The zinc-finger protein Red1 orchestrates MTREC submodules and binds the Mtl1 helicase arch domain

Abstract

Cryptic unstable transcripts (CUTs) are rapidly degraded by the nuclear exosome in a process requiring the RNA helicase Mtr4 and specific adaptor complexes for RNA substrate recognition. The PAXT and MTREC complexes have recently been identified as homologous exosome adaptors in human and fission yeast, respectively. The eleven-subunit MTREC comprises the zinc-finger protein Red1 and the Mtr4 homologue Mtl1. Here, we use yeast two-hybrid and pull-down assays to derive a detailed interaction map. We show that Red1 bridges MTREC submodules and serves as the central scaffold. In the crystal structure of a minimal Mtl1/Red1 complex an unstructured region adjacent to the Red1 zinc-finger domain binds to both the Mtl1 KOW domain and stalk helices. This interaction extends the canonical interface seen in Mtr4-adaptor complexes. In vivo mutational analysis shows that this interface is essential for cell survival. Our results add to Mtr4 versatility and provide mechanistic insights into the MTREC complex.

Fig. 1. Detailed analysis of MTREC submodule interactions.

a Organization of the MTREC complex. The Mtl1–Red1 core interacts via Red1 with all submodules, comprising Cbc1–Cbc2–Ars2, Iss10–Mmi1, Red5–Pab2–Rmn1, and Pla1. Arrows indicate all direct interactions identified in this study (see b and c). b Yeast two-hybrid analysis identified direct interactions between Red1 and Ars2, Mtl1, Iss10, Rmn1, Pla1, and Red5. For the CBC complex, Cbc1 is fused to Gal4 AD and an untagged version of Cbc2 is expressed from a third plasmid, pRS426. Gal4 BD, Gal4 DNA-binding domain; Gal4 AD, Gal4 activation domain; SDC-Leu-Trp (SDC) and SDC-Leu-Trp-His (SDC-His). Auto-activation controls are shown in Supplementary Fig. 1a. c Yeast three-hybrid analysis shows that interactions between Ars2-Rmn1, Ars2-Iss10, Pla1-Ars2, Pla1-Rmn1, and Pla1-Iss10 are bridged by Red1. SDC-Leu-Trp-Ura (SDC) and SDC-Leu-Trp-Ura-His (SDC-His). d Yeast two-hybrid analysis shows that the different submodules interact with various Red1 truncation variants. e Scheme of the interacting regions of the MTREC submodules with Red1 analyzed in d. The inset shows a multiple sequence alignment of the Ars2-binding region present in spRed1, hsZFC3H1, hsNHN1, and hsFLASH. IBR Iss10-binding region, NLS nuclear localization sequence.

Fig. 2. Interaction analysis of Red5–Pab2–Rmn1 and Mmi–Iss10 submodules.

a Y2H analysis identified interactions within the Red5–Pab2–Rmn1 submodule. b Y2H analysis identified interactions between the Iss10–Mmi1 and Red5–Pab2–Rmn1 submodules. c Scheme summarizing the interactions of Red1, Iss10, and Mmi1. The minimal interacting regions identified in this study are depicted. IBR Iss10-binding region, RBR Red1-binding region, SID self-interaction domain, Gal4 BD Gal4 DNA-binding domain, Gal4 AD Gal4 activation domain, SDC-Leu-Trp (SDC) and SDC-Leu-Trp-His (SDC-His).

Fig. 3. Characterization of the Mtl1–Red1 core complex.

a Domain organization of Red1 and Mtl1. The arch construct contains the full stalk and the KOW domain (green), whereas the short arch contains a shortened stalk (missing the first and the last helices, yellow) and the KOW domain (top panel). The relevant boundaries of the constructs used in the current study are shown with residue numbers for both proteins. Yeast two-hybrid analysis of Red1 and Mtl1 shows an interaction of the Mtl1 short arch and the Red1 C-terminal region (lower panel). A scheme with all constructs used here is provided in Supplementary Fig. 2a. b Coomassie stained SDS-PAGE of an in vitro-binding assay of Mtr4 and Red1 from Chaetomium thermophilum (ct). ctMtr4 (full-length) interacts with GST-ctRed1_1040–1091. This interaction is stable under high salt conditions (600 mM NaCl). c ITC measurement of ctRed1_1040–1091 (cell) and ctMtr4_SA (syringe). K_D and thermodynamic parameters are shown. The ITC measurement was performed in duplicate.

Fig. 4. Crystal structure of ctMtr4–ctRed1 and interface characterization.

a Domain organization of the crystallization constructs of ctMtr4 and ctRed1 are shown. The ctMtr4_SA and ctRed1_pep are linked with a GS (glycine–serine) linker to form the single-chain construct. b Crystal structure of the ctMtr4_SA–ctRed1_pep complex superimposed on the structure of the full-length Mtr4 from Saccharomyces cerevisiae (gray; PDB: 2XGJ). The stalk (light-orange) and KOW (green) domain are structurally highly similar. c Overview of the ctMtr4_SA–ctRed1_pep complex. The ctRed1 peptide (light blue) interacts with the stalk and the KOW domain of ctMtr4. The ctRed1 Zn-finger is located close to the C-terminus (inset). The Cys- and His-residues required for Zn-finger formation are conserved. d ctRed1_pep N-terminal region interacts with the stalk helices through hydrogen bonds and van der Waals contacts. e Red1 forms a U-shaped structure stabilized by hydrophobic residues, which inserts between the stalk helices and the KOW domain. f Multiple hydrophobic residues form a tight interaction between ctMtr4 KOW domain and ctRed1_pep. Rotation angles relative to the view in c are shown at the top left corner of d–f.

Fig. 5. Mutation analysis of the Mtr4/Mtl1–Red1 interface.

a Coomassie stained SDS-PAGE analysis of co-expression and purification of T4L-ctRed1_pep wild type (wt) and mutant variants. While wt T4L-ctRed1_pep co-purifies untagged ctMtr4_SA, the I1050R mutant does not. F1024R and the double S1043R, T1045R mutants had no effect on the ctMtr4–ctRed1 interaction. T Total, S soluble, E eluate. b Y2H analysis of spRed1 I612R (I1050R in ctRed1) and spMtl1 short arch or full-length variants. While the short arch shows no binding, the full-length spMtl1 retained binding. c Table showing the mutated residues in Mtl1/Red1 and Mtr4/Red1 in S. pombe (sp) and C. thermophilum (ct), respectively (color code is as in Fig. 4). d The spRed1 I612R mutant binds like wt. Mutations at I612R, F586A, and K587D (ADR) weakened the interaction. Addition of S581D and F583E mutations (DEADR variant) completely abolished binding. The DEADR variant retained binding to the remaining interaction partners in MTREC, which confirmed proper protein expression. e Y2H analysis of spMtl1 mutants for spRed1 binding. The single-residue I730R, F758R, and L781R mutants showed no reduction in binding. The L784R, E788R double mutant did not interact with spRed1, similar to our observation for the short arch deletion (∆SA).

Fig. 6. In vivo analysis of red1 DEADR and mtl1 RR variants.

a Tetrad dissection of red1 DEADR variant and red1∆. The wild type (wt) colonies grew after 3 days, whereas the DEADR variant (circles) appeared after 6 days. red1∆ colonies (triangles) grew similarly to wt colonies. b Tetrad dissection of mtl1 L784R/E788R (RR) variant. The wt colonies grew after 3 days, whereas mtl1 RR (squares) appeared after 6 days. c Phenotypic growth assay after 4 days at different temperatures. The red1 DEADR variant, red1∆, mtl1 RR, and respective wt, were spotted after serial dilutions. The growth of the mutant strains at 30 °C is comparable to wt; however, at 23 °C all mutants exhibit a slow growth phenotype. d qPCR analysis of the red1 mRNA expression level. For the analysis, red1 DEADR (dotted circles) and mtl1 RR (dotted squares) were used together with wild type and red1∆ strains. Two biological replicates were performed (labeled with c1 and c2), n = 2. Source data are provided as a Source Data file.

Fig. 7. Comparison of the ctMtr4–ctRed1 crystal structure with known Mtr4 complexes.

Crystal structure of the ctMtr4_SA–ctRed1_pep complex (Red1 in blue, stalk helices in dark gray and KOW domain in light gray) is superimposed with the structures of Mtr4-Air2 (PDB: 4U4C,violet), Mtr4-NVL (PDB: 6RO1 (ref. ), pink), Mtr4-Nop53 (PDB: 5OOQ, orange), and hMTR4-NRDE-2 (PDB: 6IEH, green). For simplicity only the Mtr4 interacting peptides of these complexes are shown. A detailed comparison is shown in Supplementary Fig. 14.