Journal: bioRxiv

Article Title: Ultra-sensitive FLORA-seq links cell-type-specific tRNAome dynamics to differentiation trajectories guiding therapeutic suppressor tRNA candidate selection

doi: 10.64898/2026.04.14.718460

Figure Lengend Snippet: a, Cloverleaf structure of tRNA with some modifications. m 1 A, 1-methyladenosine; m 1 G, 1-methylguanosine; acp 3 U, 3-(3-amino-3-carboxypropyl) uridine; m G, N , N -dimethylguanosine; m 3 C, 3-methylcytidine; ncm 5 U, 5-carbamoylmethyluridine; mcm 5 U, 5-methoxycarbonylmethyluridine; mcm 5 s U, 5-methoxycarbonylmethyl-2-thiouridine; I, inosine; i A, N -isopentenyladenosine; t A, N -threonylcarbamoyladenosine; m t A, N -methyl- N -threonylcarbamoyladenosine; ms t A, 2-methylthio- N -threonylcarbamoyladenosine; m 1 I, 1-methylinosine. b, Chemical structures of some tRNA modifications. c, The ligation efficiency of Poly(A) polymerase and T4 RNA ligase 2. Using an equal amount of total tRNA without any adapter ligation or poly(A) addition as a control, the ligation efficiency is calculated as the ratio of RNA molecules with successfully added adapters or poly(A) tails to those in the control. d, A pool of RNA oligonucleotides with identical sequences but varying 5’-terminal nucleotides (A, C, G, or U) was subjected to the template-switching reverse transcription library preparation under optimized conditions. The bar graph shows the proportion of each oligonucleotide recovered in the final sequencing libraries. e, Schematic overview illustrating the steps required for tRNA library preparation using FLORA-seq. f, Raw read coverage of mito_tRNA Leu (TAA) and cyto_tRNA Leu (AAG) from 20 HEK 293T. g, Raw read coverage of mito_tRNA Leu (TAG) and cyto_tRNA Leu (TAG) from 5 mouse morulae. h, Raw read coverage of 3’ and 5’tdR from 5 mouse morulae. i, Raw read coverage of internal tdRs from 5 mouse morulae. j, Pie chart illustrating the distribution of distinct RNA species identified from non-ribosomal genome-aligned reads in mouse morula sequencing data. Reads with mapped lengths below 15 bp were excluded from the analysis.

Article Snippet: The ligation reaction was performed in a 20 μL reaction containing 1 × T4 RNA ligase buffer, 200 ng tRNA, 10 μM pre-adenylated DNA adapter (Synthesized by Novazyme), and 200 U of T4 RNA ligase 2 (NEB) at 25°C for 2 h

Techniques: Ligation, Adapter Ligation, Control, Reverse Transcription, Sequencing

Journal: bioRxiv

Article Title: Structural basis for substrate recruitment by AMBRA1 E3 ligase receptor

doi: 10.1101/2022.12.04.519012

Figure Lengend Snippet: a. Annotated DDB1 and AMBRA1 domain schematics. AMBRA1 WD40 construct for cryo-EM was defined. BPA-BPC, β -propeller A-C; CTD, C-terminal helical domain; FL, full length; IDRs, intrinsically disordered regions. b. The coomassie blue stained SDS-PAGE analysis of purified AMBRA1 FL alone and AMBRA1 FL -DDB1-Cullin4A-RBX1 complex which were used for HDX-MS measurements. TSF, twin-strep-flag. c. Plot representing deuterium uptake by full length AMBRA1, with each data point representing the calculated central residue of an individual peptide. The Y-axis represents % deuteration for a given peptide, at each time point. HDX data statistics is given in . d. Isotopic envelopes for the selected peptides from AMBRA1, either alone or in E3 ligase complex following 3 s of incubation in D 2 O.

Article Snippet: The ubiquitination assays were performed in a 25 μl reaction volume with the following components: 100 nM UBE1 (Boston Biochem E-304), 1.5 μM UBCH5C (Boston Biochem E2-627), 0.3 μM purified AMBRA1-DDB1-Cullin4-RBX1 complex, 20 μM HA-ubiquitin (Boston Biochem U-110), 0.16 μM myc-Cyclin D1-CDK4 complex and 10 mM MgATP solution (Boston Biochem B-20) in E3 ligase reaction buffer (Boston Biochem B-71).

Techniques: Construct, Cryo-EM Sample Prep, Staining, SDS Page, Purification, Residue, Incubation

Journal: bioRxiv

Article Title: Structural basis for substrate recruitment by AMBRA1 E3 ligase receptor

doi: 10.1101/2022.12.04.519012

Figure Lengend Snippet: Bimodal isotopic envelopes for the AMBRA1 N-terminal helix (peptide spanning residues 2-28), either alone or in E3 ligase complex. Deuteration time points are indicated. Close to open transition kinetics for AMBRA1 helix-loop-helix motif is calculated by fit of two gaussians to the high and low mass subpopulation. Relative amount of the open state is plotted against deuteration time. Structure of the AMBRA1-DDB1 complex interface showing the peptide spanning residues 2-28.

Article Snippet: The ubiquitination assays were performed in a 25 μl reaction volume with the following components: 100 nM UBE1 (Boston Biochem E-304), 1.5 μM UBCH5C (Boston Biochem E2-627), 0.3 μM purified AMBRA1-DDB1-Cullin4-RBX1 complex, 20 μM HA-ubiquitin (Boston Biochem U-110), 0.16 μM myc-Cyclin D1-CDK4 complex and 10 mM MgATP solution (Boston Biochem B-20) in E3 ligase reaction buffer (Boston Biochem B-71).

Techniques:

Journal: bioRxiv

Article Title: Structural basis for substrate recruitment by AMBRA1 E3 ligase receptor

doi: 10.1101/2022.12.04.519012

Figure Lengend Snippet: a. The size exclusion profile (Superdex 200 Increase 10/300) and the coomassie blue stained SDS-PAGE analysis of the purified protein Cullin4A-DDB1-RBX1(E3) ligase which were used for in vitro ubiquitination assay. b. The size exclusion profile (Superose 6 Increase 10/300) and the coomassie blue stained SDS-PAGE analysis of the DDB1 which was used for MBP pull down assay. c. The coomassie blue stained SDS-PAGE analysis of the WT/truncated/mutated AMBRA1. d. The coomassie blue stained SDS-PAGE analysis of the Cyclin D1-CDK4 complex.

Article Snippet: The ubiquitination assays were performed in a 25 μl reaction volume with the following components: 100 nM UBE1 (Boston Biochem E-304), 1.5 μM UBCH5C (Boston Biochem E2-627), 0.3 μM purified AMBRA1-DDB1-Cullin4-RBX1 complex, 20 μM HA-ubiquitin (Boston Biochem U-110), 0.16 μM myc-Cyclin D1-CDK4 complex and 10 mM MgATP solution (Boston Biochem B-20) in E3 ligase reaction buffer (Boston Biochem B-71).

Techniques: Staining, SDS Page, Purification, In Vitro, Ubiquitin Assay, Pull Down Assay