Journal: bioRxiv

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

doi: 10.64898/2026.01.22.698655

Figure Lengend Snippet: ( A ) The number of LTR-associated 3’-tRF target sites in GENCODE transcripts (left) and the enrichment at each position relative to expectation (right). Error bars show 95% confidence intervals. Asterisks (*) indicate p -values < 0.05 from Fisher’s exact tests. ( B ) Schematic illustrating alternative splicing outcomes at LTR-initiated protein-coding transcripts. The PBS is either retained in the 5’ UTR or spliced out, depending on the position of the 5’ splice site. Percentages reflect the proportion of LTR-associated target sites located within 200 bp upstream (retained) or downstream (spliced out) of a splice donor site. ( C ) Genome browser view of the Csta2 locus showing an LTR-initiated transcript with a predicted 3’-tRF target site in its 5’ UTR. The FANTOM5 track shows total CAGE read counts across FANTOM5 datasets, which include diverse somatic tissues. RNA-seq tracks show expression in the 2-cell embryo (GSE66582) and in wild-type ( Setdb1 +/+ ) or Setdb1 knockout ( Setdb1 -/- ) mESCs (GSE29413). The start site of the LTR-retrotransposon derived transcript and the position of the predicted 3’-tRF target site are shown in detail on the right. ( D ) Schematic of the transcriptome assembly method. ( E ) The number of LTR-associated 3’-tRF target sites in assembled transcripts (left) and the enrichment at each position relative to expectation (right). Error bars show 95% confidence intervals. Asterisks (*) indicate p -values < 0.05 from Fisher’s exact tests.

Article Snippet: 500 pg total RNA was used as input for the SMARTer Stranded Total RNA-Seq Pico Input Kit v2 (Takara; 634412) or the NEBNext Single Cell/Low Input RNA Library Prep Kit (New England Biolabs; E6420L) to generate random-primed and poly(A)-primed RNA-seq libraries, respectively.

Techniques: Alternative Splicing, RNA Sequencing, Expressing, Knock-Out, Derivative Assay

Journal: bioRxiv

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

doi: 10.64898/2026.01.22.698655

Figure Lengend Snippet: ( A ) Distribution of LTR-associated 3’-tRF target sites in the 3’ UTR of protein-coding genes across LTR-retrotransposon families. Highlighted is the number of sites for which the top scoring 3’-tRF is derived from a Leu-TAA tRNA. ( B ) Genome browser view of the Mplkipl1 locus in the C57BL/6J reference strain. RNA-seq tracks show expression in the pre-implantation embryo (GSE66582). BLAST track shows the region of homology with Mplkip , with mismatches indicated in dark grey. ( C ) Repression of luciferase reporters containing 3’ UTR target sites from the indicated genes by a Leu-TAA tRF3b mimic. Relative repression was calculated by normalizing first to a no target site reporter, and then to a non-targeting tRF3b. The 3009b reporter serves as a positive control with a perfectly complementary site. For each target site, red highlighting indicates mismatches to the tRF3b sequence. Error bars show propagated standard error from technical replicates. Asterisks (*) indicate p -values < 0.05 from one-sided, one-sample t -tests. ( D ) Genome browser view of the Cyp2b23 locus showing a StringTie assembled transcript (yellow) initiated in an LTR with a predicted 3’-tRF target site in the 5’ UTR, alongside the canonical GENCODE-annotated transcript (blue). RNA-seq tracks show expression in the pre-implantation embryo (GSE66582), and in wild-type or Setdb1 knockout (GSE29413) mESCs.

Article Snippet: 500 pg total RNA was used as input for the SMARTer Stranded Total RNA-Seq Pico Input Kit v2 (Takara; 634412) or the NEBNext Single Cell/Low Input RNA Library Prep Kit (New England Biolabs; E6420L) to generate random-primed and poly(A)-primed RNA-seq libraries, respectively.

Techniques: Derivative Assay, RNA Sequencing, Expressing, Luciferase, Positive Control, Sequencing, Knock-Out

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: 500 pg total RNA was used as input for the SMARTer Stranded Total RNA-Seq Pico Input Kit v2 (Takara; 634412) or the NEBNext Single Cell/Low Input RNA Library Prep Kit (New England Biolabs; E6420L) to generate random-primed and poly(A)-primed RNA-seq libraries, respectively.

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