Journal: bioRxiv

Article Title: tRNA thiolation defects disrupt cellular proteostasis and tissue homeostasis in mammals

doi: 10.1101/2025.10.24.684405

Figure Lengend Snippet: (a) Western blot analysis of doxycycline (dox)-inducible CTU2 knockdown in HeLa cells over time. Duration of dox treatment is indicated in days. scrambled, non-targeting shRNA control. (b) APM-northern blot validation of tRNA thiolation loss in HeLa and RPE1 cells after 4 days of dox treatment. is shown representatively. (c) Viability analysis of CTU2 knockdown cell lines by Annexin V staining. +CTU2 res indicates shRNA-resistant CTU2 overexpression. One-way ANOVA, WT vs. CTU2 res p = 0.264. n = 3 technical replicates. (d) Western blot analysis of unfolded protein response (UPR) checkpoints during progressive tRNA hypothiolation in RPE1 cells. Time points indicate days of CTU2 depletion. (e) Western blot validation of selected proteins identified in DREAM-PL patient fibroblasts, analyzed under acute CTU2 knockdown conditions. (f) Ribosome profiling in 96 h CTU2 -depleted RPE1 cells compared with untreated controls. Normalized codon occupancy is shown for A- and G-ending codons of the indicated amino acids. Codons are divided into quartiles according to relative library abundance. n = 2 technical replicates. (g) Cumulative A-ending codon frequency (AAA, CAA, GAA, and AGA) in coding sequences (CDSs) of proteins quantified in CTU2 -depleted RPE1 cells after 96 hours. The dashed line indicates the average A-ending codon content (7.6%) across 19,085 analyzed human CDSs. Two-tailed unpaired t-test, p > 0.001 (down and up). (h) Gene Ontology (GO) enrichment analysis on the top 5% of human coding sequences ranked by A-ending codon content (AAA, CAA, GAA, AGA). Enriched pathways were clustered by GO Biological Process (2023). Cilium-associated terms are highlighted in orange. (i) Ribosome occupancy analysis of all mRNAs measured by ribosome profiling (f). Transcripts were ranked by A-ending codon frequency (AAA, CAA, GAA and AGA) and divided into ten equal bins (bin 1 = lowest, bin 10 = highest). A negative correlation was observed between A-ending codon frequency and ribosome abundance. ANOVA with post hoc test. Adjusted p-values are indicated. (j) Ciliogenesis assay in CTU2 -knockdown and wildtype RPE1 cells upon serum starvation. Representative immunofluorescence images show primary cilia (green) and nuclei (blue). The treatment scheme is shown above. Quantification of fractions of cilia-forming RPE1 cells under tRNA hypothiolation is provided. Two-tailed unpaired t-test, p-values are indicated. n = 3 technical replicates. Scale bars 10µm.

Article Snippet: For CTU2 knockdown lines we utilized the GLTR system, an all-in-one lentiviral, doxycycline inducible short hairpin RNA (shRNA) expression vector (Addgene 55790, 58246).

Techniques: Western Blot, Knockdown, shRNA, Control, Northern Blot, Biomarker Discovery, Staining, Over Expression, Two Tailed Test, Immunofluorescence

Journal: bioRxiv

Article Title: Precision RNAi in Tomato Using Synthetic Trans-Acting Small Interfering RNAs Derived From Minimal Precursors

doi: 10.1101/2025.04.10.648111

Figure Lengend Snippet: B/c -based vectors for direct cloning of syn-tasiRNAs downstream SlmiR482b target site (TS). ( A ) TS is in orange, the spacer sequence derived from AtTAS1c is in blue. Top, diagram of the Gateway-compatible entry vectors. Bottom, diagram of binary vector for in plant expression of syn-tasiRNAs. RB: right border; 35S: Cauliflower mosaic virus promoter; Bsa I: Bsa I recognition site, ccd B: gene encoding the gyrase toxin, in pink; LB: left border; attL1 and attL2: GATEWAY recombination sites. Kan R : kanamycin resistance gene; Hyg R : hygromycin resistance gene. ( B ) Organization of SlmiR482b-based syn-tasiRNA constructs. Base pairing between SlmiR482b (dark orange) and its target site (light orange) nucleotides is shown. Curved black arrows indicate DCL4 processing sites. Black linear arrows indicate sRNA-guided cleavage sites. TS refers to target site. In the example diagram, one single 21-nt guide syn-tasiRNA sequence was introduced at the 3’D2[+] position in SlmiR482bTS -based precursors.

Article Snippet: These new B/c vectors pENTR-SlmiR482bTS-B/c (Addgene plasmid #234368) and pMDC32B-SlmiR482bTS-B/c (Addgene plasmid #234369) were deposited in Addgene.

Techniques: Cloning, Sequencing, Derivative Assay, Plasmid Preparation, Expressing, Virus, Construct

Journal: bioRxiv

Article Title: Precision RNAi in Tomato Using Synthetic Trans-Acting Small Interfering RNAs Derived From Minimal Precursors

doi: 10.1101/2025.04.10.648111

Figure Lengend Snippet: Functional analysis of Solanum lycopersicum T1 transgenic lines expressing syn-tasiR-SlSFT, a syn-tasiRNA targeting SINGLE FLOWER TRUSS ( SlSFT ). ( A ) Schematic representation of the anti- SlSFT syn-tasiRNA construct, 35S:SlmiR482bTS-SlSFT , engineered to express syn-tasiR-SlSFT (light blue) from a minimal precursor containing the SlmiR482b target site (TS) (orange) and a 11-nt spacer derived from AtTAS1c (dark blue). Other details are as described in . ( B ) Photographs taken at 5 weeks post-transplanting (wpt) of representative transgenic tomato lines expressing anti- SlSFT syn-tasiRNA compared to a non-transgenic control (NTC) plant. Top panel: whole plants. Bottom panel: detail of the apical region, with arrows marking the first emerging inflorescence (I1) and the last leaf (L), numbered, before it. ( C ) Phenotypic analysis of flowering time in NTC and syn-tasiRNA transgenic lines, showing the number of leaves present at the time of the first emerging inflorescence in each plant. ( D ) Accumulation of SlSFT mRNA in tomato plants. Data are presented as the mean +SE relative expression levels of SlSFT mRNA at 12 wpt after normalization to ACTIN (SlACT), as determined by RT–qPCR (NTC=1 in all comparisons). The asterisk indicates a significant difference from the NTC samples (P<0.05; pairwise Student’s t -test comparison). The NTC sample corresponds to a pooled sample from five NTCs. ( E ) 5′-RLM-RACE analysis of syn-tasiR-SlSFT-guided cleavage of SlSFT . Upper panel: predicted base pairing between syn-tasiR-SlSFT and SlSFT mRNA, with the expected cleavage site indicated by an arrow. The proportion of cloned 5′-RLM-RACE products at the expected cleavage site is shown for syn-tasiR-SlSFT-expressing lines. Lower panel: ethidium bromide-stained gel showing 5′-RLM-RACE products corresponding to the 3′ cleavage product from syn-tasiR-SlSFT-guided cleavage (top), along with RT– PCR products for the target SlSFT (middle) and the control SlACT genes (bottom). The position and expected sizes of syn-tasiRNA-based 5′-RLM-RACE products and control RT-PCR products are indicated.

Article Snippet: These new B/c vectors pENTR-SlmiR482bTS-B/c (Addgene plasmid #234368) and pMDC32B-SlmiR482bTS-B/c (Addgene plasmid #234369) were deposited in Addgene.

Techniques: Functional Assay, Transgenic Assay, Expressing, Construct, Derivative Assay, Control, Quantitative RT-PCR, Comparison, Clone Assay, Staining, Reverse Transcription Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Precision RNAi in Tomato Using Synthetic Trans-Acting Small Interfering RNAs Derived From Minimal Precursors

doi: 10.1101/2025.04.10.648111

Figure Lengend Snippet: Accumulation and processing of syn-tasiR-SlSFT expressed from SlmiR482bTS -based precursors in Solanum lycopersicum T1 transgenic lines. ( A ) Northern blot detection of syn-tasiR-SlSFT in RNA preparations from apical leaves collected 12 weeks post-transplanting from six independent transgenic lines and one non-transgenic control (NTC). Each sample represents a pool of two apical leaves. Other details are as described in . ( B ) sRNA profile of 19-24 nt [+] reads mapping to each of the 54 nucleotide positions within the SlmiR482bTS-SlSFT precursor from plants expressing 35S:SlmiR482bTS-SlSFT . Orange, dark blue and light blue boxes represent nucleotides corresponding to NbmiR482aTS , the AtTAS1c -derived spacer and syn-tasiR-SlSFT, respectively. ( C ) Pie chart showing the percentage of reads corresponding to accurately processed 21-nt authentic syn-tasiR-SlSFT (blue) versus other 19-24-nt sRNAs (gray). ( D ) Radar plot displaying the distribution of 21-nt reads across the 21 registers of the precursor transcripts, with position 1 designated immediately after the SlmiR482b-guided cleavage site.

Article Snippet: These new B/c vectors pENTR-SlmiR482bTS-B/c (Addgene plasmid #234368) and pMDC32B-SlmiR482bTS-B/c (Addgene plasmid #234369) were deposited in Addgene.

Techniques: Transgenic Assay, Northern Blot, Control, Expressing, Derivative Assay

Journal: bioRxiv

Article Title: Precision RNAi in Tomato Using Synthetic Trans-Acting Small Interfering RNAs Derived From Minimal Precursors

doi: 10.1101/2025.04.10.648111

Figure Lengend Snippet: Functional analysis of potato virus X (PVX) constructs expressing syn-tasiRNAs against tomato spotted wilt virus (TSWV) in S. lycopersicum . ( A ) Schematic representation of PVX-based constructs. Nucleotides of anti-TSWV art-sRNA sequences syn-tasiR-TSWV-1, syn-tasiR-TSWV-2, syn-tasiR-TSWV-3 and syn-tasiR-TSWV-4 are in red, dark brown, light brown and yellow, respectively. Nucleotides of control anti-GUS art-sRNA sequences syn-tasiR-GUS Sl -1 and syn-tasiR-GUS Sl -2 are in dark and light grey, respectively. Nucleotides of AtmiR173a target site (TS) are in red and brown, respectively. Other details are as in . ( B ) Diagram of PVX-based constructs expressing anti-TSWV or anti- GUS syn-tasiRNAs. Color coding for the syn-tasiRNA sequences is consistent with panel (A). Other details are as in . ( C ) Two-dimensional line graph showing, for each of the six-plant sets listed, the percentage of symptomatic plants per day during 28 days. ( D ) Photographs taken at 14 days post-inoculation (dpi) of plants agroinoculated with the different constructs and inoculated (+TSWV) or not (mock) with TSWV. ( E ) Western blot detection of TSWV in protein extracts from apical leaves collected at 14 dpi. A Ponceau-stained membrane is shown as a loading control, highlighting the large subunit of Rubisco (ribulose1,5-biphosphate carboxylase/oxygenase). ( F ) RT-PCR detection at 14 dpi of SlmiR482bTS -based precursors and PVX coat protein fragment (PVX-CP) in apical leaves agroinoculated plants. RT-PCR amplification of SlTYP41 is included as a control. ( G ) Northern blot detection of anti-TSWV art-sRNAs in RNA preparations from apical leaves collected at 14 dpi. A cocktail of probes to simultaneously detect syn-tasiR-TSWV-1, syn-tasiR-TSWV-2, syn-tasiR-TSWV-3 and syn-tasiR-TSWV-4 was used. Other details are as in .

Article Snippet: These new B/c vectors pENTR-SlmiR482bTS-B/c (Addgene plasmid #234368) and pMDC32B-SlmiR482bTS-B/c (Addgene plasmid #234369) were deposited in Addgene.

Techniques: Functional Assay, Virus, Construct, Expressing, Control, Western Blot, Staining, Membrane, Reverse Transcription Polymerase Chain Reaction, Amplification, Northern Blot

Journal: bioRxiv

Article Title: Precision RNAi in Tomato Using Synthetic Trans-Acting Small Interfering RNAs Derived From Minimal Precursors

doi: 10.1101/2025.04.10.648111

Figure Lengend Snippet: Transgene-free gene silencing through PVX-based syn-tasiR-VIGS in Solanum lycopersicum . ( A ) Schematic representation of PVX-based constructs. Nucleotides of anti- SlSu syn-tasiRNAs (syn-tasiR-SlSu-1 and syn-tasiR-SlSu-2) are shown in dark and light blue, respectively. Nucleotides of anti- SlDXS syn-tasiRNAs (syn-tasiR-SlDXS-1 and syn-tasiR-SlDXS-2) are shown in dark and light green, respectively. Other details are as in and . ( B ) Diagram of PVX-based constructs expressing anti- SlSu , anti- SlDXS or anti- GUS syn-tasiRNAs. Color coding for the syn-tasiRNA sequences is consistent with panel (A). Other details are as in and . ( C ) Experimental procedure for transgene-free syn-tasiR-VIGS in S. lycopersicum . Left: crude extracts are prepared from Nicotiana benthamiana plants previously agroinfiltrated with the corresponding syn-tasiR-VIGS construct. Right: young tomato plants are spray-inoculated with syn-tasiR-VIGS crude extracts to induce bleaching associated with SlSu or SlDXS silencing. ( D ) Representative photographs of tomato leaves at 14 days post-spray (dps), from plants sprayed with different crude extracts obtained from agroinoculated N. benthamiana plants. ( E ) Accumulation of SlSu and SlDXS mRNA in tomato plants treated with syn-tasiR-VIGS crude extracts. Data are presented as the mean +SE relative expression levels of SlSu or SlDXS mRNA at 14 dps after normalization to ACTIN ( SlACT ), as determined by RT–qPCR (Mock=1 in all comparisons). The asterisk indicates a significant difference from the mock samples (P<0.05; pairwise Student’s t -test comparison). ( F ) RT-PCR detection at 14 dps of SlmiR482bTS -based precursors and PVX coat protein fragment (PVX-CP) in apical leaves of sprayed plants. RT-PCR amplification of SlACT is included as a control. ( G ) Northern blot detection of anti- SlSu and anti- SlDXS syn-tasiRNAs in RNA preparations from apical leaves collected at 14 dps. A cocktail of probes to simultaneously detect syn-tasiR-SlSu-1, syn-tasiR-SlSu-2, syn-tasiR-SlDXS-1 and syn-tasiR-SlDXS-2 was used. Other details are as described in .

Article Snippet: These new B/c vectors pENTR-SlmiR482bTS-B/c (Addgene plasmid #234368) and pMDC32B-SlmiR482bTS-B/c (Addgene plasmid #234369) were deposited in Addgene.

Techniques: Construct, Expressing, Quantitative RT-PCR, Comparison, Reverse Transcription Polymerase Chain Reaction, Amplification, Control, Northern Blot