromide staining signal during gel electrophoresis and alkaline transfer. G, Relative polyadenylation of transcripts was assessed by qRT-PCR of the enriched or unbound fraction after oligo-dT bead isolation. The relative target sites of LINE-1 qPCR primers (A, B, C, and D) are schematized in C . Results are shown as mean ± SD ( N ≥ 3 replicates). P values were calculated by an unpaired Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. H, Left: Experimental scheme of 4sU pulse-chase experiments in MCF7 cells . Right: Chromatin dissociation kinetics of various RNAs. SRY is a control for cytosol-localized noncoding RNA, whereas α-satellite RNA is a control for chromatin-associated noncoding RNA. Chr, chromatin bound; Cyto, cytosolic; LTR, long terminal repeat; Nuc, nuclear soluble or chromatin released; RPM, reads per million.

Article Snippet: Custom Hybridization Chain Reaction (HCR) probe sets targeting the LINE-1 5′UTR promoter (lot #RTH492) were designed by Molecular Instruments, Inc. using the full-length L1HS consensus sequence (RepBase).

Techniques: Western Blot, Expressing, Control, Quantitative RT-PCR, Sequencing, Staining, Binding Assay, Northern Blot, Nucleic Acid Electrophoresis, Isolation, Pulse Chase

Journal: Cancer Discovery

Article Title: LINE-1 Locus Transcription Nucleates Oncogenic Chromatin Architecture

doi: 10.1158/2159-8290.CD-25-1085

Figure Lengend Snippet: Chromatin-associated LINE-1 RNAs are expressed primarily from cell type–specific young LINE-1 loci. A, Cell type–dependent and locus-specific LINE-1 expression repertoire. Schematic depicts hypothetical LINE-1 loci 1, 2, and 3 and their variable expression levels across cell types A, B, and C. B, LINE-1 locus expression distributions, quantified as TPM, from chrRNA-seq data are shown for the three youngest human LINE-1 subfamilies (L1HS, L1PA2, and L1PA3). Read counts aligned to sense-oriented LINE-1 loci in hg38 are discarded. Numbers ( N ) of remaining full-length (>5 kb) LINE-1 loci quantified are shown. C, Hierarchical clustering of the TPM values of locus expression from full-length copies of L1HS, L1PA2, and L1PA3. D, Venn diagram intersecting the top 10% expressed L1HS loci from the five highest LINE-1–expressing cell lines. E, Genome browser view of representative L1HS loci among the top 10% expressed copies. F, Motif analysis of TF binding and ChromHMM enrichment comparing expressed vs. nonexpressed LINE-1 loci. G, FOXA1 ChIP-seq signal at expressed vs. nonexpressed L1HS loci in MCF7 cells, represented as metagene plots spanning the 6-kb length of intact L1HS sequences and their 5 kb flanking regions. The difference in peak strength between two groups was calculated using an unpaired two-sided Student t test. The FOXA1 binding motif detected by HOMER is shown. RPKM, reads per kilobase million. H, Relative transcript and protein abundance of FOXA1 and LINE-1 5′UTR as measured by qRT-PCR and Western blotting with or without dox-induced FOXA1 overexpression in MCF7 cells. Results are shown as mean ± SD ( N ≥ 3 replicates). P values were calculated using an unpaired Student t test. I, ChromHMM feature annotations enriched at expressed LINE-1 loci compared against nonexpressed LINE-1 loci. P values were calculated using a Fisher exact test.

Article Snippet: Custom Hybridization Chain Reaction (HCR) probe sets targeting the LINE-1 5′UTR promoter (lot #RTH492) were designed by Molecular Instruments, Inc. using the full-length L1HS consensus sequence (RepBase).

Techniques: Expressing, Binding Assay, ChIP-sequencing, Quantitative Proteomics, Quantitative RT-PCR, Western Blot, Over Expression

Journal: Cancer Discovery

Article Title: LINE-1 Locus Transcription Nucleates Oncogenic Chromatin Architecture

doi: 10.1158/2159-8290.CD-25-1085

Figure Lengend Snippet: LINE-1 transcription is necessary and sufficient to form long-range chromatin interactions. A, L1HS transcription unit is schematized along with relative positions of 5′UTR-targeting ASO1, ASO2, and smRNA-FISH probes. Subfamily expression of LINE-1 loci by RNA-seq in MCF7 cells upon ASO treatment. SCR, scrambled control. Results are shown as mean ± SD ( N ≥ 3 replicates). TPM values for individual loci were summed per subfamily and normalized by subfamily full-length copy number. P values were calculated using two-way ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, NS, not significant. B, Expression changes of individual full-length LINE-1 loci upon LINE-1 ASO2 treatment in MCF7 cells. HILLs are marked in large red dots, whereas non-HILLs are marked in small pink dots. C, Representative fluorescent micrographs of smRNA-FISH foci in MCF7 cells treated with SCR or LINE-1 ASO2. Pink pseudo-colors, LINE-1 Cy5 probes. Yellow pseudo-colors, β-actin Cy3 probes. Quantification of the numbers of LINE-1 smRNA-FISH foci from N = 30 cells. P values were calculated using an unpaired Student t test. **, P < 0.01; ***, P < 0.001. D, Representative genome browser view of L1CaP-C interactions in MCF7 cells treated with SCR or LINE-1 ASO2. E, Circos plots depicting intrachromosomal and interchromosomal multi-way chromunities identified by the Chromunity algorithm as orange arcs using MCF7 L1CaP-C data. Green boxes depict positions of full-length LINE-1 loci in hg38. The top 50 genes with the highest number of supporting concatemers are shown. F, Schematic of cell engineering to test sufficiency of LINE-1 transcription in generating de novo chromatin interactions. Donor cassette encodes mScarlet to enrich for knock-in MCF7 cells by FACS. Capture probe was designed to target the 3′ end of the TRE3GS promoter to ensure equal enrichment across conditions and transgenes. G, Top: genotyping validation of knock-in cassettes in enriched MCF7 cells engineered to harbor EV control transgene or a 6-kb LINE-1 reporter. Primers span the Rogi2 locus. “+/−” denotes successful knock-in and “−/−” denotes the empty allele. Bottom: expression analysis of reporter transgenes by qRT-PCR with or without dox induction. H, Violin plots of the distance span ( d ) of L1CaP-C–detected interactions with the reporter locus. P values were calculated based on combined replicates using a Mann-Whitney rank-sum test. ****, P < 0.0001. Cyto, cytosolic; Nuc, nuclear; WT, wild type.

Article Snippet: Custom Hybridization Chain Reaction (HCR) probe sets targeting the LINE-1 5′UTR promoter (lot #RTH492) were designed by Molecular Instruments, Inc. using the full-length L1HS consensus sequence (RepBase).

Techniques: Expressing, RNA Sequencing, Control, Knock-In, Biomarker Discovery, Quantitative RT-PCR, MANN-WHITNEY

Journal: Cancer Discovery

Article Title: LINE-1 Locus Transcription Nucleates Oncogenic Chromatin Architecture

doi: 10.1158/2159-8290.CD-25-1085

Figure Lengend Snippet: LINE-1 transcripts interact with chromatin architecture–associated RNA-binding proteins. A, ChIRP-MS experimental scheme. B, ChIRP-qPCR after pull-down of RNAs from formaldehyde-crosslinked MCF7 cells, with or without RNase A treatment, using LINE-1 5′UTR–targeting biotinylated probes. Two pairs of LINE-1 5′UTR primers were used to detect on-target enrichment. ACTB primers were used as nontargeting control. % recovery indicates the proportion of the input RNA recovered after pull-down and elution. Results are shown as mean ± SD ( N ≥ 3 replicates). P values were calculated by an unpaired Student t test. C, Volcano plot of enriched proteins relative to RNase A–treated control conditions. Significant hits are marked as red dots. D, qRT-PCR of SAFB, SAFB2, and SLTM triple knockdown (TKD) with inducible CRISPR inhibition versus AAVS1-targeting control sgRNA. Results are shown as mean ± SD ( N ≥ 3 replicates). P values were calculated by an unpaired Student t test. E, Venn diagrams intersecting significantly downregulated genes in LINE-1 ASO2–treated MCF7 cells or TKD cells relative to controls (sgAAVS1). P values were calculated by hypergeometric distribution. The top enriched CPDB pathways are shown. F, Schematic model of the LINE-1 transcription–mediated formation of chromatin interaction hubs that nucleate target genes and cis -regulatory elements (CRE) through chromatin architecture–regulating RNA-binding proteins, whereas locus-specific LINE-1 transcription is influenced by lineage-specific TFs. BH, Benjamini–Hochberg.

Article Snippet: Custom Hybridization Chain Reaction (HCR) probe sets targeting the LINE-1 5′UTR promoter (lot #RTH492) were designed by Molecular Instruments, Inc. using the full-length L1HS consensus sequence (RepBase).

Techniques: RNA Binding Assay, Control, Quantitative RT-PCR, Knockdown, CRISPR, Inhibition

Journal: bioRxiv

Article Title: Genetic variation shapes human mRNA translation and disease risk

doi: 10.64898/2026.02.10.705206

Figure Lengend Snippet: ( A ) Overview of ribosome profiling and RNA-seq data processing. Transcripts per million (TPM) values were calculated from Ribo-seq and RNA-seq data, and translation efficiency (TE) was calculated as TPM ribo-seq /TPM RNA-seq . mRNAs were classified into high-, intermediate-, and low-TE categories, with “individual-sharing” transcripts defined by consistent classification across samples after filtering (see Methods). ( B ) Architecture of the convolutional-recurrent hybrid neural network for TE prediction. Full-length mRNA sequences were one-hot encoded, with a fifth channel labeling RNA regions (5’UTR, CDS, and 3’UTR), and fed into the CNN and BiLSTM layers, followed by fully connected layers to output the predicted TE-high probabilities (referred to as pTE). ( C ) Model performance metrics (AUC, PRAUC, Precision, Recall, Accuracy, and F1-score) from 10-fold cross-validation; each point represents one-fold. ( D ) Receiver operating characteristic (ROC) curves for the 10 folds; the x-axis shows specificity; the y-axis shows sensitivity. ( E ) Relationship between predicted pTE categories and actual TE values. The x-axis shows predicted score categories; the y-axis shows log 10 (TE+1) across samples to accommodate zero values.

Article Snippet: To evaluate the impact of 5’UTR variants on translation efficiency, we synthesized variant-containing 5’UTR sequences (GENEWIZ) and cloned them into the Fluc-Nluc dual luciferase reporter vector ( ).

Techniques: RNA Sequencing, Labeling, Biomarker Discovery

Journal: bioRxiv

Article Title: Genetic variation shapes human mRNA translation and disease risk

doi: 10.64898/2026.02.10.705206

Figure Lengend Snippet: Sequence and structural correlates of translation efficiency captured by TEFL-mRNA. ( A-B ) Distribution of pTE across mRNAs with or without (A) 3’UTR miRNA binding sites or (B) 5’UTR upstream open reading frame (uORF). Y-axis shows the fraction of mRNAs in each pTE category. P-value from Fisher’s Exact test indicates significant shifts in pTE distributions. ( C ) Correlation between estimated mRNA half-life and predicted TE. ( D ) Correlations between pTE and the length of the 5’UTR, CDS, and 3’UTR. ( E-G ) Correlations between pTE and (E) GC ratio, (F) codon adaptation index (CAI), or (G) adjusted minimum free energy (AMFE; minimum free energy normalized by mRNA length). For all scatter plots, points represent bin-averaged (10 mRNAs per bin) values of sequence features (x-axis) and pTE (y-axis); Pearson’s correlation coefficients (R) and p values are shown.

Article Snippet: To evaluate the impact of 5’UTR variants on translation efficiency, we synthesized variant-containing 5’UTR sequences (GENEWIZ) and cloned them into the Fluc-Nluc dual luciferase reporter vector ( ).

Techniques: Sequencing, Binding Assay

Journal: bioRxiv

Article Title: Genetic variation shapes human mRNA translation and disease risk

doi: 10.64898/2026.02.10.705206

Figure Lengend Snippet: The effects of single-nucleotide variants on translation efficiency. ( A ) Overview of TEFL-mRNA variant analysis. Genetic variants from gnomAD were annotated with VEP, and mRNA transcripts containing 5’UTR, 3’UTR, or CDS were extracted. Full-length mRNA sequences with reference and alternative alleles were input into TEFL-mRNA to calculate TE changes (ΔpTE = pTE ALT – pTE REF ). ( B ) Distribution of pTE categories for mRNAs with uAUG-creating SNVs versus all gnomAD 5’UTR variants; enrichment tested by Fisher’s Exact test. X-axis shows the fraction of mRNAs in each pTE category. ( C ) Cumulative distribution of pTE for uAUG-creating (red line) versus gnomAD 5’UTR (grey line) variants; p-value from Kolmogorov-Smirnov test. ( D ) Regional distribution of TE effects for TE-altering variants. Left: boxplot of |ΔpTE| of variants with |ΔpTE| > 0.1 in different regions. The number of such variants is shown above each box. Right: fraction of SNVs passing different |ΔpTE| cutoffs. ( E ) Relationship between allele frequency (AF) and |ΔpTE| for 5’UTR SNVs. Brown points mark variants with |pTE| > 0.1. ( F ) Percentage of transcripts with 5’UTR SNVs passing the specified |ΔpTE| cutoff within the allele frequency (AF) range among all transcripts with 5’UTR SNVs that pass the |ΔpTE| cutoff. ( G ) Schematic of the Fluc-Nluc dual luciferase reporter used to assay SNV effects on TE. ( H ) Reporter assay results for six gnomAD SNVs. Left: SNVs whose variant alleles increased TE; right: SNVs whose variant alleles decreased TE. Bars show mean ± standard deviation (SD) from biological triplicates. Y-axis shows the normalized luciferase ratio. P values from one-tailed t-tests are shown. TEFL-mRNA predicted pTE values are shown below each panel.

Article Snippet: To evaluate the impact of 5’UTR variants on translation efficiency, we synthesized variant-containing 5’UTR sequences (GENEWIZ) and cloned them into the Fluc-Nluc dual luciferase reporter vector ( ).

Techniques: Variant Assay, Luciferase, Reporter Assay, Standard Deviation, One-tailed Test

Journal: bioRxiv

Article Title: Genetic variation shapes human mRNA translation and disease risk

doi: 10.64898/2026.02.10.705206

Figure Lengend Snippet: TE-altering SNVs in coding and noncoding regions. ( A ) Predicted ΔpTE for SNVs located within ±1000nt of the start or stop codon. Each point indicates an individual SNV; colors indicate the alternative allele. X-axis shows the relative distance to the start or stop codon. ( B ) Fractions of transcripts with 4 alternative allele types (A/C/G/U) among 5’UTR SNVs exceeding different ΔpTE cutoffs. ( C ) Counts of TE-increasing and TE-decreasing synonymous SNVs with |ΔpTE| > 0.05 (corresponding to a false positive rate of 1%, similarly in panels D-G below). Variants changing codons toward non-optimal (orange) or optimal (yellow) are shown separately. P-value from Fisher’s exact test. ( D-E ) ΔpTE for missense SNVs introducing specific amino acids. (D) TE-increasing variants frequently introduce hydrophobic residues (Phe, Leu). (E) TE-decreasing variants more often introduce Gly, Thr, and Pro, linked to reduced elongation efficiency. ( F ) Proportion of TE-increasing versus TE-decreasing effects among all missense SNVs or Pro-substituting SNVs; enrichment tested by Fisher’s Exact test. Y-axis shows the fraction of transcripts with the SNVs in each category. ( G ) Relationship between ΔpTE and poly-proline tract length created by missense SNVs. n denotes the number of SNVs per group.

Article Snippet: To evaluate the impact of 5’UTR variants on translation efficiency, we synthesized variant-containing 5’UTR sequences (GENEWIZ) and cloned them into the Fluc-Nluc dual luciferase reporter vector ( ).

Techniques: Introduce

Journal: bioRxiv

Article Title: Genetic variation shapes human mRNA translation and disease risk

doi: 10.64898/2026.02.10.705206

Figure Lengend Snippet: Contribution of mRNA structure and RBP binding to translation alteration. ( A ) Absolute changes in adjusted minimum free energy (|ΔAMFE|) for TE-decreasing, TE-increasing, and TE-unchanged SNVs in the 5’UTR and CDS. ( B ) Comparison of |ΔAMFE| between TE-decreasing and TE-increasing SNVs in the 5’UTR and CDS; p-values from Student’s t-test are shown. ( C ) Structures of the mRNA NM_001320334 with the reference (C, left) or alternative allele (T, right) at an SNV (rs758379451) in the 5’ UTR. Structures were predicted using LinearFold and visualized with foRNA. ( D ) Heatmaps showing RBPs whose binding was significantly changed by TE-altering SNVs (compared to TE-unchanged SNVs) in 5’UTR (left) and CDS (right). Binding changes were predicted by DeepBind; p-values from Student’s t-test are color-coded. ( E-F ) Representative RBP binding motifs in the 5’UTR (E) and CDS (F). For each motif, bar plots show counts of TE-altering SNVs that map to specific motif positions.

Article Snippet: To evaluate the impact of 5’UTR variants on translation efficiency, we synthesized variant-containing 5’UTR sequences (GENEWIZ) and cloned them into the Fluc-Nluc dual luciferase reporter vector ( ).

Techniques: Binding Assay, Comparison

Journal: bioRxiv

Article Title: Genetic variation shapes human mRNA translation and disease risk

doi: 10.64898/2026.02.10.705206

Figure Lengend Snippet: Disease associations of genetic variants that alter translation. ( A ) Construction and evaluation of processes for variants with |ΔpTE| > 0.1. Dot size indicates the number of genes the GO term contains; colors denote parent terms summarized by rrvgo . Axes represent the first two components from PCoA of the semantic similarity matrix. ( B ) Disease ontology enrichment for the same variant set as in (A). Dot size indicates gene counts; color scale shows adjusted p-values. ( C ) Fractions of transcripts with 5’UTR SNVs that passed various |ΔpTE| cutoffs in GWAS (red) and gnomAD (blue). P-values from Fisher’s exact test. ( D ) GWAS diseases (left) and non-disease traits (right) enriched for 5’UTR TE-altering SNVs (|ΔpTE| > 0.1). Bars show counts; color scale indicates the fraction of transcripts in each trait carrying such variants. The enriched traits with top 10 SNVs counts were filtered using p < 0.05 from Fisher’s Exact test. ( E ) Top 10 ClinVar immune-related SNVs ranked by |ΔpTE| in the 5’UTR (top) and CDS (bottom). ( F-G ) Luciferase reporter assays for GWAS disease-related SNVs. Bars show mean ± SD from biological triplicates; points indicate replicates; y-axis shows the normalized luciferase ratio. P-values from one-tailed t-tests are shown. TEFL-mRNA predicted pTE values for reference (REF) and alternative (ALT) alleles, along with associated GWAS traits, are listed below each plot.

Article Snippet: To evaluate the impact of 5’UTR variants on translation efficiency, we synthesized variant-containing 5’UTR sequences (GENEWIZ) and cloned them into the Fluc-Nluc dual luciferase reporter vector ( ).

Techniques: Variant Assay, Luciferase, One-tailed Test

Journal: bioRxiv

Article Title: Genetic variation shapes human mRNA translation and disease risk

doi: 10.64898/2026.02.10.705206

Figure Lengend Snippet: Translation-altering SNVs across multiple cell types. ( A ) Receiver-operating characteristic (ROC) curves for TEFL-mRNA models trained separately in nine human cell types. Performance was assessed by 10-fold cross-validation for the TE-high vs others classification; each fold is shown, and the per-model AUC is annotated. ( B ) Cross-cell-type agreement for 5’UTR SNVs. Tiles show the Spearman correlations of ΔpTE between each pair of cell types, computed on ExAC 5’UTR SNVs with |ΔpTE|>0.02 in both cell types (two-sided test; *** p < 0.001). Overlaid pie charts indicate the proportion of shared SNVs with concordant positive (red), concordant negative (blue), or discordant (salmon) signs of TE effects. ( C ) Sharing of TE effects across cell lines by region. Stacked bars give the percentage of SNVs that are “shared” across cell types (orange), defined by a concordance score >0.8 (the fraction of profiled cell types in which the ΔpTE sign matches the majority sign among cell types where |ΔpTE|>0.02), for 5’UTR, CDS missense, CDS synonymous, and 3’UTR SNVs. Count and percentage of SNVs contributing to each bar are shown. ( D ) Disease enrichment of TE-altering GWAS 5’UTR SNVs across cell types. Heatmap shows the enrichment of TE-altering SNVs (|ΔpTE|>0.02) associated with disease traits (EFO:0000408) in at least two cell types using Fisher’s exact test (p < 0.05); values are z-scores of the odds ratio in each trait across cell types. Row/column dendrograms indicate hierarchical clustering of traits and cell types (*** p<0.001, ** p<0.01, * p<0.05).

Article Snippet: To evaluate the impact of 5’UTR variants on translation efficiency, we synthesized variant-containing 5’UTR sequences (GENEWIZ) and cloned them into the Fluc-Nluc dual luciferase reporter vector ( ).

Techniques: Biomarker Discovery