ABT1 modifies SMARD1 pathology via interactions with IGHMBP2 and stimulation of ATPase and helicase activity
Gangadhar P. Vadla, … , Kamal Singh, Monique A. Lorson
Gangadhar P. Vadla, … , Kamal Singh, Monique A. Lorson
Published December 8, 2022
Citation Information: JCI Insight. 2023;8(2):e164608. https://doi.org/10.1172/jci.insight.164608.
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Research Article Genetics Neuroscience

ABT1 modifies SMARD1 pathology via interactions with IGHMBP2 and stimulation of ATPase and helicase activity

Abstract

SMA with respiratory distress type 1 (SMARD1) and Charcot-Marie-Tooth type 2S (CMT2S) are results of mutations in immunoglobulin mu DNA binding protein 2 (IGHMBP2). IGHMBP2 is a UPF1-like helicase with proposed roles in several cellular processes, including translation. This study examines activator of basal transcription 1 (ABT1), a modifier of SMARD1-nmd disease pathology. Microscale thermophoresis and dynamic light scattering demonstrate that IGHMBP2 and ABT1 proteins directly interact with high affinity. The association of ABT1 with IGHMBP2 significantly increases the ATPase and helicase activity as well as the processivity of IGHMBP2. The IGHMBP2/ABT1 complex interacts with the 47S pre-rRNA 5′ external transcribed spacer and U3 small nucleolar RNA (snoRNA), suggesting that the IGHMBP2/ABT1 complex is important for pre-rRNA processing. Intracerebroventricular injection of scAAV9-Abt1 decreases FVB-Ighmbp2nmd/nmd disease pathology, significantly increases lifespan, and substantially decreases neuromuscular junction denervation. To our knowledge, ABT1 is the first disease-modifying gene identified for SMARD1. We provide a mechanism proposing that ABT1 decreases disease pathology in FVB-Ighmbp2nmd/nmd mutants by optimizing IGHMBP2 biochemical activity (ATPase and helicase activity). Our studies provide insight into SMARD1 pathogenesis, suggesting that ABT1 modifies IGHMBP2 activity as a means to regulate pre-rRNA processing.

Authors

Gangadhar P. Vadla, Sara M. Ricardez Hernandez, Jiude Mao, Mona O. Garro-Kacher, Zachary C. Lorson, Ronin P. Rice, Sarah A. Hansen, Christian L. Lorson, Kamal Singh, Monique A. Lorson

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Figure 4

The association of ABT1 with IGHMBP2 increases the ATPase and helicase activity as well as the processivity of IGHMBP2.

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The association of ABT1 with IGHMBP2 increases the ATPase and helicase a...
(A) ATPase activity measured with increasing concentrations of ABT1. (B) Increasing concentrations of IGHMBP2. (C) IGHMBP2 (100 nM) incubated with increasing concentrations of ABT1. The data represent mean values from 3 independent experiments and 9 readings. (D) Helicase activity measured with increasing concentrations of ABT1. (E) Increasing concentrations of IGHMBP2. (F) IGHMBP2 (100 nM) incubated with increasing concentrations of ABT1. The data represent mean values from 3 independent experiments and 9 readings. (G) Lanes 1–4 show unwinding analyses of TP31-18mer incubated with 100 nM IGHMBP2 after 1, 3, 5 and 7 minutes, respectively. Lanes 5–8 show unwinding analysis of TP31-18mer incubated with 100 nM IGHMBP2 + 100 nM ABT1 after 1, 3, 5, and 7 minutes, respectively. Lane 9 shows heat-induced TP31-18mer DNA separation (95°C for 5 seconds) used as a positive control. Lane 10 shows Cy3 labeled DNA TP31-18mer alone (negative control). (H) Lanes 1–4 show unwinding analysis of TP31-18mer incubated with 100 nM IGHMBP2 after 1, 3, 5, and 7 minutes, respectively. Lane 5 shows unwinding analysis of TP31-18mer incubated with 100 nM IGHMBP2 + 20 nM ABT1 with no incubation. Lanes 6–10 show unwinding analysis of TP31-18mer with 100 nM IGHMBP2 + increasing concentrations of ABT1 (20, 40, 50, 75, 100 nM) after 7 minutes of incubation. (I) Quantification of 3 independent experiments of unwinding analyses as shown in G. (J) Quantification of 3 independent experiments of unwinding analyses as shown in H. Arrow indicates the double-stranded duplex, and the arrowhead indicates the resolved duplex. DNA substrate was used at a final concentration of 10 nM. iM, free phosphate produced in μM; RFU, relative fluorescence units. Data are represented as mean ± SD; 1-tailed paired t test was used to calculate statistical significance.