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( A ) Genome browser view of the murine Peg3 locus. RNA-seq tracks show expression in placenta (ENCFF516KLL), E14.5 whole brain (ENCFF570RBK), and embryonic fibroblasts (ENCFF918FWL). The Peg3 5’ UTR is enlarged with predicted 3’-tRF target sites colored by alignment score. For each 3’-tRF, abundance in HeLa cells estimated from small RNA-seq data (GSE82199) is shown in reads per million (RPM). Error bars show standard error from n = 3 biological replicates. ( B ) Luciferase reporter assay in HeLa cells testing Peg3 5’ UTR target site variants. Fold change was calculated by normalizing relative light units measured for each target site variant to those of the wild-type (wt) reporter. Red highlighting indicates mutated nucleotides in each variant. The alignment of top scoring tRF3b sequences at each site is shown above the wild-type sequence. Error bars show propagated standard error from technical replicates. Asterisks (*) indicate p -values < 0.05 from one-sided, one-sample t -tests. ( C ) Western blot showing endogenous PEG3 expression in mouse embryonic fibroblasts transfected with the indicated tRF3b mimics. Probing for tubulin and Ponceau S staining served as loading controls. ( D ) Peg3 mRNA levels in <t>P19</t> embryonal teratocarcinoma cells transfected with the indicated siRNAs or tRF3b mimics. Expression was calculated relative to samples transfected with a non-targeting (NT) siRNA. Error bars show propagated standard error from technical replicates. Asterisks (*) indicate p -values < 0.05 from one-sided, one-sample t -tests. ( E ) Peg3 mRNA levels in mESCs of the indicated genotype. Fold change was calculated relative to wild-type. Error bars show standard error. Asterisks (*) indicate p -values < 0.05 from differential expression analysis. Data are from GSE122627, GSE110942, GSE78971 and GSE78974.
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( A ) Genome browser view of the murine Peg3 locus. RNA-seq tracks show expression in placenta (ENCFF516KLL), E14.5 whole brain (ENCFF570RBK), and embryonic fibroblasts (ENCFF918FWL). The Peg3 5’ UTR is enlarged with predicted 3’-tRF target sites colored by alignment score. For each 3’-tRF, abundance in HeLa cells estimated from small RNA-seq data (GSE82199) is shown in reads per million (RPM). Error bars show standard error from n = 3 biological replicates. ( B ) Luciferase reporter assay in HeLa cells testing Peg3 5’ UTR target site variants. Fold change was calculated by normalizing relative light units measured for each target site variant to those of the wild-type (wt) reporter. Red highlighting indicates mutated nucleotides in each variant. The alignment of top scoring tRF3b sequences at each site is shown above the wild-type sequence. Error bars show propagated standard error from technical replicates. Asterisks (*) indicate p -values < 0.05 from one-sided, one-sample t -tests. ( C ) Western blot showing endogenous PEG3 expression in mouse embryonic fibroblasts transfected with the indicated tRF3b mimics. Probing for tubulin and Ponceau S staining served as loading controls. ( D ) Peg3 mRNA levels in <t>P19</t> embryonal teratocarcinoma cells transfected with the indicated siRNAs or tRF3b mimics. Expression was calculated relative to samples transfected with a non-targeting (NT) siRNA. Error bars show propagated standard error from technical replicates. Asterisks (*) indicate p -values < 0.05 from one-sided, one-sample t -tests. ( E ) Peg3 mRNA levels in mESCs of the indicated genotype. Fold change was calculated relative to wild-type. Error bars show standard error. Asterisks (*) indicate p -values < 0.05 from differential expression analysis. Data are from GSE122627, GSE110942, GSE78971 and GSE78974.
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( A ) Genome browser view of the murine Peg3 locus. RNA-seq tracks show expression in placenta (ENCFF516KLL), E14.5 whole brain (ENCFF570RBK), and embryonic fibroblasts (ENCFF918FWL). The Peg3 5’ UTR is enlarged with predicted 3’-tRF target sites colored by alignment score. For each 3’-tRF, abundance in HeLa cells estimated from small RNA-seq data (GSE82199) is shown in reads per million (RPM). Error bars show standard error from n = 3 biological replicates. ( B ) Luciferase reporter assay in HeLa cells testing Peg3 5’ UTR target site variants. Fold change was calculated by normalizing relative light units measured for each target site variant to those of the wild-type (wt) reporter. Red highlighting indicates mutated nucleotides in each variant. The alignment of top scoring tRF3b sequences at each site is shown above the wild-type sequence. Error bars show propagated standard error from technical replicates. Asterisks (*) indicate p -values < 0.05 from one-sided, one-sample t -tests. ( C ) Western blot showing endogenous PEG3 expression in mouse embryonic fibroblasts transfected with the indicated tRF3b mimics. Probing for tubulin and Ponceau S staining served as loading controls. ( D ) Peg3 mRNA levels in <t>P19</t> embryonal teratocarcinoma cells transfected with the indicated siRNAs or tRF3b mimics. Expression was calculated relative to samples transfected with a non-targeting (NT) siRNA. Error bars show propagated standard error from technical replicates. Asterisks (*) indicate p -values < 0.05 from one-sided, one-sample t -tests. ( E ) Peg3 mRNA levels in mESCs of the indicated genotype. Fold change was calculated relative to wild-type. Error bars show standard error. Asterisks (*) indicate p -values < 0.05 from differential expression analysis. Data are from GSE122627, GSE110942, GSE78971 and GSE78974.
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( A ) Genome browser view of the murine Peg3 locus. RNA-seq tracks show expression in placenta (ENCFF516KLL), E14.5 whole brain (ENCFF570RBK), and embryonic fibroblasts (ENCFF918FWL). The Peg3 5’ UTR is enlarged with predicted 3’-tRF target sites colored by alignment score. For each 3’-tRF, abundance in HeLa cells estimated from small RNA-seq data (GSE82199) is shown in reads per million (RPM). Error bars show standard error from n = 3 biological replicates. ( B ) Luciferase reporter assay in HeLa cells testing Peg3 5’ UTR target site variants. Fold change was calculated by normalizing relative light units measured for each target site variant to those of the wild-type (wt) reporter. Red highlighting indicates mutated nucleotides in each variant. The alignment of top scoring tRF3b sequences at each site is shown above the wild-type sequence. Error bars show propagated standard error from technical replicates. Asterisks (*) indicate p -values < 0.05 from one-sided, one-sample t -tests. ( C ) Western blot showing endogenous PEG3 expression in mouse embryonic fibroblasts transfected with the indicated tRF3b mimics. Probing for tubulin and Ponceau S staining served as loading controls. ( D ) Peg3 mRNA levels in <t>P19</t> embryonal teratocarcinoma cells transfected with the indicated siRNAs or tRF3b mimics. Expression was calculated relative to samples transfected with a non-targeting (NT) siRNA. Error bars show propagated standard error from technical replicates. Asterisks (*) indicate p -values < 0.05 from one-sided, one-sample t -tests. ( E ) Peg3 mRNA levels in mESCs of the indicated genotype. Fold change was calculated relative to wild-type. Error bars show standard error. Asterisks (*) indicate p -values < 0.05 from differential expression analysis. Data are from GSE122627, GSE110942, GSE78971 and GSE78974.
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( A ) Genome browser view of the murine Peg3 locus. RNA-seq tracks show expression in placenta (ENCFF516KLL), E14.5 whole brain (ENCFF570RBK), and embryonic fibroblasts (ENCFF918FWL). The Peg3 5’ UTR is enlarged with predicted 3’-tRF target sites colored by alignment score. For each 3’-tRF, abundance in HeLa cells estimated from small RNA-seq data (GSE82199) is shown in reads per million (RPM). Error bars show standard error from n = 3 biological replicates. ( B ) Luciferase reporter assay in HeLa cells testing Peg3 5’ UTR target site variants. Fold change was calculated by normalizing relative light units measured for each target site variant to those of the wild-type (wt) reporter. Red highlighting indicates mutated nucleotides in each variant. The alignment of top scoring tRF3b sequences at each site is shown above the wild-type sequence. Error bars show propagated standard error from technical replicates. Asterisks (*) indicate p -values < 0.05 from one-sided, one-sample t -tests. ( C ) Western blot showing endogenous PEG3 expression in mouse embryonic fibroblasts transfected with the indicated tRF3b mimics. Probing for tubulin and Ponceau S staining served as loading controls. ( D ) Peg3 mRNA levels in <t>P19</t> embryonal teratocarcinoma cells transfected with the indicated siRNAs or tRF3b mimics. Expression was calculated relative to samples transfected with a non-targeting (NT) siRNA. Error bars show propagated standard error from technical replicates. Asterisks (*) indicate p -values < 0.05 from one-sided, one-sample t -tests. ( E ) Peg3 mRNA levels in mESCs of the indicated genotype. Fold change was calculated relative to wild-type. Error bars show standard error. Asterisks (*) indicate p -values < 0.05 from differential expression analysis. Data are from GSE122627, GSE110942, GSE78971 and GSE78974.
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( A ) Genome browser view of the murine Peg3 locus. RNA-seq tracks show expression in placenta (ENCFF516KLL), E14.5 whole brain (ENCFF570RBK), and embryonic fibroblasts (ENCFF918FWL). The Peg3 5’ UTR is enlarged with predicted 3’-tRF target sites colored by alignment score. For each 3’-tRF, abundance in HeLa cells estimated from small RNA-seq data (GSE82199) is shown in reads per million (RPM). Error bars show standard error from n = 3 biological replicates. ( B ) Luciferase reporter assay in HeLa cells testing Peg3 5’ UTR target site variants. Fold change was calculated by normalizing relative light units measured for each target site variant to those of the wild-type (wt) reporter. Red highlighting indicates mutated nucleotides in each variant. The alignment of top scoring tRF3b sequences at each site is shown above the wild-type sequence. Error bars show propagated standard error from technical replicates. Asterisks (*) indicate p -values < 0.05 from one-sided, one-sample t -tests. ( C ) Western blot showing endogenous PEG3 expression in mouse embryonic fibroblasts transfected with the indicated tRF3b mimics. Probing for tubulin and Ponceau S staining served as loading controls. ( D ) Peg3 mRNA levels in P19 embryonal teratocarcinoma cells transfected with the indicated siRNAs or tRF3b mimics. Expression was calculated relative to samples transfected with a non-targeting (NT) siRNA. Error bars show propagated standard error from technical replicates. Asterisks (*) indicate p -values < 0.05 from one-sided, one-sample t -tests. ( E ) Peg3 mRNA levels in mESCs of the indicated genotype. Fold change was calculated relative to wild-type. Error bars show standard error. Asterisks (*) indicate p -values < 0.05 from differential expression analysis. Data are from GSE122627, GSE110942, GSE78971 and GSE78974.

Journal: bioRxiv

Article Title: 3’-tRNA Fragments Target Domesticated LTR-Retrotransposons

doi: 10.64898/2026.01.22.698655

Figure Lengend Snippet: ( A ) Genome browser view of the murine Peg3 locus. RNA-seq tracks show expression in placenta (ENCFF516KLL), E14.5 whole brain (ENCFF570RBK), and embryonic fibroblasts (ENCFF918FWL). The Peg3 5’ UTR is enlarged with predicted 3’-tRF target sites colored by alignment score. For each 3’-tRF, abundance in HeLa cells estimated from small RNA-seq data (GSE82199) is shown in reads per million (RPM). Error bars show standard error from n = 3 biological replicates. ( B ) Luciferase reporter assay in HeLa cells testing Peg3 5’ UTR target site variants. Fold change was calculated by normalizing relative light units measured for each target site variant to those of the wild-type (wt) reporter. Red highlighting indicates mutated nucleotides in each variant. The alignment of top scoring tRF3b sequences at each site is shown above the wild-type sequence. Error bars show propagated standard error from technical replicates. Asterisks (*) indicate p -values < 0.05 from one-sided, one-sample t -tests. ( C ) Western blot showing endogenous PEG3 expression in mouse embryonic fibroblasts transfected with the indicated tRF3b mimics. Probing for tubulin and Ponceau S staining served as loading controls. ( D ) Peg3 mRNA levels in P19 embryonal teratocarcinoma cells transfected with the indicated siRNAs or tRF3b mimics. Expression was calculated relative to samples transfected with a non-targeting (NT) siRNA. Error bars show propagated standard error from technical replicates. Asterisks (*) indicate p -values < 0.05 from one-sided, one-sample t -tests. ( E ) Peg3 mRNA levels in mESCs of the indicated genotype. Fold change was calculated relative to wild-type. Error bars show standard error. Asterisks (*) indicate p -values < 0.05 from differential expression analysis. Data are from GSE122627, GSE110942, GSE78971 and GSE78974.

Article Snippet: P19 embryonal teratocarcinoma cells (ATCC; CRL-1825) were grown in MEM α (Gibco; 12571063) with 2.5% fetal bovine serum and 7.5% bovine calf serum (VWR; 10158-358) or, for P19 RNA-seq, 10% fetal bovine serum only.

Techniques: RNA Sequencing, Expressing, Luciferase, Reporter Assay, Variant Assay, Sequencing, Western Blot, Transfection, Staining, Quantitative Proteomics