Journal: Nature Communications

Article Title: Structures and mechanisms of U6 snRNA m 6 A modification by METTL16

doi: 10.1038/s41467-025-63021-0

Figure Lengend Snippet: a Schematic diagram of S. pombe METTL16 (spMETTL16) compared with human METTL16 (hsMETTL16) and C. elegans METT10 (ceMETT10). The N-terminal methyltransferase domain (spMTD, magenta) and the C-terminal KA-1 domain (spKA-1, cyan) of S. pombe , used in the experiments, are depicted. b In vitro methylation of the S. pombe U6 snRNA (spU6 snRNA) transcript by spMETTL16 and spMTD under standard conditions. spU6 snRNA (0.5 μM) was incubated with 200 nM spMETTL16 or spMTD in the presence of 1 mM SAM for 4 min at 37 °C. Error bars represent the standard deviation (SD) of three independent experiments ( N = 3), and the center of the error bands indicates the mean of the measured values. c Steady-state kinetics of methylation of the spU6 snRNA transcript by spMETTL16 and spMTD. spU6 snRNA (0–10 μM) was incubated with 200 nM spMETTL16 or spMTD in the presence of 1 mM SAM at 37 °C. All experiments were independently repeated three times with similar results. d , e Semi-quantitative RT-PCR analyses of two introns: d SPAC18B11.09 C and e ckn1 . The nucleotide sequences of the respective 5′ splice sites (−3 to +4) are shown. The upper and lower bands on the gel represent retained and spliced introns, respectively. Intron retention in the Δ mtl16 strain was rescued by ectopic expression of plasmid-encoded full-length METTL16 (spMETTL16), but not by the methyltransferase domain (spMTD) alone. All experiments were independently repeated three times with similar results (Supplementary Fig. ). f Nucleotide sequence of S. pombe U6 snRNA (spU6 snRNA, left). The nucleotide sequence of the spISL used for crystallization of the spKA-1–spISL complex (spISL, right). g Gel retardation assay of spISL by spKA-1. spISL RNAs were incubated with various concentrations of spKA-1 (0–10 μM). The fractions of the shifted RNA in the gel (left) were quantified (right). The experiments were independently repeated two times with similar results. Source data for b , c , g are provided as a file.

Article Snippet: Synthetic S. pombe U6 snRNA internal stem-loop (spISL) RNA was purchased from Fasmac, Japan.

Techniques: In Vitro, Methylation, Incubation, Standard Deviation, Quantitative RT-PCR, Expressing, Plasmid Preparation, Sequencing, Crystallization Assay, Electrophoretic Mobility Shift Assay

Journal: Nature Communications

Article Title: Structures and mechanisms of U6 snRNA m 6 A modification by METTL16

doi: 10.1038/s41467-025-63021-0

Figure Lengend Snippet: a – d Detailed views of the interactions between spKA-1 and spISL. The colors for nucleosides are the same as in Fig. . e In vitro methylation of the spU6 snRNA transcript by spMETTL16 and its variants with mutations in the KA-1 domain under standard conditions. spU6 snRNA (0.5 μM) was incubated with 200 nM spMETTL16 or its variants in the presence of 1 mM SAM for 4 min at 37 °C. Error bars represent the standard deviation (SD) of three independent experiments ( N = 3), and the center of the error bands indicates the mean of the measured values. Source data are provided as a file. Semi-quantitative RT-PCR analyses of two introns: f SPAC18B11.09 C and g ckn1 . The upper and lower bands on the gel represent retained and spliced introns, respectively. Intron retention in the Δ mtl16 strain was rescued by ectopic expression of plasmid-encoded full-length METTL16 (spMETTL16), but not by the spMETTL16 variants with mutations in the KA-1 domain shown in ( e ) (Supplementary Fig. ).

Article Snippet: Synthetic S. pombe U6 snRNA internal stem-loop (spISL) RNA was purchased from Fasmac, Japan.

Techniques: In Vitro, Methylation, Incubation, Standard Deviation, Quantitative RT-PCR, Expressing, Plasmid Preparation

Journal: Nature Communications

Article Title: Structures and mechanisms of U6 snRNA m 6 A modification by METTL16

doi: 10.1038/s41467-025-63021-0

Figure Lengend Snippet: a Cryo-EM map (left) and model (right) of the spMETTL16–spU6 snRNA complex in the absence of SAM (spMETTL16–spU6). MTD, KA-1 domain, and modeled RNA are colored magenta, blue and orange, respectively. The N-terminal extension (residues 1–61) and the RNA binding loop (residues 172–200) are not modeled, and only nucleotides 41–82 of spU6 snRNA are modeled. b Cryo-EM map (left) and model (right) of the spMETTL16–spU6 snRNA complex in the presence of SAM (spMETTL16–SAM–spU6). MTD, KA-1 domain, and modeled RNA are colored as in ( a ). The N-terminal extension and the RNA binding loop (residues 187–194) are not modeled, and only nucleotides 35–83 of spU6 snRNA are modeled. c Structural comparison between spMETTL16–spU6 (gray) and spMETTL16–SAM–spU6. In the presence of SAM, α4 in the MTD becomes resolved, and the ACA*GAGA motif in spU6 snRNA shifts toward the catalytic site of the MTD. d The secondary structure of spMETTL16. The MTD and KA-1 domain are colored magenta and blue, respectively. The regions enclosed by the dashed lines were not visible in the spMETTL16–spU6 structure. α4 was visible in the spMETTL16–SAM–spU6 complex. The N-terminal extension was predicted by AlphaFold2 (Data base: AF- O42662 -F1-v4) .

Article Snippet: Synthetic S. pombe U6 snRNA internal stem-loop (spISL) RNA was purchased from Fasmac, Japan.

Techniques: Cryo-EM Sample Prep, RNA Binding Assay, Comparison

Journal: Nature Communications

Article Title: Structures and mechanisms of U6 snRNA m 6 A modification by METTL16

doi: 10.1038/s41467-025-63021-0

Figure Lengend Snippet: a Nucleotide sequences of human MAT2A hairpin (MAT2A-hp) and S. pombe U6 snRNA and its variants used for the assays in ( b ). b In vitro methylation of spU6 snRNA and its variants by spMETTL16 under standard conditions. spU6 snRNA or its variants (0.5 μM) was incubated with 200 nM spMETTL16 in the presence of 1 mM SAM for 4 min at 37 °C. Error bars represent the standard deviation (SD) of three independent experiments ( N = 3), and the center of the error bands indicates the mean of the measured values. c Superimposition of the spMETTL16–SAM–spU6 structure onto the human METTL16 MTD (hsMETTL16_MTD, gray) in complex with MAT2A hairpin RNA (MAT2A-hp: gray, PDB ID: 6DU4) . The regions corresponding to the red regions (N-terminal extension and RNA binding loop), enclosed by dashed lines, in hsMETTL16_MTD were not visible in the present spMETTL16–SAM–spU6 structure. d Superimposition of the structure of the catalytic pocket of spMETTL16–SAM–spU6 and that of hsMETTL16_MTD–MAT2A-hp (gray). A17 in MAT2A-hp in the hsMETTL16–MAT2A-hp complex is deeply docked in the pocket and closer to SAM (cyan) than A37 in spU6 snRNA in the spMETTL16–SAM–spU6 complex. e In vitro methylation of the spU6 snRNA transcript by spMETTL16 and its N-terminal extension deletion mutant (Δ_NTE: amino acids 1–50 were deleted) and RNA binding loop deletion mutant (Δ_RBL: amino acid 186–189 were deleted), under standard conditions as in Fig. . Error bars represent the standard deviation (SD) of three independent experiments ( N = 3), and the center of the error bands indicates the mean of the measured values. Source data for b , e are provided as a file.

Article Snippet: Synthetic S. pombe U6 snRNA internal stem-loop (spISL) RNA was purchased from Fasmac, Japan.

Techniques: In Vitro, Methylation, Incubation, Standard Deviation, RNA Binding Assay, Mutagenesis

Journal: Nature Communications

Article Title: Structures and mechanisms of U6 snRNA m 6 A modification by METTL16

doi: 10.1038/s41467-025-63021-0

Figure Lengend Snippet: a Initial binding stage: U6 snRNA initially binds to METTL16 through interactions between the ISL and KA-1 domain (blue). The ACA*GAGA motif, which contains the methylation site A37 in U6 snRNA, is highlighted in red. b Pre-productive stage: SAM (cyan) binding to the MTD (magenta) triggers a conformational change in the MTD, shifting the ACA*GAGA motif of U6 snRNA closer to the catalytic site via the RNA-binding loop. At this stage, the motif is specifically recognized. c Productive stage: The interactions of the N-terminal extension (NTE) and the RNA-binding loop with U6 snRNA promote the transition to the productive stage. U6 snRNA undergoes structural rearrangements, adopting productive conformation. This process leads to the formation of the transition region and base-pairing within the telestem in U6 snRNA, shifting A37 deeper into the catalytic site, and thereby facilitating efficient m 6 A modification by MTD.

Article Snippet: Synthetic S. pombe U6 snRNA internal stem-loop (spISL) RNA was purchased from Fasmac, Japan.

Techniques: Binding Assay, Methylation, RNA Binding Assay, Modification

Journal: Nucleic Acids Research

Article Title: Assembly of SARS-CoV-2 nucleocapsid protein with nucleic acid

doi: 10.1093/nar/gkae256

Figure Lengend Snippet: Schematic organization of N-protein and hypothetical architectures of NA complexes. (A) Organization of N-protein chain with folded domains NTD (green cylinder) and CTD (blue square), and the intrinsically disordered N-arm, C-arm, and linker, the latter containing the transient helix in the leucine rich sequence (LRS) capable of oligomerization (cylinder). (B) N-protein in solution is a dimer linked with high affinity at the CTD. (C) Occupation of NA binding sites in the NTD induces folding in the LRS and causes compaction (magenta) and LRS oligomerization in dimers, trimers, tetramers, and higher oligomers. (D) Configuration of two N-protein dimers independently scaffolded on NA T40 (red bar) without further LRS oligomerization. (E) Two N-protein dimers scaffolded on NA and stabilized through LRS oligomerization. (F) Similar to (E) with crosslink between NA strands allowing the formation of higher oligomers. (G) N-protein dimer with two SL7 stem–loop RNA ({2N/2SL7}, grey), depicted in alternate configurations occupying all major NA interfaces in the NTD and CTD creating different intra-dimer crosslinks. (H) Possible architecture of N-protein/stem–loop complexes allowing {2N/2SL7} units to oligomerize via LRS and simultaneous multivalent binding of SL7 in inter-dimer crosslinks. Dotted lines belong to neighboring {2N/2SL7} units not fully drawn. (I) The top view of a 6x{2N/2SL7} hexamer of dimers. The dashed lines indicates two levels of inter-dimer crosslinks of neighboring {2N/2SL7} subunits, via contacts of the LRS interfaces (dark red dashed) and via multivalent RNA binding of CTD and/or scaffolding of NTD (light red dashed).

Article Snippet: The oligonucleotides T 40 , U 40 and stem–loop RNA SL7 were purchased from Integrated DNA Technologies (Skokie, IL) and purified by HPLC and lyophilized by the vendor.

Techniques: Sequencing, Binding Assay, RNA Binding Assay, Scaffolding

Journal: Nucleic Acids Research

Article Title: Assembly of SARS-CoV-2 nucleocapsid protein with nucleic acid

doi: 10.1093/nar/gkae256

Figure Lengend Snippet: Complex mass distributions of NWT with T40, U40 and SL7 in mass photometry. Shown are mass histograms of NWT with T40 (A), U40 (B) and SL7 (C). The inset in (C) shows peak mass values vs peak number of the 0.25 μM NWT with 0.3 μM SL7 mixture and linear fit leading to a mass increment of 118 kDa.

Article Snippet: The oligonucleotides T 40 , U 40 and stem–loop RNA SL7 were purchased from Integrated DNA Technologies (Skokie, IL) and purified by HPLC and lyophilized by the vendor.

Techniques:

Journal: Nucleic Acids Research

Article Title: Assembly of SARS-CoV-2 nucleocapsid protein with nucleic acid

doi: 10.1093/nar/gkae256

Figure Lengend Snippet: Concentration-dependent RNP assembly of NWT with stem–loop SL7. (A) Sedimentation coefficient distributions from SV-AUC experiments recorded at 260 nm. Mixtures of NWT and SL7 at concentrations indicated in B65K (or B75Na for the two most concentrated mixtures). (B) Autocorrelation data from DLS of the highest concentration mixture. The best-fit single-species model leads to a diffusion coefficient of 2.466 × 10−7 cm2/s or a Stokes radius of 8.5 nm.

Article Snippet: The oligonucleotides T 40 , U 40 and stem–loop RNA SL7 were purchased from Integrated DNA Technologies (Skokie, IL) and purified by HPLC and lyophilized by the vendor.

Techniques: Concentration Assay, Sedimentation, Diffusion-based Assay

Journal: Nucleic Acids Research

Article Title: Assembly of SARS-CoV-2 nucleocapsid protein with nucleic acid

doi: 10.1093/nar/gkae256

Figure Lengend Snippet: RNP formation of N-protein with SL7 depends on LRS oligomerization in vitro and in viral assembly. (A) Mass distributions from MP of mixtures of 0.25 μM N-protein with 0.3 μM SL7 in moderate ionic strength buffer B65K for NWT and the mutants inhibiting LRS oligomerization N:L222P and N:L222P/R226P. (B) Sedimentation coefficient distributions from SV-AUC experiments at 2.5 μM N-protein with 3.0 μM SL7 in buffer B65K recorded at 260 nm. Shown are results with NWT, N:L222P and N:L222P/R226P.

Article Snippet: The oligonucleotides T 40 , U 40 and stem–loop RNA SL7 were purchased from Integrated DNA Technologies (Skokie, IL) and purified by HPLC and lyophilized by the vendor.

Techniques: In Vitro, Sedimentation

Journal: Nucleic Acids Research

Article Title: Assembly of SARS-CoV-2 nucleocapsid protein with nucleic acid

doi: 10.1093/nar/gkae256

Figure Lengend Snippet: NTD and CTD binding to T40, U40 and SL7. Sedimentation coefficient distributions c(s) from SV-AUC of NTD (A) or CTD (B) alone and in mixtures with T40, U40 or SL7, respectively. Mixture experiments are carried out in low ionic strength buffer B10Na to promote formation of the complexes with maximum stoichiometry. Distributions of free NA are reduced by a factor 2. For NTD alone, the molecular weight determined from c(s) analysis and best-fit frictional ratio is 15.1 kDa, which compares well to the theoretically expected value of 15.2 kDa. For CTD alone, the experimental molecular weight is 27.3 kDa, which compares to the theoretical value of 26.6 kDa for a CTD dimer.

Article Snippet: The oligonucleotides T 40 , U 40 and stem–loop RNA SL7 were purchased from Integrated DNA Technologies (Skokie, IL) and purified by HPLC and lyophilized by the vendor.

Techniques: Binding Assay, Sedimentation, Molecular Weight