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 5

IGHMBP2 and ABT1 associate with the 5′ external transcribed spacer and U3 snoRNA.

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IGHMBP2 and ABT1 associate with the 5′ external transcribed spacer and U...
Binding isotherms with normalized fluorescence (Fnorm[%]) plotted. IGHMBP2 is RED-Tris-NTA labeled. The 5′ ETS + U3 snoRNA and scramble RNA + U3 snoRNA were annealed. Single RNAs were analyzed with starting concentrations of 1 μM, and 2 RNAs were analyzed with starting concentrations of 500 nM each. (A) 100 nM IGHMBP2 + 5′ ETS RNA. (B) 100 nM IGHMBP2 + 5′ ETS RNA + U3 snoRNA. (C) 100 nM IGHMBP2 + 100 nM ABT1 + 5′ ETS RNA; KD ≥ 697 nM. (D) 100 nM IGHMBP2 + 100 nM ABT1 + U3 snoRNA; KD = 35 nM. (E) 100 nM IGHMBP2 + 100 nM ABT1 + 5′ ETS RNA + U3 snoRNA; KD = 28 nM. (F) 100 nM IGHMBP2 + scramble RNA. (G) 100 nM IGHMBP2 + scrambled RNA + U3 snoRNA. (H) 100 nM IGHMBP2 + 100 nM ABT1 + scramble RNA + U3 snoRNA; KD = 1 nM. Each panel is taken from 9 readings of 3 independent experiments; data are shown as mean ± SD. Values are plotted using MO.Affinity Analysis software, and the data were fit to a quadratic equation using nonlinear regression. The data are fit to a quadratic equation to determine the KD. (I) SDS PAGE gel of UV crosslinked products. Samples were crosslinked for 60 minutes unless stated. Lane 1 shows protein marker; lane 2 shows 5′ ETS RNA; lane 3 shows IGHMBP2, ABT1, 5′ ETS RNA; lane 4 shows ABT1, 5′ ETS RNA; lane 5 shows IGHMBP2, ABT1, 5′ ETS RNA crosslinked 30 minutes; lane 6 shows IGHMBP2, 5′ ETS RNA; lane 7 shows IGHMBP2, ABT1, U3 snoRNA; lane 8 shows ABT1, U3 snoRNA; lane 9 shows IGHMBP2, ABT1, U3 snoRNA crosslinked 30 minutes; and lane 10 shows IGHMBP2, U3 snoRNA. Boxed area in lanes 3 and 7 represent the samples submitted for mass spectrometry. (J) Mass spectrometry IGHMBP2/ABT1 ratio with the 5′ ETS or U3 snoRNA. Three biological samples were normalized to total intensity to quantify IGHMBP2 and ABT1 ± SD.