Fig 2: Three dimensional modeling of a putative ternary complex, showing shEL86 cleavage product binding simultaneously to Dicer and TRBP dsRBD12 (Left) Spatial arrangement of Dicer's RNase III (yellow, PDB id 3C4B; Du et al, 2008) and PAZ-Platform (cyan, PDB id 4NHA; Tian et al, 2014) domains resulting from their docking onto EL86 (black). Each domain interacts with a distinct RNA region without any steric clash. (Right) Side-view showing the proximity of Dicer's dsRBD (gold) with TRBP dsRBD2 (red). dsRBD1 is shown in blue.The distance between TRBP dsRBD2 and dsRBD3 was estimated by docking within the EM envelope of apo-Dicer (EMD-5601; Taylor et al, 2013) our 3D model for the dsRBD12-EL86-PAZ-Platform-RNase III, RIG-I domains 1,3 (PDB id 4A36; Kowalinski et al, 2011) and TRBP-dsRBD3-Dicer-PBD (PDB id 4WYQ; Wilson et al, 2015). Orthogonal views are shown.

Fig 3: Dicing assay: shEL86 cleavage by Dicer Secondary structure of shEL86. The 5′ and 3′ arms are colored blue and red, respectively. The 32P‐radiolabel incorporated at the 5′ end is indicated by a blue asterisk. Dicer's cleavage sites experimentally determined are indicated by black arrows. Gray arrows indicate Dicer's cleavage sites inferred from the cleavage sites positions on the 5′‐arm.(Left) Representative denaturing polyacrylamide gel showing uncleaved RNA substrate (57 nucleotides) and two cleavage products of 21 and 22 nucleotides. Lane 1: negative control in the absence of Dicer; lanes 2–9: Dicer cleavage in the presence of increasing concentration of dsRBD12 (0, 20, 50, 100, 500, 200, 1,000, 2,000 fmol). (Right) Quantification of shEL86 cleavage for increasing dsRBD12 concentrations, calculated as 100× RNA‐product: total‐RNA. Each datapoint represents the average ± SD of three experimental replicates. The cleavage yields in the absence and in the presence of dsRBD12 were compared with a Student's t‐test. The asterisk denotes a P‐value of < 0.05.

Fig 4: BCDIN3D regulates tRNAHis 3’ fragment processing.A. Dicer assay with 5’P and 5’Pme2 tRNAHis using 0, 1, 2 and 4 μL of human Dicer at 1 μg/μL. Note that the ladder is the ss20 ssDNA Ladder and its migration is offset by 10–20 nt compared to RNA. The graph on the right shows the quantification by ImageQuant of the indicated bands. B. RNAs from two independent in vitro Dicer assays using mock or 2 μl of human Dicer at 1 μg/μl and 20 pmol of tRNAHis-5’P or -5’Pme2 were sequenced by TGIRT-seq. The bars represent mean ± SD (n = 2) of the ratio of normalized full length tRNAHis reads (>73 nt) over the mock control (**, p-value of ~0.0095). C. The graph represents mean ± SD (n = 2) of the ratio of normalized tRNAHis reads ending at the indicated position. The deduced Dicer cuts are indicated on the diagram of tRNAHis and the sequence of tRNAHis aligned to hsa-miR-4454. The red and grey arrows indicate the position of Dicer cuts that are sensitive to tRNAHis phospho-methylation. D. The graph on the left shows the RTqPCR analysis of the BCDIN3D mRNA normalized to ALAS1 and B2M, and the images on the right show the western blot analysis of the BCDIN3D protein from MDA-MB-231shNC and shBCDIN3D (shB3D) cells. E. Average ± SEM (n = 2) of tRNAHis 3’ fragment reads normalized to total mapped reads relative to shNC. F. Analysis of tRNAHis aminoacylation in MDA-MB-231shNC and shBCDIN3D cells. 1 μg of RNAs purified under acidic conditions, which preserve the aminoacyl-tRNA ester bond, was analyzed by acidic gel and northern blot. As a control, 1 μg of RNA was deacylated in vitro prior to gel migration. The slower migrating band corresponds to the aminoacylated tRNAHis (charged), while the faster migrating band corresponds to the un-aminoacylated tRNAHis (uncharged). G. Quantification of acidic northern blot with tRNAHis probe from 2 biological replicates, including the acidic northern blot in Fig 4F. The graph shows average ± SEM (n = 2). The total levels of tRNAHis are normalized to shNC, and the aminoacylation ratios of tRNAHis are calculated as the ratio of the signal of the aminoacylated band over the sum of both bands.

Fig 5: RNA secondary structure of SARS-CoV-2 miR-O7a precursor and sequence conservation among different SARS coronaviruses and within SARS-CoV-2 variants Predicted RNA secondary structure for the CoV2-miR-O7a and flanking sequence using the first 70 nt of the open reading frame of the ORF7a. The arrows indicate the sites of the miRNAs possibly cleaved by Dicer. The stem–loop structure is not conserved in SARS-CoV. The colors indicate the base pair probabilities.Conservation of the first 70 nt of the ORF7a sequence among different SARS coronaviruses. The underlined sequences are related to the position of the SARS-CoV-2 miR-O7a. The conserved ribonucleotides of the CoV2-miR-O7a sequence are marked in red and in blue all the non-conserved ribonucleotides across the 70nt sequence. The bat and pangolin coronaviruses closely related to SARS-CoV-2 are marked in purple.Percentage of conservation along the nucleotide positions in the ORF7a among 4,055,609 sequenced SARS-CoV-2 genomes. The first 70 nt are shown in red and show a higher percentage of conservation compared to the rest of the sequence of ORF7a. Boxplot shows the distribution of conservation percentage for each nucleotide either in the first 70 nt or 71–366 nt of ORF7a among 4,055,609 sequenced SARS-CoV-2 genomes. Box plots display median (line), first and third quartiles (box), and 5th /95th percentile value (whiskers). Each dot represents the outliers. Two-tailed P values were calculated using Student’s t-test.