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Structure and Alternative Splicing of the Antisense FMR1 (ASFMR1) Gene

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Abstract

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder caused by an expansion of 55–200 CGG repeats (premutation) in the 5'-UTR of the FMR1 gene. Bidirectional transcription at FMR1 locus has been demonstrated and specific alternative splicing of the Antisense FMR1 (ASFMR1) gene has been proposed to have a contributing role in the pathogenesis of FXTAS. The structure of ASFMR1 gene is still uncharacterized and it is currently unknown how many isoforms of the gene are expressed and at what level in premutation carriers (PM) and if they may contribute to the premutation pathology. In this study, we characterized the ASFMR1 gene structure and the transcriptional landscape by using PacBio SMRT sequencing with target enrichment (IDT customized probe panel). We identified 45 ASFMR1 isoforms ranging in sizes from 523 bp to 6 Kb, spanning approximately 59 kb of genomic DNA. Multiplexing and sequencing of six human brain samples from PM samples and normal control (HC) were carried out on the PacBio Sequel platform. We validated the presence of these isoforms by qRT-PCR and Sanger sequencing and characterized the acceptor and donor splicing site consensus sequences. Consistent with previous studies conducted in other tissue types, we found a high expression of ASFMR1 isoform Iso131bp in brain samples of PM as compared to HC, while no differences in expression levels were observed for the newly identified isoforms IsoAS1 and IsoAS2. We investigated the role of the splicing regulatory protein Sam68 which we did not observe in the alternative splicing of the ASFMR1 gene. Our study provides a useful insight into the structure of ASFMR1 gene and transcriptional landscape along with the expression pattern of various newly identified novel isoforms and on their potential role in premutation pathology.

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Data Availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank the participants of the community‐based studies who donated their time and sample for this study. This paper is dedicated to the memory of Matteo.

Funding

This work was supported by the NIH grants RO1HD036071 and RO1MH07804.

Author information

Authors and Affiliations

  1. Department of Biochemistry and Molecular Medicine, University of California Davis, School of Medicine, Sacramento, CA, 95817, USA

    Marwa Zafarullah & Flora Tassone

  2. Bioinformatics Core, Genome Center, University of California Davis, Davis, CA, 95616, USA

    Jie Li

  3. Pacific Biosciences, Inc, Menlo Park, CA, 94025, USA

    Elizabeth Tseng

  4. MIND Institute, University of California Davis Medical Center, Sacramento, CA, 95817, USA

    Flora Tassone

Authors
  1. Marwa Zafarullah
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  2. Jie Li
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  4. Flora Tassone
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Contributions

Designed Research.

Conception: F. Tassone

Organization: M. Zafarullah, and F. Tassone

Execution: M. Zafarullah, F. Tassone

Analyzed Data

Design: F. Tassone, E. Tseng

Execution: J.LI, E. Tseng,

Review and Critique: M. Zafarullah, J. Li, E. Tseng, F. Tassone

Manuscript Preparation

Writing the first draft: M. Zafarullah

Review and Critique: M. Zafarullah, J. Li, E. Tseng, F. Tassone

All authors read and approved the final manuscript.

Corresponding author

Correspondence to Flora Tassone.

Ethics declarations

Ethics Approval

All samples were collected under approved Institutional Review Board (IRB) protocols and all methods were carried out in accordance with the relevant guidelines and regulations.

Consent to Participate

Informed consent was obtained from all individual participants included in the study.

Consent for Publication

Not applicable.

Competing Interests

M.Z.: No disclosures to report.

J.L.: No disclosures to report.

E.T: E.T. is an employee of Pacific Biosciences.

F.T. has received the funding from Azrieli Foundation and Zynerba for studies in Fragile X syndrome.

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Zafarullah, M., Li, J., Tseng, E. et al. Structure and Alternative Splicing of the Antisense FMR1 (ASFMR1) Gene. Mol Neurobiol 60, 2051–2061 (2023). https://doi.org/10.1007/s12035-022-03176-9

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  • DOI: https://doi.org/10.1007/s12035-022-03176-9

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