Journal: bioRxiv

Article Title: RNA-binding proteins provide specificity to the PAN2–PAN3 mRNA deadenylation complex

doi: 10.1101/2025.09.27.678968

Figure Lengend Snippet: (a) Domain diagram of MEX3C and MEX3C(418-600). MEX3C comprises an N-terminal intrinsically disordered region (IDR), two tandem KH domains which bind a specific RNA sequence, a C-terminal IDR, and a RING domain at the extreme C-terminus. (b) NMR analysis of MEX3C(418-600). 1 H, 15 N 2D HSQC spectra of 45 μM 15 N-labelled MEX3C(418-600) alone (grey), with 2.25 μM PAN3 PKC (blue), or 4.5 μM PAN3 PKC (purple). Insets show chemical shift perturbations for resonances of the LARR motif upon addition of PAN3 PKC (grey to blue to purple). The arrows indicate the direction of shift upon titration of PAN3 PKC . (c) Weighted chemical shift perturbations (CSP) of MEX3C(418-600). Histogram plots of chemical shift perturbation in 1 H, 15 N 2D HSQC upon addition of PAN3 PKC at 20:1 (blue, top) and 10:1 (purple, bottom) ratios. Prolines and unassigned residues are indicated with a circle. (d) The LARR motif contributes to targeted deadenylation by PAN2–PAN3. Deadenylation assays with 40 nM PAN2–PAN3 ΔN278 -SII were performed with MBP-MEX3C(222-600)-lipoyl or with the same construct containing a deletion of the LARRV sequence. Reactions were analyzed by denaturing PAGE. The sizes of the substrate with various poly(A) tail lengths are indicated.

Article Snippet: For pull-down assays between MBP-tagged MEX3C truncations and SII-tagged PAN2–PAN3, 50 μl 2 μM MBP-tagged MEX3C truncation was immobilized on 15 μl amylose sepharose resin (NEB) pre-incubated in PB for 1 h, 4°C.

Techniques: Sequencing, Titration, Construct

Journal: bioRxiv

Article Title: RNA-binding proteins provide specificity to the PAN2–PAN3 mRNA deadenylation complex

doi: 10.1101/2025.09.27.678968

Figure Lengend Snippet: (a) Many RBP interactors of PAN3 are also proximal to CCR4–NOT. Prey RBPs from N- and C-terminally averaged BirA*-tagged CNOT9 (n=49) were compared to high-confidence prey RBPs from N- and C-terminally averaged BirA*-tagged PAN3 (green, n=46). Many RBP interactors of PAN3 (33/46) were also proximal to CNOT9. (b) Sequence-specific RBPs are proximal to PAN2–PAN3 and CCR4–NOT. Data from were used to generate a bait-bait scatter plot comparing RBP proximal interactors of PAN3 and of CNOT9; each dot represents a high-confidence proximal prey RBP (BFDR ≤ 0.01) and selected dots are labelled. Red: previously known interactors of PAN2–PAN3; green: direct interactors of PAN2–PAN3 characterized in this study; blue: proximal RBP interactors of PAN3. (c) MEX3C accelerates CCR4–NOT deadenylation in vitro. Deadenylation assays were performed with 10 nM human CCR4–NOT with synthetic RNA substrates containing a 30-adenosine poly(A) tail downstream of 19 non-poly(A) ribonucleotides containing the MEX3 Recognition Element (MRE). Full-length MEX3C, or the C-terminal half of MEX3C without the RING domain (222-600) containing or lacking (ΔLARRV) the LARR motif were added at saturating RBP:RNA ratios. Reactions were analyzed by denaturing PAGE. RNA sizes with various poly(A) tail lengths are indicated. (d) Tethering model of deadenylation specificity. RNA-binding proteins (blue, purple, pink) can act as RNA adapters for PAN2–PAN3 and CCR4–NOT. These RNA adapters bind specific sequence motifs to effectively tether the deadenylation complexes to their substrates. PAN2–PAN3 and CCR4–NOT may target different or the same mRNAs for specific deadenylation via unique mechanisms.

Article Snippet: For pull-down assays between MBP-tagged MEX3C truncations and SII-tagged PAN2–PAN3, 50 μl 2 μM MBP-tagged MEX3C truncation was immobilized on 15 μl amylose sepharose resin (NEB) pre-incubated in PB for 1 h, 4°C.

Techniques: Sequencing, In Vitro, RNA Binding Assay