Journal: Redox Biology

Article Title: LRPPRC regulates redox homeostasis via the circANKHD1/FOXM1 axis to enhance bladder urothelial carcinoma tumorigenesis

doi: 10.1016/j.redox.2021.102201

Figure Lengend Snippet: SLIRP forms a complex with LRPPRC to enhance its stability and to prevent LRPPRC from degradation. (A) LRPPRC interaction partners were detected in UM-UC-3 and T24 cells. Silver staining is used for the detection of differential protein bands. The band of LRPPRC and SLIRP are indicated by arrows. (B) Mass spectrometry identified SLIRP, which was pulled down from T24 cell lysates by LRPPRC (Top). The interaction between LRPPRC and SLIRP was confirmed by co-immunoprecipitation in T24 cells (Bottom). (C) Confocal staining presented the co-localization of LRPPRC and SLIRP in T24 cells. (D) Western blots revealed the regulatory relationship between LRPPRC and SLIRP. (E) siNC and siSLIRP were transfected in T24 cells, and 48 h later, the cells were treated with 10 μM of MG132 for various number of times and the cell lysates were immunoblotted as indicated. (F) T24-NC and T24-siSLIRP cells were treated with 50 μg/mL cycloheximide. Whole-cell lysates were harvested at the indicated times and the cell lysates were immunoblotted as indicated. (G) Flag-LRPPRC and HA-ubiquitin were transfected to T24-NC and T24-siSLIRP cells. The cells were treated with 10 μM MG132 for 8 h before harvest. Cell lysates were immunoprecipitated with Flag antibody and immunoblotted as indicated. (H) Colony formation assays show that knockdown of LRPPRC inhibited UCB cell proliferation capacity which was reversed by SLIRP overexpression. Error bars: mean ± SD of three independent experiments. *** P < 0.001; **** P < 0.0001.

Article Snippet: Immunoprecipitation (IP) of LRPPRC, SLIRP, or FLAG was performed using rabbit anti-human LRPPRC antibody (1:100, 21175-1-AP, Proteintech, Chicago, USA) or rabbit anti-human SLIRP antibody (1:100, 26006-1-AP, Proteintech, Chicago, USA) or mouse anti-human FLAG (1:100, 66008-2-Ig, Proteintech, Chicago, USA) at 4 °C for 2 h, followed by incubation with protein A/G magnetic beads (Thermo Fisher Scientific, Waltham, MA, USA) at 4 °C overnight and washing with lysis buffer.

Techniques: Silver Staining, Mass Spectrometry, Immunoprecipitation, Staining, Western Blot, Transfection, Ubiquitin Proteomics, Knockdown, Over Expression

Journal: Redox Biology

Article Title: LRPPRC regulates redox homeostasis via the circANKHD1/FOXM1 axis to enhance bladder urothelial carcinoma tumorigenesis

doi: 10.1016/j.redox.2021.102201

Figure Lengend Snippet: LRPPRC regulates the circANKHD1/FOXM1 axis to promote cell growth in UCB. (A–B) The expression of FOXM1 and PRDX3 decreased after the knockdown of LRPPRC and were restored by the overexpression of circANKHD1 in orthotopic xenograft bladder tumors. Representative images of hematoxylin and eosin staining and IHC staining of FOXM1 and PRDX3 of orthotopic xenograft bladder tumors. Scale bar, 100 μm (A). IHC score of FOXM1 (Left) or PRDX3 (Right) in orthotopic xenograft bladder tumors as indicated. Data shown are the means ± SD, n = 5 (B). (C–D) The expression level of LRPPRC was positively correlated with that of circANKHD1 and FOXM1, and the expression level of circANKHD1 was positively correlated with that of FOXM1 in UCBs of the SYSUCC cohort. Representative images of IHC staining of LRPPRC, FOXM1 and PRDX3 in two UCB tissues with low or high expression of the three proteins. Scale bar, 100 μm (C). Spearman's correlation demonstrating that LRPPRC expression is positively correlated with circANKHD1 (detected by qRT-PCR) and FOXM1 (D). (E) Proposed model for the regulatory landscape of the LRPPRC/SLIPR/circANKHD1/FOXM1 signaling axis in promoting the pathogenesis of UCB. SLIRP forms a stable complex with LRPPRC to protect it from degradation, LRPPRC modulates ROS balance and protects UCB cells from oxidative stress via mt-mRNA metabolism and the circANKHD1/FOXM1 axis, thereby resulting in UCB cell growth.

Article Snippet: Immunoprecipitation (IP) of LRPPRC, SLIRP, or FLAG was performed using rabbit anti-human LRPPRC antibody (1:100, 21175-1-AP, Proteintech, Chicago, USA) or rabbit anti-human SLIRP antibody (1:100, 26006-1-AP, Proteintech, Chicago, USA) or mouse anti-human FLAG (1:100, 66008-2-Ig, Proteintech, Chicago, USA) at 4 °C for 2 h, followed by incubation with protein A/G magnetic beads (Thermo Fisher Scientific, Waltham, MA, USA) at 4 °C overnight and washing with lysis buffer.

Techniques: Expressing, Knockdown, Over Expression, Staining, Immunohistochemistry, Quantitative RT-PCR

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