Journal: bioRxiv

Article Title: RNA length and receptor usage define innate immune recognition across species

doi: 10.64898/2026.05.21.726451

Figure Lengend Snippet: Induction of antiviral response in human (A549), chicken (DF-1), and duck (CCL-141) cells following transfection with an in vitro transcribed mvRNA. The mvRNA used in this study is a 60 nt RNA forming an approximately 28 bp duplex and bearing a 5’-triphosphate (5’-ppp) or dephosphorylated (5’-OH) end. Luciferase activity driven by interferon-responsive promoters is shown. Cells were transfected with mvRNA for 24 h prior to measurement. Bars represent mean ± SEM from three independent biological replicates (each with three technical replicates), and individual points indicate technical replicates. Statistical significance: ****P < 0.0001; ns, not significant (one-way ANOVA with Tukey’s multiple comparisons test).

Article Snippet: Human lung epithelial A549 cells (CCL-185) and chicken embryo fibroblast DF-1 cells (CRL-3586) were obtained from ATCC.

Techniques: Transfection, In Vitro, Luciferase, Activity Assay

Journal: bioRxiv

Article Title: RNA length and receptor usage define innate immune recognition across species

doi: 10.64898/2026.05.21.726451

Figure Lengend Snippet: (A) Induction of antiviral response in human (A549), chicken (DF-1), and duck (CCL-141) cells following transfection with in vitro transcribed dsRNAs of defined lengths (50, 200, 550, and 1600 bp). dsRNAs carried either a 5’-triphosphate (5’-ppp) or a dephosphorylated (5’-OH) end. Luciferase activity driven by interferon-responsive promoters is shown. Cells were transfected with the indicated dsRNA for 24 h prior to measurement. Bars represent mean ± SEM from three independent biological replicates (each with three technical replicates), and individual points indicate technical replicates. Statistical significance is shown relative to mock-treated samples: **P < 0.01; ****P < 0.0001; ns, not significant (one-way ANOVA with Tukey’s multiple comparisons test). (B) Western blot analysis of MX1 protein expression in human and chicken cells following transfection with mvRNA and 5’-triphosphorylated dsRNAs of increasing lengths (50-550 bp).

Article Snippet: Human lung epithelial A549 cells (CCL-185) and chicken embryo fibroblast DF-1 cells (CRL-3586) were obtained from ATCC.

Techniques: Transfection, In Vitro, Luciferase, Activity Assay, Western Blot, Expressing

Journal: bioRxiv

Article Title: RNA length and receptor usage define innate immune recognition across species

doi: 10.64898/2026.05.21.726451

Figure Lengend Snippet: (A) Schematic overview of RNA fractionation from virus (H5N8 or H1N1)-infected, dsRNA-treated, and mock-treated A549 cells using HPLC. Total RNA was fractionated, and RNA species shorter than 200 nt were collected. The short RNA fraction, either untreated or dephosphorylated with alkaline phosphatase, was used for transfection of human (A549) and chicken (DF-1) cells. (B) Northern blot analysis confirming the presence of endogenous short RNA, SNORD44 (63 nt), in the collected fraction. (C) Induction of antiviral response in human and chicken cells following transfection with the isolated short RNA fraction, assessed by western blot analysis of MX1 protein expression. (D) Analysis of total RNA isolated from virus-infected, dsRNA-treated, and mock-treated A549 cells. rRNA integrity was assessed in a denaturing agarose gel.

Article Snippet: Human lung epithelial A549 cells (CCL-185) and chicken embryo fibroblast DF-1 cells (CRL-3586) were obtained from ATCC.

Techniques: Fractionation, Virus, Infection, Transfection, Northern Blot, Isolation, Western Blot, Expressing, Agarose Gel Electrophoresis

Journal: bioRxiv

Article Title: RNA length and receptor usage define innate immune recognition across species

doi: 10.64898/2026.05.21.726451

Figure Lengend Snippet: (A) Western blot analysis of antiviral responses assessed by MX1 expression in human (A549) cells lacking RIG-I or MDA5 following transfection with 5’-triphosphorylated in vitro transcribed mvRNA and 5’-triphosphorylated dsRNAs of increasing lengths. Lanes labeled “A549 WT” represent wild-type A549 cells transfected with 5’-ppp mvRNA and are included as positive controls for immune activation. (B) Induction of antiviral response in human cells lacking RIG-I or MDA5 following transfection with the isolated short RNA fraction, assessed by western blot analysis of MX1 expression. Lanes labeled “A549 WT” represent wild-type A549 cells transfected with with 5’-ppp mvRNA and are included as positive controls for immune activation. (C) Induction of antiviral response in chicken cells lacking MDA5 following transfection with 5’-triphosphorylated in vitro transcribed mvRNA and 5’-triphosphorylated dsRNAs of increasing lengths, assessed by western blot analysis of MX1 expression. Lanes labeled “DF-1 WT” represent wild-type DF-1 cells transfected with 5’-ppp 550 bp dsRNA and are included as positive controls for immune activation.

Article Snippet: Human lung epithelial A549 cells (CCL-185) and chicken embryo fibroblast DF-1 cells (CRL-3586) were obtained from ATCC.

Techniques: Western Blot, Expressing, Transfection, In Vitro, Labeling, Activation Assay, Isolation

Journal: bioRxiv

Article Title: RNA length and receptor usage define innate immune recognition across species

doi: 10.64898/2026.05.21.726451

Figure Lengend Snippet: (A) Western blot analysis of PKR and eIF2α phosphorylation in human (A549), chicken (DF-1), and duck (CCL-141) cells following transfection with short (mvRNA) or long (1600 bp) dsRNA carrying either a 5’-triphosphate (5’-ppp) or a dephosphorylated (5’-OH) end. Human and chicken cells were transfected for 5 h, whereas duck cells were transfected for 7.5 h prior to harvesting. PKR phosphorylation was assessed in human samples. (B) Puromycin incorporation assay to assess global translation in human, chicken, and duck cells following transfection with short or long dsRNA. Reduced puromycin signal indicates translational inhibition.

Article Snippet: Human lung epithelial A549 cells (CCL-185) and chicken embryo fibroblast DF-1 cells (CRL-3586) were obtained from ATCC.

Techniques: Western Blot, Phospho-proteomics, Transfection, Inhibition

Journal: bioRxiv

Article Title: RNA length and receptor usage define innate immune recognition across species

doi: 10.64898/2026.05.21.726451

Figure Lengend Snippet: (A) Schematic overview of the pull-down assay. Human (A549), chicken (DF-1), and duck (CCL-141) cells were transfected with a mixture of 5’-triphosphorylated short (mvRNA) and long (1600 bp) dsRNA or mock-treated prior to lysis. Cell lysates were incubated with biotinylated short or long dsRNA carrying either a 5’-triphosphate (5’-ppp) or a dephosphorylated (5’-OH) end. RNA-protein complexes were isolated using streptavidin beads and analyzed by mass spectrometry (MS/MS). (B) Heatmaps showing enrichment of RNA-binding proteins in pull-down assays from human, chicken, and duck cell lysates. Protein enrichment is displayed as a composite score calculated as log2(FC) multiplied by -log10(p-value), relative to beads-only control samples. Color scales indicate relative protein enrichment. The top 30 enriched proteins are shown; full datasets are provided in the supporting information (Fig. S11).

Article Snippet: Human lung epithelial A549 cells (CCL-185) and chicken embryo fibroblast DF-1 cells (CRL-3586) were obtained from ATCC.

Techniques: Pull Down Assay, Transfection, Lysis, Incubation, Isolation, Mass Spectrometry, Tandem Mass Spectroscopy, RNA Binding Assay, Protein Enrichment, Control

Journal: bioRxiv

Article Title: RNA length and receptor usage define innate immune recognition across species

doi: 10.64898/2026.05.21.726451

Figure Lengend Snippet: Analysis of total RNA integrity in human (A549), chicken (DF-1), and duck (CCL-141) cells following transfection with short (mvRNA) or long (1600 bp) dsRNA carrying either a 5’-triphosphate (5’-ppp) or a dephosphorylated (5’-OH) end. RNA profiles were assessed using Agilent 2100 Bioanalyzer.

Article Snippet: Human lung epithelial A549 cells (CCL-185) and chicken embryo fibroblast DF-1 cells (CRL-3586) were obtained from ATCC.

Techniques: Transfection