Molecular insight into the specific enzymatic properties of TREX1 revealing the diverse functions in processing RNA and DNA/RNA hybrids

Abstract In various autoimmune diseases, dysfunctional TREX1 (Three prime Repair Exonuclease 1) leads to accumulation of endogenous single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and DNA/RNA hybrids in the cytoplasm and triggers immune activation through the cGAS–STING pathway. Although inhibition of TREX1 could be a useful strategy for cancer immunotherapy, profiling cellular functions in terms of its potential substrates is a key step. Particularly important is the functionality of processing DNA/RNA hybrids and RNA substrates. The exonuclease activity measurements conducted here establish that TREX1 can digest both ssRNA and DNA/RNA hybrids but not dsRNA. The newly solved structures of TREX1–RNA product and TREX1–nucleotide complexes show that 2′-OH does not impose steric hindrance or specific interactions for the recognition of RNA. Through all-atom molecular dynamics simulations, we illustrate that the 2′-OH-mediated intra-chain hydrogen bonding in RNA would affect the binding with TREX1 and thereby reduce the exonuclease activity. This notion of higher conformational rigidity in RNA leading TREX1 to exhibit weaker catalytic cleavage is further validated by the binding affinity measurements with various synthetic DNA–RNA junctions. The results of this work thus provide new insights into the mechanism by which TREX1 processes RNA and DNA/RNA hybrids and contribute to the molecular-level understanding of the complex cellular functions of TREX1 as an exonuclease.


Expression and purification of mHMGB2
The wild-type mouse HMGB2 (mHMGB2, 1-210 a.a) gene was cloned into a pET28a vector and expressed in E. coli BL21-CodonPlus (DE3)-RIPL strain.E. coli cells were cultured in Luria Broth (LB) medium at 37 °C supplemented with 35 μg/ml chloramphenicol, 25 μg/ml streptomycin, and 50 μg/ml kanamycin to OD 600 of 0.5-0.6 and then induced by 1 mM isopropyl β-D-1-thiogalactopyranoside at 18 °C for 20 h.The cells were collected through centrifugation at 8,000 rpm for 30 min at 4 °C and further lysed through sonication in 50 mM Tris-HCl pH 8.0, 300 mM NaCl.The cell debris was clarified through centrifugation at 13,000 rpm at 4 °C for 30 min, and the supernatant was loaded into an affinity column (HiTrap TALON crude 5 ml, GE Healthcare) and purified by standard protocol.Target proteins were further purified by an anion-exchange column (HiTrap Heparin HP 5 ml, GE Healthcare) and a size-exclusion column (HiLoad TM 16/60 Superdex TM 75 prep grade, GE Healthcare).Purified mHMGB2 was concentrated to at least 10mg/mL in 50 mM Tris-HCl pH 7.0, 300 mM NaCl, and stored at -20 °C until use.

Expression and purification of mRNaseH1 and mRNaseH2
The truncated mouse RNaseH1 (mRNaseH1, 27-285 a.a) gene was cloned into a pET22b vector and expressed in E. coli B834 (DE3) pLysS strain.E. coli cells were cultured in Luria Broth (LB) medium at 37 °C supplemented with 35 mg/ml chloramphenicol and 100 mg/ml ampicillin to OD 600 of 0.4-0.6 and then induced by 1 mM isopropyl β-D-1-thiogalactopyranoside at 18 °C for 18 h.The cells were collected through centrifugation at 6,000 rpm for 30 min at 4 °C, and further lysed through sonication in 50 mM Tris-HCl pH 8.0, 300 mM NaCl.The cell debris was clarified through centrifugation at 13,000 rpm at 4 °C for 30 min and the supernatant was loaded into an affinity column (HisTrapFF 5 ml, GE Healthcare) and purified by standard protocol.Target proteins were further purified by an ionexchange column (HiTrap SP FF 5 ml, GE Healthcare) and a size-exclusion column (HiLoad TM 16/60 Superdex TM 75 prep grade, GE Healthcare).Purified truncated mRNaseH1 was concentrated to at least 7 mg/mL in 50 mM CH 3 COONa pH 4.5, 500 mM NaCl, and stored at -20 °C until use.
The wild-type mouse RNaseH2A and RNaseH2BC genes were cloned into a pETDuet vector and pRSFDuet vector, respectively, and co-expression in E. coli B834(DE3) pLysS strain.E. coli cells were cultured in LB medium at 37 °C supplemented with 50 mg/ml kanamycin, 35 mg/ml chloramphenicol, and 100 mg/ml ampicillin to an OD 600 of 0.6-0.7 and then induced by 1 mM isopropyl β-D-1-thiogalactopyranoside at 18 °C for 18 h.The cells were harvested through centrifugation and further lysed through sonication in 50 mM Tris-HCl, 300 mM NaCl, pH 8.0.The lysate was clarified through centrifugation at 13 000 rpm at 4°C for 20 min.The supernatant was loaded into an affinity column (HiTrap™ TALON crude 5 ml, GE Healthcare) and purified by standard protocol.Target proteins were further purified by an ion-exchange column (HiTrap™ HP 5 ml, GE Healthcare), an ion-exchange column (HiTrap™ SP HP 5 ml, GE Healthcare), and a size-exclusion column (HiLoad™ 16/60 Superdex 200 prep grade, GE Healthcare).Purified wild-type mRNaseH2ABC complex was concentrated to at least 7.5 mg/mL in 50 mM Tris-HCl pH 7.0, 300 mM NaCl, and stored at -20 °C until use.

Electrophoretic Mobility Shift Assay
Gel shift assays were conducted to determine the DNA and RNA binding affinities of RNase T. After incubating RNase T in a solution containing 120 mM NaCl, 50 mM EDTA, and 20 mM Tris-HCl, pH 7.0 for 10 minutes on ice, 5'-FAM-labeled ssDNA and ssRNA substrates (0.5 µM) were added and further incubated for an additional 10 minutes on ice.The concentrations of RNase T used were 2.5, 5, 10, 20, and 40 µM.The resulting samples were separated using 10% TBE native PAGE and visualized with a fluorescence detector.
(A) The domain structure of mTREX1.(B)(C) The recombinant full-length, truncated, and mutated mTREX1, mHMGB-2, mRNase H1, and mRNase H2 were purified and protein purity was analyzed by SDS-PAGE.(D) Nuclease activity assays of full-length mTREX1 on digesting ssDNA (20 mer), ssRNA (20 mer), and dsRNA (20 bp).The activity property is similar to truncated mTREX1.between mTREX1 and various nucleotides in the presence of 2 mM MgCl 2 .(A) (B) (D) (E) Quantification of the affinity (K d ) between mTREX1 and various ribonucleotides and deoxyribonucleotides, such as CMP, dCMP, GMP, and dGMP.Dissociation constants (K d ) were calculated using a one-site binding mode.(C) (F) The comparison of the binding between CMP and dCMP, or GMP, and dGMP.X in the log [X] is the concentration of CMP, dCMP, GMP, or dGMP.