Journal: Genes & development

Article Title: Uncovering a mammalian neural-specific poly(A) binding protein with unique properties.

doi: 10.1101/gad.350597.123

Figure Lengend Snippet: Figure 1. PABPC1L2 (neuPABP) displays a neural-specific ex- pression pattern. (A) Schematic representation of PABPC1 and PABPC1L2 domain organization. (B) Schematic diagram of a hu- man X chromosome showing the position of the Pabpc1l2 ampli- conic gene. (C) Semiquantitative RT-PCR analysis of Pabpc1l2 and Actin mRNAs from multiple adult mouse tissues (C57BL/ 6J; age: 5 mo). (D) Western blotting of PABPC1, neuPABP, GAPDH, and Actin on lysates prepared from select adult mouse tissues (C57BL/6J; age: 5 mo).

Article Snippet: Furthermore, neuPABP and PABPC1 expression clones were generated by recombining the attL sites in the pDONR221-neuPABP and PABPC1 entry clones and the attR sites in the pLEX-307 destination vector (Addgene).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Genes & development

Article Title: Uncovering a mammalian neural-specific poly(A) binding protein with unique properties.

doi: 10.1101/gad.350597.123

Figure Lengend Snippet: Figure 3. neuPABP specificity for poly(A) RNA. (A) Summary of RNAcompete experi- ments for GST-neuPABP. The sequence logo of the neuPABP RNA binding motif is shown, along with a scatter plot displaying the Z scores and motifs for the two halves of the RNA pool (set A and set B). (B) Recombinant PABPC1 and neuPABP were analyzed by SDS-PAGE and Coomassie blue staining. (C) High-affinity binding of neuPABP to oligo(A) RNA. EMSA was carried out as described in the Materials and Methods. A constant amount of 32P-oligo (A)25 RNA was incubated with specific concen- trations of neuPABP or PABPC1. The KD value of ∼50 nM was calculated from three biological experiments for both PABPC1 and neuPABP. Recombinant GST (control) did not lead to a gel shift of radiolabeled oligo.

Article Snippet: Furthermore, neuPABP and PABPC1 expression clones were generated by recombining the attL sites in the pDONR221-neuPABP and PABPC1 entry clones and the attR sites in the pLEX-307 destination vector (Addgene).

Techniques: Sequencing, RNA Binding Assay, Recombinant, SDS Page, Staining, Binding Assay, Incubation, Control, Gel Shift

Journal: Genes & development

Article Title: Uncovering a mammalian neural-specific poly(A) binding protein with unique properties.

doi: 10.1101/gad.350597.123

Figure Lengend Snippet: Figure 4. neuPABP is expressed during neuronal maturation. (A) Subcellular frac- tionation of an adult mouse brain cortex (C57BL/6J; age: 2 mo) shows cytoplasmic lo- calization of both neuPABP and PABPC1. GAPDH and hnRNPA1 were used as mark- ers for cytoplasmic and nuclear fractions, respectively. (B) Western blot analysis of PABPC1, neuPABP, Actin, and β-tubulin III on lysates prepared from mouse brain cortices isolated at different stages of em- bryonic and postnatal development. (C) Western blot analysis of neuPABP, PSD- 95, and β-tubulin III on lysates prepared from mouse primary cortical neurons. Neu- rons were isolated from P0 pups and cul- tured for defined days in vitro (DIV). (D) Western blot analysis of subcellular frac- tions of an adult mouse cortex (C57BL/6J; age: 6 mo) prepared by synaptosome frac- tionation. Lysates were probed with the postsynaptic (PSD) marker PSD-95 and the presynaptic marker synaptophysin (Syn), as well as neuPABP, PABPC1, and GAPDH. Cortex homogenates (H) were generated, and supernatant (S2) and the crude synapto- somal pellet (P2) were acquired after high- speed centrifugation of the S1 supernatant. The crude synaptosomal fraction was fur- ther fractionated into a Triton X-100-solu- ble non-PSD fraction (extrasynaptic) and a Triton X-100-insoluble PSD-containing fraction (synaptic).

Article Snippet: Furthermore, neuPABP and PABPC1 expression clones were generated by recombining the attL sites in the pDONR221-neuPABP and PABPC1 entry clones and the attR sites in the pLEX-307 destination vector (Addgene).

Techniques: Western Blot, Isolation, In Vitro, Marker, Generated, Centrifugation

Journal: Genes & development

Article Title: Uncovering a mammalian neural-specific poly(A) binding protein with unique properties.

doi: 10.1101/gad.350597.123

Figure Lengend Snippet: Figure 5. neuPABP localizes with early RNP fractions on polysome gradients and in- teracts with specific RNAs. (A) Polysome profile traces of lysates prepared from mouse cortices (C57BL/6J; age: P9). (B) Lysates were fractionated by sucrose gradient centrifuga- tion. Fractions were subsequently collected, TCA-precipitated, and resolved by SDS- PAGE, and Western blotting was subse- quently performed using antibodies against neuPABP, PABPC1, and a ribosomal protein marker (RPS6). (C) Immunoprecipitation of neuPABP from an adult mouse hippocampus (C57BL/6J; age: 6 mo). Immunoprecipitated complexes were subjected to SDS-PAGE, and Western blotting was performed using anti-neuPABP and anti-GAPDH antibodies. neuPABP-enriched RNAs were isolated us- ing RNA purification kit (Qiagen) and identi- fied by RNA-seq. (D) Volcano scatter plot showing most significantly enriched RNAs with neuPABP (threshold set at log2 FC≥ 1.5 and P-value set at <1× 10−20). BC1 RNA and mRNAs encoding ribosomal proteins (red) and nuclear-encoded mitochondrial proteins (blue) were enriched. (E) Top Wiki- pathway (WP) and associated gene ontology (GO) terms (cellular component) signifi- cantly enriched among proteins coded for by neuPABP-enriched mRNAs (FC ≥2). The number above each column represents the number of genes associated with its cor- responding term. (F) RT-qPCR analyses of neuPABP-enriched transcripts identified by RNA-seq. neuPABP was immunoprecipitat- ed from adult mouse hippocampi (C57BL/6J; age: 6 mo), and associated RNAs were Tri- zol-extracted. Error bars represent SEM from biological replicates (n = 3). Data points for biological replicates are shown as solid circles. Data were normalized to an in vitro transcribed RLuc spiked-in RNA.

Article Snippet: Furthermore, neuPABP and PABPC1 expression clones were generated by recombining the attL sites in the pDONR221-neuPABP and PABPC1 entry clones and the attR sites in the pLEX-307 destination vector (Addgene).

Techniques: SDS Page, Western Blot, Marker, Immunoprecipitation, Isolation, Purification, RNA Sequencing, Quantitative RT-PCR, In Vitro

Journal: Genes & development

Article Title: Uncovering a mammalian neural-specific poly(A) binding protein with unique properties.

doi: 10.1101/gad.350597.123

Figure Lengend Snippet: Figure 6. neuPABP associates with untranslated mRNAs present in early RNP fraction. Cortices of adult mice (C57BL/6J; age: 6 mo) were triturated and formaldehyde-cross-linked. Lysates were prepared and fractionated by sucrose gradient centrifugation. (A) Ribosome traces of lysates prepared from formalde- hyde-cross-linked adult mouse cortices (C57BL/6J; age: 6 mo). (B) Free RNP fractions (depleted of ribosomal subunits) were collected from the polysome gradient and resolved by SDS-PAGE, and Western blotting was performed using antibodies against RPS6, neuPABP, PABPC1, and GAPDH. (C) Immunoprecipitation of neuPABP from free RNP fractions from B. Immunopre- cipitated complexes were resolved by SDS-PAGE, and Western blotting was performed using antibodies against neuPABP, PABPC1, and GAPDH. (D) RT- qPCR analysis of neuPABP-associated RNAs isolated from C. Error bars represent SEM from biological repli- cates (n = 3), which are shown as solid circles. A mito- chondrial mRNA (mt.ND1) was used as a negative control. Data were normalized to an in vitro tran- scribed RLuc spiked-in RNA.

Article Snippet: Furthermore, neuPABP and PABPC1 expression clones were generated by recombining the attL sites in the pDONR221-neuPABP and PABPC1 entry clones and the attR sites in the pLEX-307 destination vector (Addgene).

Techniques: Gradient Centrifugation, SDS Page, Western Blot, Immunoprecipitation, Quantitative RT-PCR, Isolation, Negative Control, In Vitro

Journal: Genes & development

Article Title: Uncovering a mammalian neural-specific poly(A) binding protein with unique properties.

doi: 10.1101/gad.350597.123

Figure Lengend Snippet: Figure 8. neuPABP has been selected to not bind eIF4G. (A) Schematic diagrams of PABPC1 and neuPABP domain organiza- tion, along with a comparative sequence analysis of human (Hs) and mouse (Mm) PABPC1 with human, mouse, and bat (Pk) neuPABP RRM2. Amino acids that play a role in PABPC1 binding to eIF4G are denot- ed by a green dot. Corresponding amino ac- ids or those in proximity to eIF4G- intearcting residues are red. (B) Recombi- nant glutathione-S-transferase (GST)-tagged eIF4G41–244 was incubated with maltose-bind- ing protein (MBP)-tagged PABPC1RRM1+2, neuPABPWT, or neuPABPMUT (Ile221Thr, Phe265Leu, and Tyr268Asp). Precipitated proteins were separated by SDS-PAGE and visualized by Coomassie staining. (C) Re- combinant GST, GST-tagged neuPABPWT, and neuPABPMUT proteins were analyzed by SDS-PAGE and Coomassie blue staining. (D) Capped poly(A)+ luciferase reporter RNA was incubated in Krebs-2 extract. Reactions were supplemented with either recombi- nant GST, GST-neuPABPWT, or GST- neuPABPMUT, as indicated. Normalized lu- ciferase activity was measured relative to buffer alone (control). Error bars represent SEM from biological replicates (n = 3). A two-tailed Student t-test (equal variance) was conducted (vs. GST) to access signifi- cance. (∗∗) P-value <0.004 was calculated in GST-neuPABPWT treatment.

Article Snippet: Furthermore, neuPABP and PABPC1 expression clones were generated by recombining the attL sites in the pDONR221-neuPABP and PABPC1 entry clones and the attR sites in the pLEX-307 destination vector (Addgene).

Techniques: Sequencing, Binding Assay, Incubation, SDS Page, Staining, Luciferase, Activity Assay, Control, Two Tailed Test

Journal: Genes & development

Article Title: Uncovering a mammalian neural-specific poly(A) binding protein with unique properties.

doi: 10.1101/gad.350597.123

Figure Lengend Snippet: Figure 9. Model for the biological role of neuPABP. (Panel i) neuPABP binds to BC1 RNA and select translationally dormant mRNAs that may be transported to postsyn- aptic compartments. As neuPABP also lacks the PABPC1 MLLE domain, it may protect mRNAs from mRNA decay factors that can interact with this domain, including the PAN2–PAN3 complex and Tob, which interacts with the CCR4–NOT deadenylase complex. (Panel ii) It is possible that in spe- cific contexts (depicted as a question mark), PABPC1 may displace neuPABP from mRNA poly(A) tails, bind eIF4G, and stimu- late their mRNA translation.

Article Snippet: Furthermore, neuPABP and PABPC1 expression clones were generated by recombining the attL sites in the pDONR221-neuPABP and PABPC1 entry clones and the attR sites in the pLEX-307 destination vector (Addgene).

Techniques:

Journal: Journal for Immunotherapy of Cancer

Article Title: Enhanced antigen cross-presentation in human colorectal cancer-associated fibroblasts through upregulation of the lysosomal protease cathepsin S

doi: 10.1136/jitc-2021-003591

Figure Lengend Snippet: Cathepsin S enhances cross-presentation in fibroblasts. (A) Relative expression of cathepsin S (CTSS) in NBS wildtype (gray bar), vector control (gray bar) and two CTSS overexpressing NBS fibroblasts (#1 and #2, red bars) normalized for expression of β-actin. Student’s t-test ***p≤0.0001 (B) Western blot analysis of cathepsin S expression in NBS wildtype, vector control and CTSS overexpressing fibroblasts. The CTSS/β-actin ration is shown for quantification. (C) IFNγ production by neoantigen-specific T cells after 24 hours coincubation with wildtype (gray bar), vector control (gray bar) and CTSS overexpressing NBS fibroblasts (red bars). Means with SD are plotted from representative experiments (n=4). Two-tailed ANOVA with correction for multiple testing *p≤0.05, **p≤0.01. (D) Expression of cathepsin S in CRC-conditioned fibroblasts with shRNA-mediated cathepsin S knockdown. Student’s t test *p≤0.05. (E) IFNγ production by neoantigen-specific T cells after 24 hours coincubation with cathepsin-S KD (blue bars) and vector control CRC-conditioned NBS fibroblasts (gray bar). Means with SD are plotted from representative experiments (n=2). Two-tailed ANOVA with correction for multiple testing *p≤0.05. ANOVA, analysis of variance; CRC, colorectal cancer; NBS, Nijmegen breakage syndrome.

Article Snippet: The lentiviral cDNA expression vector expressing cathepsin S was generated through Gateway cloning, using the cathepsin S entry vector (pDNR223 backbone) from the human ORF library (Sigma) and pLex307 (addgene: #41392) as the destination vector.

Techniques: Expressing, Plasmid Preparation, Western Blot, Two Tailed Test, shRNA

Journal: Journal for Immunotherapy of Cancer

Article Title: Enhanced antigen cross-presentation in human colorectal cancer-associated fibroblasts through upregulation of the lysosomal protease cathepsin S

doi: 10.1136/jitc-2021-003591

Figure Lengend Snippet: Detection of cathepsin S expression in colorectal cancer CAFs. (A) Relative expression of cathepsin S in primary tumor and liver-metastasis-derived CRC CAFs compared with matched normal fibroblasts. Mean and SD are plotted of a technical triplicate. Student’s t test *p≤0.05. (B) IHC staining of primary CRC tissue for cathepsin S. Black arrows point to cathepsin S positive spindle-shaped cells. (C) IF staining of primary CRC tissue for vimentin (red), DAPI (green) and cathepsin S (pink). CAFs, cancer-associated fibroblasts; CRC, colorectal cancer; IHC, immunohistochemistry; IF, immunohistochemistry.

Article Snippet: The lentiviral cDNA expression vector expressing cathepsin S was generated through Gateway cloning, using the cathepsin S entry vector (pDNR223 backbone) from the human ORF library (Sigma) and pLex307 (addgene: #41392) as the destination vector.

Techniques: Expressing, Derivative Assay, Immunohistochemistry, Staining