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: Nucleic acids research

Article Title: 2'-O-Methylation of the second transcribed nucleotide within the mRNA 5' cap impacts the protein production level in a cell-specific manner and contributes to RNA immune evasion.

doi: 10.1093/nar/gkac722

Figure Lengend Snippet: Figure 1. Tetranucleotide cap analogues act as initiators of in vitro transcription reactions. (A) Structure of the cap analogues used in this study; newly synthesized tetranucleotide and previously obtained trinucleotide (14) cap analogues are presented on the left and right side, respectively. (B) Comparison of the major transcription initiation events during the in vitro transcription reaction when either no cap analogue, a trinucleotide cap analogue or a tetranucleotide cap analogue was used as an initiator. Capped RNA obtained in the in vitro transcription reaction with a tri- or tetranucleotide cap analogue is 27 nt long, as uncapped RNA is 25 nt long. (C) Analysis of short RNAs obtained by in vitro transcription using T7 RNA polymerase in the presence of different cap analogues (DNAzyme-trimmed and HPLC-purified transcripts). The capping efficiency values (percentage) determined by densitometric quantification of the major bands corresponding to capped and uncapped RNA are shown at the top of the gel. Minor extra bands most probably arise from unspecific addition of nucleotides during in vitro transcription.

Article Snippet: Biotinylated pAp (pAp N6-PEG-biot) (22) was ligated to RNAs by T4 RNA ligase 1 (M0204, New England Biolabs).

Techniques: Analogues, In Vitro, Synthesized, Comparison, Purification

Journal: Nucleic acids research

Article Title: 2'-O-Methylation of the second transcribed nucleotide within the mRNA 5' cap impacts the protein production level in a cell-specific manner and contributes to RNA immune evasion.

doi: 10.1093/nar/gkac722

Figure Lengend Snippet: Figure 3. 2′-O-Methylation within the cap structure does not influence RNA affinity for the translational machinery. (A) Relative affinities of transcripts bearing different cap analogues for murine eIF4E determined using MST. Bars represent the mean value ± SEM from three independent replicates. Statistical significance: **P <0.01 (one-way ANOVA with Turkey’s multiple comparisons test). (Representative MST curves and competitive binding curves obtained in the experiment are presented in Supplementary Figure S8.) (B) Comparison of apparent binding constant values KD,app for capped RNAs in complexes with murine eIF4E measured with MST with dissociation constants of eIF4E–cap complexes obtained using time-synchronized fluorescence quenching titration (ts-FQT) (14). For all RNAs, three independent replicates were performed, besides cap2(A) and cap2-1(A) RNAs, for which data from two replicates are presented. (C) Competition of differently capped IVT mRNAs encoding Gaussia luciferase with endogenous mRNAs for the translation machinery. HEK 293 Flp-In T-REx cells expressing shmiRs targeting CMTR1, CMTR2 or both (a negative control was utilized in parallel) were transfected with IVT mRNAs, and medium was collected after 72 h for luciferase activity analysis. Bars represent the mean value ± SEM normalized to Gaussia luciferase activity measured for transcripts with cap0(A). Data for three independent experiments are presented (each biological replicate consisted of three transfections).

Article Snippet: Biotinylated pAp (pAp N6-PEG-biot) (22) was ligated to RNAs by T4 RNA ligase 1 (M0204, New England Biolabs).

Techniques: Methylation, Analogues, Binding Assay, Comparison, Fluorescence, Titration, Luciferase, Expressing, Negative Control, Transfection, Activity Assay

Journal: Nucleic acids research

Article Title: 2'-O-Methylation of the second transcribed nucleotide within the mRNA 5' cap impacts the protein production level in a cell-specific manner and contributes to RNA immune evasion.

doi: 10.1093/nar/gkac722

Figure Lengend Snippet: Figure 4. 2′-O-Methylation of the second transcribed nucleotide prevents RNA from decapping by DXO but not by DCP2. (A–C) Short capped RNAs were subjected to treatment with hDCP2 (wild type or mutant) over a 60 min time-course. Reactions without enzyme served as controls. Aliquots from the indicated time points were resolved on a polyacrylamide gel and bands corresponding to capped transcripts (27 nt long) and to RNAs decapped by hDCP2 action (26 nt long) were quantified densitometrically. (A) Representative polyacrylamide gel analyses obtained for all tested capped RNAs (two additional repetitions with wild-type hDCP2 of this experiment are shown in Supplementary Figure S12). (B and C) Quantitative results for all studied RNAs. The fraction of capped RNA remaining in the total RNA was plotted as a function of time. Data points represent mean values ± SD from triplicate experiments. (D) Short capped RNAs were subjected to treatment with hDXO (wild type or mutant) over a 60 min time-course. Reactions without enzyme served as controls. Aliquots from the indicated time points were resolved on a polyacrylamide gel. (E) Experimental set-up as in (D); however, a 2.5-fold higher hDXO concentration was used.

Article Snippet: Biotinylated pAp (pAp N6-PEG-biot) (22) was ligated to RNAs by T4 RNA ligase 1 (M0204, New England Biolabs).

Techniques: Methylation, Mutagenesis, Concentration Assay