Sen1 architecture: RNA-DNA hybrid resolution, autoregulation, and insights into SETX inactivation in AOA2

Abstract

The senataxin (SETX, Sen1 in yeasts) RNA-DNA hybrid resolving helicase regulates multiple nuclear transactions, including DNA replication, transcription, and DNA repair, but the molecular basis for Sen1 activities is ill defined. Here, Sen1 cryoelectron microscopy (cryo-EM) reconstructions reveal an elongated inchworm-like architecture. Sen1 is composed of an amino terminal helical repeat Sen1 N-terminal (Sen1N) regulatory domain that is flexibly linked to its C-terminal SF1B helicase motor core (Sen1^Hel) via an intrinsically disordered tether. In an autoinhibited state, the Sen1^Sen1N domain regulates substrate engagement by promoting occlusion of the RNA substrate-binding cleft. The X-ray structure of an activated Sen1^Hel engaging single-stranded RNA and ADP-SO₄ shows that the enzyme encircles RNA and implicates a single-nucleotide power stroke in the Sen1 RNA translocation mechanism. Together, our data unveil dynamic protein-protein and protein-RNA interfaces underpinning helicase regulation and inactivation of human SETX activity by RNA-binding-deficient mutants in ataxia with oculomotor apraxia 2 neurodegenerative disease.

Keywords: DNA repair; Helicase; R-loop; RNA-DNA hybrid; SETX; SF1B; Sen1; X-ray crystallography; cryo-EM; senataxin; transcription.

Figure 1. Cryo-EM structure of Sen1^FL

(A) 2D cryo-EM class averages of full-length Sen1 show an asymmetric elongated assembly. (B) Cryo-EM volume of Sen1^FL is displayed showing the Sen1N domain in brown, and the helicase core in blue. (C) The Sen1N helical repeat (tan). Extensive interactions with helical repeat are mediated by RecA1 1b (orange) and 1c (turquoise) insertion elements. The LZZ (RecA1 1c insertion) reinforces the interface.

Figure 2. Crystal structure of the Sen1^Hel–ssRNA–ADP–SO₄ complex

(A) Left: RNA-DNA unwinding substrates. Right: Sen1^FL and Sen1^Hel at the amounts indicated were incubated with 50 nmol of Sub7 (Table S2). Reactions were initiated by addition of a mixture containing ATP (1 mM) and MgCl₂ (2 mM). FAM signal was detected at 520 nm every 10 sec over 30 min at 25 °C. Initial rates were determined by linear fit of the first 5 min of the unwinding reaction. Error bars are SD from 5 replicates. (B) Top: gel-based RNA-DNA unwinding assay. Bottom: Effect of RNA overhang length on Sen1^Hel RNA-DNA unwinding activity. Sub1-Sub5 (Supplementary Table 3) at 20 nM, Sen1^Hel (50 nM), 19-Trap DNA (Table 1) at 200 nM, ATP (1 mM), MgCl₂ (2 mM) were incubated for 15 min at 37 °C. (C) X-ray structure of the Sen1^Hel–RNA–ADP–SO₄ complex. The extended LZZ coiled coil is not shown. (D) Composite Omit 2Fo-Fc electron density at 1.0 σ, carved 2.0 Å around the RNA chain. (E) Sen1^Hel RNA-protein interactions show an extensive RNA binding interface. (F) APBS surface electrostatic surface representation (blue=electropositive, red= electronegative) of the Sen1 RNA binding surface. Sen1 encircles the RNA via its RecA1 and RecA2 domains. (G) Representative HADDOCK RNA-DNA poses displayed show secondary putative RNA-DNA hybrid binding electropositive surface flanks the ssRNA binding site and is assembled by the 1b and 1c RecA1 insertions.

Figure 3.. A phosphate sensor is a lynchpin in the Sen1 power stroke

(A) Structural superposition of the Sen1^Hel–RNA–ADP–SO₄ complex (tan) with the HsUpf1-RNA-ADP-AlF₄ complex (grey). (B) Structural superposition of the Sen1-RNA-ADP-S0₄ complex with ScSen1-ADP-Mg²⁺ complex, RSCB:5MZN. Close superposition of the RecA1 domains is observed. A 7.5 Å translocation is coincident with phosphate release. The blue sphere marks the position of Mg²⁺ in the ScSen1 -ADP-Mg²⁺ complex. (C) Sen1 active site. Four residues from the RecA1 and RecA2 domains bind SO₄, a mimic of the hydrolyzed phosphate prior to phosphate release. (D) Active site structural superposition of ScSen1-ADP and CtSen1-RNA-ADP-SO₄ complexes colored as indicated. Arrows show structural rearrangements of conserved gamma-phosphate sensing residues upon phosphate (SO₄ mimic) release.

Figure 4.. The Sen1N domain is autoinhibitory.

(A) Design of a PreScission Protease cleavable Sen1. (B) Schematic of activation of PreScission Protease cleavable Sen1^N-PP-C. (C) Purified Sen1 proteins or PreScission Protease cleaved protein (Sen1^N-PP-C – Cut) used in panels D–F. (D) Substrate stimulated ATP hydrolysis of mutant Sen1 proteins was monitored in a phosphate release assay. Sen1^FL, Sen1^N-PP-C (Uncut), PreScission Protease-cleaved Sen1N-PP-C (Cut) and Sen1Hel (5 nM) were incubated with Sub6 (Supplementary Table 3) at 5 μM and ATP (1 mM) and MgCl2 (2 mM) for 15 min at 37 °C. Error bars are SD from 3 replicates, ***p<0.001, **** p<0.0001. (E) Helicase assay of indicated Sen1 protein (1 nM) was performed as in Figure 2A and normalized to unwound fraction of Sen1^Hel for comparison. (F) Comparison of unwound fractions at 25 min from experiments in “E”. Error bars are SD from 3 replicates. Statistical analysis was done using paired t-test relative to Sen1^FL. *p<0.05, ***p<0.001, ****p<0.0001.

Figure 5.. Cryo-EM structure of Sen1^N-PP-C

(A) Local resolution of Sen1^N-PP-C, calculated in using the Local Resolution Estimation in CryoSparc, and displayed using a blue (high-resolution, 3.17 Å) to red (low-resolution, 4.0 Å) color-coded surface. (B) Three interaction interfaces (Int1-Int3) mediate Sen1^Sen1N interaction with Sen1^Hel. The LZZ from Sen1^Hel binds both Int1 and Int3 on Sen1^Sen1N. A ΔLZZ internal deletion construct is marked. (C) Consurf analysis of the Sen1^Sen1N surface reveals conserved surface interaction interfaces (Int1-Int3, dotted lines) are involved in interactions with Sen1^Hel. Top: Sen1N domain interaction interfaces that bind the helicase domain. Bottom: Helicase domain interaction surfaces that bind the Sen1N domain. (D) Molecular details of the Int1 interdomain Sen1^Sen1N - Sen1^Hel interaction interface. (E) Cryo-EM density for Sen1^Sen1N Int2 region displayed contoured at 11.0 σ overlaid upon a molecular surface diagram of the β-barrel domain (orange). (F) Molecular details of the Int3 interdomain Sen1^Sen1N - Sen1^Hel interaction interface. (G) MBP-pulldowns show the LZZ is critical for the Sen1^Sen1N–Sen1^Hel interaction. (H) Trans inhibition of Sen1^Hel by Sen1^Sen1N. Sen1^Hel (1 nM) was pre-incubated with Sen1^Sen1N at the indicated concentrations for 5 min on ice. Reactions were initiated by addition of 50 nM Sub7 (Supplementary Table 3) and a mixture containing ATP (1 mM) and MgCl₂ (2 mM). The percentage of unwound ssDNA was calculated from the FAM signal at 30 min. Error bars are SD from 3 replicates, **p<0.01, *p<0.05.

Figure 6.. Mechanism of Sen1 auto-inhibition

(A) Structural superposition of Sen1^N-PP-C and the Sen1^Hel RNA complex reveals a cascade of conformational rearrangements associated with autoinhibition. (B) Surface diagram of the Sen1-RNA complex and Sen1^N-PP-C shows alterations in the RNA binding channel in the closed state “C”. (D) Fluorescence polarization ssRNA binding. Binding to ssRNA was conducted using enzyme titration and monitoring fluorescence polarization from Sub8 (Supplementary Table 3. Error bars reflect SD from 3 replicates. (E) Superposition of an example HADDOCK docking binding pose and the autoinhibited Sen1^N-PP-C state.

Figure 7. AOA2 mutants Sen1 RNA binding mechanism

(A) Structural overlay of an Alphafold model of human SETX helicase domain (blue) with the CtSen1 RNA complex (tan). (B) Alphafold model of the hSETX helicase domain showing the location of mapped AOA2 and ALS4 mutations (C) Two AOA2 mutations map to the RNA binding cleft. Human equivalent positions are shown below the CtSen1 numbering in parentheses and the CtSen1 structure is displayed. (D) Fluorescence polarization ssRNA binding. Binding to ssRNA was conducted using enzyme titration and monitoring fluorescence polarization from Sub8 (Supplementary Table 3). Error bars reflect SD from 3 replicates. (E) ATPase activity. Proteins (5 nM) were incubated with Sub6 (Supplementary Table 3) and ATP (1 mM) at 5 μM for 15 min at 37 °C. The L1551W mutant had no measurable activity. Error bars are SD from 3 replicates, **p<0.01, ****p<0.0001. (F) RNA—DNA unwinding activity. Proteins (1 nM) were incubated with 50 nM Sub7 (Supplementary Table 3), ATP (1 mM) and MgCl₂ (2 mM). FAM signal at 10 min was used to compare the unwinding activities of Sen1^Hel mutants. The L1551W mutant had no measurable activity. Error bars are SD from 3 replicates, **p<0.01, ****p<0.0001. (G) Purified Sen1^Hel proteins used in panels B–D. (H) Model for Sen1 autoinhibition and RNA translocation.