Interferon-stimulated gene GBP1 protects SMN2 from degradation

doi:10.21203/rs.3.rs-2822819/v1

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Interferon-stimulated gene GBP1 protects SMN2 from degradation

https://doi.org/10.21203/rs.3.rs-2822819/v1

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Spinal muscular atrophy (SMA) is lethal autosomal recessive disease of muscle due to the pathogenic variations of the survival motor neuron 1 (SMN) encoding the motor neuron (SMN1, MIM#600354). The mutations of SMN result in insufficient full-length of SMN protein, which leads to muscle atrophy and even respiratory involvement. No effective treatments are currently available. The SMN2 gene is a regulator of the phenotype and its copy number is associated with the severity of disease. Previous studies have demonstrated that the interferons and IRF-1 induce the expression of SMN. In the current study, we found that patients with more copies of SMN2 had higher levels of GBP1 in peripheral blood and the stability of SMN2 protein was better with the presence of guanylate-binding protein 1(GBP1). Therefore, the IFN-γ-induced GBP1 protects SMN2 from degradation. Our findings provided new insights into the treatment of SMA.

Biological sciences/Genetics

Biological sciences/Molecular biology

Health sciences/Diseases

spinal muscular atrophy

guanylate-binding protein 1

survival motor neuron 2

Spinal muscular atrophy (SMA) is a hereditary disease of muscle weakness and muscle atrophy caused by degeneration of the anterior horn cells of the spinal cord, characterized by progressive, symmetrical weakness and atrophy of proximal limbs and trunk muscles. The patients may eventually die of respiratory failure and severe lung infection¹. SMA can be divided into 5 subtypes according to the age of onset, among which type 0 is infantile type and respiratory failure is the early phenotype. It is estimated that the general survival of these patients does not exceed six months. The types I, II, and III are all related to children ^2,3. Type I is the severe and the most common type, accounting for 45% of SMA and mostly die of respiratory failure before the age of 2. Type II is a moderate type, accounting for about 20% of the patients. The individuals of Type II are affected within 6 to 18 months and survive for more than 2 years. About 30% of patients are type III, a mild type and patients of this type are affected within 18 years of age. There is a good chance of long-term survival in this case. Types II and III SMA are mainly caused by the gene transformation from SMN1 to SMN2, so patients with mild SMA phenotype carry more copies of SMN2^2,4.

The frequency of SMA pathogenic variants carriers is as high as 1/54 to 1/40⁵. Drugs approved by Food and Drug administration (FDA) for the treatment of SMA include zolgensma, nusinersen, and risdiplam. Zolgensma is a therapeutic drug that utilizes non-replicating adeno-associated virus type 9 (scAAV9) as a vector to guide the correct SMN1 gene into neuronal cells to produce full-length SMN protein⁶. Nusinersen is an antisense oligonucleotides (ASO), which promotes the transcription of SMN2 gene into full-length mRNA containing exon 7 transcripts and then increases the expression of full-length SMN protein⁷. Risdiplam is an oral small-molecule drug and is a SMN2 splicing modifier that upregulates full-length SMN expression⁸. Although gene therapy has significantly developed and been used for treatment of SMA, many challenges still remain. The above-mentioned drugs are too expensive to afford for most families. Moreover, gene therapy is not effective for some patients with severe and a long history of SMA. Therefore, it is necessary to further explore new drugs to improve the treatment effect.

Guanylate-binding proteins (GBPs) belong to the GTPase family and the expression of GBPs can be induced by IFN-γ⁹. Human GBPs family can be divided into 7 members (GBP1 ~ GBP7) and GBP1 and GBP5 are the most studied in recent years¹⁰. GBP1 inhibits vesicular stomatitis virus (VSV), swine fever virus (CSFV), and encephalomyocarditis virus (EMCV) replication through its GTPase activity¹¹. It has been reported that interferon and IRF-1 promote SMN expression ¹². However, the relationship between interferon-induced downstream genes and SMN remains unknown.

Our study found that patients with more copies of SMN2 had higher expression lever of GBP1 in peripheral blood. Moreover, the transfection experiment showed that the mRNA of SMN2 in GBP1-transfected cells was not significantly different from that in the mock group. However, the protein of SMN2 was significantly higher than that of the control. Therefore, the IFN-γ-induced GBP1 may protect SMN2 from degradation. Our findings imply interferon or interferon-stimulated genes are associated with the severity of SMA, which provides new insights into the treatment of SMA.

Cells, samples and Plasmids

293T cells (ATCC, Rockefeller, MD, USA) were grown in DMEM (Gibco BRL, Gaithersburg, USA) containing 2 mmol/L L-glutamine (Amresco, Solon, USA), and 10% fetal bovine serum (FBS, Hyclone, Logan, USA), and antibiotics (100 IU/ml penicillin and 10 mg/ml streptomycin). Peripheral blood samples were obtained from patients who came to Mindong Hospital Affiliated to Fujian Medical University and volunteered for detection of SMN1 exon deletion. Plasmids pcDNA3.1-GBP1 and pcDNA3.1-SMN2 was synthesized by whole gene (Sangon Co., Ltd. Shanghai, China). The use of data and medical record was approved by the Ethics Committee of Mindong Hospital Affiliated to Fujian Medical University and conformed to the Declaration of Helsinki. All participants provided their written informed consents.

Deletion of SMN1 exons 7/8

The copy number of exon 7/8 of SMN1 was detected by PCR-melting curve method (Tianlong Technology Co., Ltd. Xian, China). Thermal cycling conditions consisted of 95℃ for 5 minutes, followed by 35 cycles of 95℃ for 10 seconds and 60℃ for 15 seconds. This was followed by a dissolution profile at 95°C for 1 minute and 40°C for 1 minute. The data is normalized by the software, and the P value and R value are calculated. When the P value is zero, it is homozygous deletion, when R≤0.70, it is heterozygous deletion, and no deletion was defined when R≥0.74.

Detection of SMN2 copies by digital PCR

The copy numbers of SMN2 were measured in each DNA sample using the QX200 Droplet Analyzer (BIO-RAD, USA). The patient's peripheral blood DNA was extracted with DNA rapid extraction kit (Sangon Co., Ltd. Shanghai, China) and its concentration was measured. Subsequent digital PCR assays, where droplets are first prepared, followed by PCR with probe: 5¢FAM-AAGGCAAGGCATTAC-3¢MGB, Thermal cycling conditions consisted of 95℃ for 10 minutes, followed by 40 cycles of 94℃ for 30 seconds and 60℃ for 1 minute, then heat inactivated at 98°C for 10 minutes. Finally, droplet analysis and detection¹³.

Transfection

Monolayer culture of 293T cells, grown overnight in a six-well dish, was transfected with different amounts of pcDNA3.1-GBP1 and pcDNA3.1-SMN2 by Lipofectamine 2000 (Invitrogen, CA, USA). Simultaneously blank group and mock group were set. The cells were incubated at 37℃ in a 5% CO2 incubator and harvested at different times for RT-qPCR and Protein quantification described later.

Quantitative real-time PCR

RT-qPCR was performed to determine SMN2 and GBP1 after transfecting 293T cells. Briefly, infected cells using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. 1.5mg RNA samples were reverse transcribed with the primers of oligo dT and at 70℃ 5min, and the cDNA was further synthesized 5 min on ice, then 5 min at 20℃, 1 h at 42℃, and 15 min at 70℃ and then stored at 4℃. The qPCR was performed by using SYBR green probe (Tiangen Biotech Co., Ltd. Beijing, China). The primers for the GBP1 gene were as follows: forward primer, 5¢- AGGAGTTCCTTCAAAGATGTGGA-3¢; reverse primer, 5¢- TTCTGAACAAAGAGACGATAGCC-3¢ and the SMN2 gene were as follows: forward primer, 5¢- CTATCATGCTGGCTGCCTCCATT-3¢; reverse primer, 5¢- TGTCTAAAACCCATATAATAGCC-3¢. Thermal cycling conditions included 15min at 95℃, followed by 45 cycles of 15 s at 95℃ and 1min at 60℃. The specificity of the qPCR products was verified by melting point analysis from 45℃ to 95℃.

Protein quantification by BCA

The protein concentrations were measured by using BCA protein assay kit (Sangon Co., Ltd. Shanghai, China) and generated standard curves using dilutions of bovine serum albumin (BSA) (Sigma, Steinheim, Germany). We normalized samples to 1gram total soluble protein from BCA-analysis. SMN2 protein levels in transfected cells were quantified using the standardized SMN2 ELISA (Enzo Life Sciences, Farmingdale, NY) and expressed as nanogram per 1 gram of total protein^14,15.

Statistical analyses

Statistical analyses were performed using GraphPad Prism 8 software (GraphPad Software, La Jolla, CA). The comparison was conducted using Student’s t-test. P < 0.05 was considered statistically significant.

Detection of deletion types in SMN1 exon 7/8.

PCR-melting showed that R and P values of patients with different deletion types of SMN1 exon 7/8. Homozygous deletion of SMN1 exon 7/8 has a P value of zero, and SMN1 exon 7/8 heterozygous deletion or non-deletion mainly depends on its R value(non-deletion R≥0.74, heterozygous deletion R≤0.70), which was shown in Figures 1(a) and 1(b). Although this method is simple, it can only be used to determine the copy number of the SMN1 gene. It cannot be used to determine the situation of two or more copies on the same chromosome and the existence of point mutations and other mutation types in the gene¹⁶. Therefore, it is necessary to use more accurate methods such as multiplex ligation dependence amplification (MLPA) or droplet digital PCR and gene sequencing^17,18.

Detection of SMN2 copies and GBP1 mRNA levels by digital PCR and RT-qPCR.

Digital PCR was used to detect the copy number of SMN2 in patients with different deletion types of SMN1 exon 7/8. The copy number of SMN2 in peripheral blood of patients with deletion of exon 7 was significantly higher than that of controls, but there was no significant difference between the patients with exon 8 deletion and controls (Figure2a-2e). Furthermore, the SMN2 copy number did not significantly increase in the neonates with homozygous deletion of exons 7 and 8 compared with the controls. We supposed that the innate immunity of infants cannot produce enough IFN-g{Li, 2020 #201}, and thus the infants cannot produce normal lever of SMN2¹⁹. We also examined GBP1 mRNA expression levels in patients with different SMN1 deletion types. It was revealed that patients with different SMN1 deletion types had higher levels of GBP1 in peripheral blood when the copy numbers of SMN2 increased (Figure 3).

RT-qPCR to detect the expression of SMN2 after cells transfected with GBP1.

To investigate the relationship between the GBP1 and SMN2, different amounts of pcDNA3.1-GBP1 and pcDNA3.1-SMN2 were transfected into 293T cells and the cells were harvested after 48 hours. No significant difference was found in the mRNA level of SMN2 in GBP1-transfected 293T cells compared with the mock group, while the protein level of SMN2 was significantly higher in GBP1-transfected 293T cells than that in the control group. The results were shown in Figures 4(a) and 4(b). At the same time, 2mg pcDNA3.1-GBP1 and 2mg pcDNA3.1-SMN2 were transfected into 293T cells and the cells were harvested at different times after transfection. The results showed that SMN2 protein was stable and at a high level from 48 hours to 72 hours after transfection, and the protein decreased significantly after 72 hours, which may be related to cell growth status (Figure 4c).

The difference between SMN1 and SMN2 lies in the C/T transition at position 6 of exon 7, whose mutation affects the activity of the exonic splicing enhancer (ESE) and causes SMN2 pre-mRNA to skip exon 7 during splicing²⁰. An unstable protein (SMN D7) was produced, and only about 20% of the SMN2 gene was able to produce full-length SMN protein (FL-SMN)²¹. In 2004, HC Chang et al. found that the degradation of SMN protein depends on the ubiquitin/protease pathway. However, the mechanisms regulating FL-SMN and SMNDD7 protein stability and degradation are rarely investigated^22,23. The interferon-stimulated gene GBP1 degrades viral proteins through its GTPase activity and ubiquitination²⁴. Sophie Baron-Delage et al. identified a candidate interferon-stimulated response element (ISRE) that overlaps the interferon regulatory factor-binding motif (IRF-E) in the promoter regions of the SMN and SMNc genes¹². In the current study, we found that SMN2 was more stable in cells with the overexpression of GBP1. This is either due to GBP1 being preferentially degraded, or due to GBP1 disrupting SMN2's ubiquitin/proteasome pathway, which should be further investigated.

The level of GBP1 in the peripheral blood of patients was associated with the copy number of SMN2. The level of SMN2 mRNA in GBP1-transfected cells was not significantly different from that in the mock group. However, the protein level of SMN2 was significantly higher in GBP1-transfected cells than the mock and blank groups. Therefore, IFN-γ-induced GBP1 may protect SMN2 from degradation. However, the mechanisms need investigation.

Data availability

The data used to support the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Author Contributions

ZL conceived of the study; MX participated in its design and drafted the manuscript; ML and JZ collected the samples; JL collected clinical data. All authors have read and approved the final manuscript.

Funding

This work was supported by the Natural Science Foundation of Fujian Province (2021J011454).

Acknowledgment

We are thankful for our patients who were voluntary to denote the peripheral blood samples.

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No competing interests reported.

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Interferon-stimulated gene GBP1 protects SMN2 from degradation

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