Fig 1: A RLRKGR motif on PD-L1 protects TβRI mRNA from degradation by competing with the RNA exosome complex(A) EXOSC10 was knocked down by two different shRNAs and the cells were collected for qRT-PCR (upper) and WB (lower) for TβRI. Knockdown of EXOSC10 led to increases of TβRI mRNA and protein level of HSCs. *p < 0.05, **p < 0.01 by ANOVA, n = 3.(B) Left: control HSCs, HSCs with PD-L1 knockdown, EXOSC10 knockdown, or knockdown of both were collected for qRT-PCR and WB for TβRI. EXOSC10 knockdown rescued TβRI mRNA and TβRI protein of PD-L1 knockdown HSCs. ***p < 0.001 by ANOVA, n = 3. Right: the stability of TβRI mRNA was assessed in the presence of Actinomycin D. PD-L1 knockdown accelerated the degradation of TβRI mRNA in HSCs and this effect was abrogated by knockdown of EXOSC10. *p < 0.05 by ANOVA, n = 3.(C) RIP assay revealed that knockdown of PD-L1 led to increased binding of TβRI mRNA to EXOSC10 in HSCs. ***p < 0.001 by ANOVA, n = 3.(D) RIP assay revealed that PD-L1 FL and PD-L1 T + C competed off EXOSC10/TβRI mRNA binding promoted by PD-L1 knockdown in HSCs. ***p < 0.001 by ANOVA, n = 3.(E) Left: the RLRKGR motif on PD-L1 T + C was changed to ALAAGA (PD-L1 T + C [4A]) or ALAAGR (PD-L1 T + C [3A]). RIP assay revealed that both mutants abrogated PD-L1 T + C/TβRI mRNA binding in HSCs. ***p < 0.001 by ANOVA, n = 5. Right: qRT-PCR and WB revealed that both mutants failed to rescue TβRI mRNA and TβRI protein of PD-L1 knockdown HSCs compared with wild-type PD-L1 T + C. ***p < 0.001 by ANOVA, n = 4.(F) RIP assay showed that PD-L1 T + C competed off EXOSC10/TβRI mRNA binding in PD-L1 knockdown HSCs, and this effect was abrogated by either mutant. **p < 0.01, ***p < 0.001 by ANOVA, n = 3. All data are represented as mean ± SEM.
Fig 2: Scan for conserved D‐box and Catalog of Somatic Mutations in Cancer (COSMIC) mutations. (A) The human EXOSC10 protein sequence (UniProt Q01780) is shown. Three matches to the extended D‐box are highlighted in yellow (top). Aligned sequences of the matches are shown and conserved amino acids are marked in yellow (bottom). (B) A multiple sequence alignment (MSA) done with the EBI's MUSCLE software of yeast (UniProt Q12149) and human (UniProt Q01780) proteins is given. Conserved (*) and similar (:) amino acids are indicated at the bottom. (C) A phylogenetic tree of eight Rrp6/EXOSC10 orthologs created by the EBI's MUSCLE software is shown. (D) An MSA of D‐box matches in eight orthologs named via their UniProt unique identifiers is shown. Amino acids critical for D‐box function are highlighted in blue; S402 is highlighted in green; amino acids within the match are highlighted in yellow. (E) Data for EXOSC10 from COSMIC are given. (F) An EXOSC10 protein structure prediction from AlphaFold is shown (AF:Q01780). A red box marks the helix and the position of serine 402 (S402). An enlarged image of the helix is given and the position of S402 is indicated.
Fig 3: S402 position and atomic interactions within EXOSC10 and phenotypic analysis of Exosc10 S402T. (A) A schematic shows the positions of conserved amino acids (interpro) and the corresponding missense mutations identified in the human population (gnomad). (B) Atomic interactions of serine, threonine, alanine, and proline at Position 402 created using dynamut are shown. The target amino acid is given in light green. Dotted lines symbolize interactions. (C) The structure of EXOSC10's catalytic subunit provided by Protein Databank is shown. A red box marks the position of S402. An enlarged image shows the interactions of S402 with neighboring residues. (D) A flow chart shows the experimental approach to generating a transgenic gene deletion model. (E) A gel displays the outcome of a cleavage assay. Lane 1 contains molecular weight markers (MW); Lanes 2–6 contain different amounts in nanograms (ng) of sgRNA and Cas9 protein as indicated at the top. The sizes of DNA fragments are shown in base pairs (bp) to the left. A schematic at the bottom represents the target DNA and cleavage fragments. (F) Data showing the outcome of the gene editing experiment are summarized for postnatal (top) and embryonic (bottom) samples as indicated.
Fig 4: Molecular biological analysis of EXOSC10 stability under different conditions. (A) A western blot assay of whole cell extracts (WCE) and EXOSC10 co‐immunoprecipitated samples (Co‐IP) is shown (n = 1). Loading control (ACTB) and target protein (C1D) are indicated to the right. Relevant molecular weight markers (MW) are shown to the left. WCE samples from cells transfected with control siRNA (Cont, green) and target siRNA (C1D, red) are compared with Co‐IP samples from cells transfected with wild‐type (EXOSC10WT, green) and mutant (EXOSC10S402T, red) alleles as shown at the top. A color‐coded bar diagram at the bottom plots relative signal intensities (y‐axis) against samples as indicated (x‐axis). (B) A western blot of triplicate samples is shown (n = 3). Target and loading control proteins are indicated to the right; the target gene alleles are shown at the top and the cell line used is given at the bottom. Molecular weight markers (MW) are indicated. (C) Representative images of an immunocytochemistry assay revealing DNA (DAPI in blue) and proteins (Anti‐MYC) for tagged wild‐type (EXOSC10WT) and mutant (EXOSC10S402T, EXOSC10K583R) variants are shown in green as indicated in the top left corner. The number of cells analyzed for each allele (n) is given. Scale bar = 10 μm. (D) Color‐coded bar diagrams plot the number of pixels (left) or signal intensity units (right, y‐axis) against EXOSC10 wild‐type (green) and mutant (red) alleles (x‐axis). The error bars indicate standard deviation. (E) A western blot is shown like in panel B. A bar diagram to the left plots normalized intensity units (EXOSC10/ACTB; y‐axis) against triplicate samples (x‐axis; n = 3). A bar diagram to the right plots averaged intensity units (y‐axis) against control and experimental samples (x‐axis). The error bars indicate standard deviation. The target protein is given at the top. (F–G) Western blots, quantified band intensity signals, and averaged signals are shown like in panel B for representative duplicate (panel F, total n = 4; anova followed by Tukey's post hoc range test, Lanes 1 versus 3: p = 0.05 and Lanes 1 versus 4: p = 0.04; * = significant) and quadruplicate (panel G, n = 4) samples.
Fig 5: Mass spectrometry (MS)‐analysis of EXOSC10 and interacting proteins. (A) A schematic summarizes the experimental approach. The sample was injected twice (technical replicates n = 2). (B) The amino acid sequence of human EXOSC10 is shown at the top. Peptides containing phosphorylated and ubiquitinated residues are highlighted in green and blue, respectively. Modified amino acids are highlighted in yellow, the D‐box match is marked in light gray, S402 is shown in blue and K583 is shown in red. Peptide sequences and modifications are given at the bottom. (C) A lollipop plot shows post‐translational modifications (PTMs; y‐axis) within the protein sequence covering EXOSC10's N‐terminal protein interaction domain (NTD; PMC2NT), catalytic domain (CAT, EXO1, and HRDC) and C‐terminal domain (CTD; x‐axis). Modified amino acids are shown in green (phosphorylation) and blue (ubiquitination). (D) A table summarizes the output of selected proteins that co‐purified with EXOSC10. RNA exosome subunits are shown in green. Anaphase promoting complex/cyclosome (APC/C) subunits and co‐factors are shown in red. An enzyme that SUMOylates EXOSC10 is shown in blue. Identifiers are given as indicated at the top. The numbers (#) of peptide spectrum matches (PSMs) and unique peptides are shown. Protein network data from the String database are given for subunits of nuclear and cytoplasmic RNA exosomes, APC/C subunits and the EXOSC10 interactor USP36. Nodes and color‐coded edges are shown. A legend summarizes the color code of different types of interactions.
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