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Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay EMSA and DNA-affinity Precipitation Assay DAPA

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Identification and Validation of Putative Nesprin Variants.

Flavia Autore^1,2, Catherine M Shanahan², Qiuping Zhang³

¹Randall Division of Cell and Molecular Biophysics, King's College London, London, UK.

Methods in Molecular Biology (Clifton, N.J.)

|May 6, 2016

Summary

Nesprins are scaffolding proteins forming the LInker of Nucleoskeleton-and-Cytoskeleton (LINC) complex. New methods help identify nesprin variants, clarifying their complex biology and functions.

Keywords:

Alternative splicing EST database Multi-tissue RT-PCR Nesprins RACE

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Area of Science:

Cell Biology
Molecular Biology
Biochemistry

Background:

Nesprins are multi-isomeric scaffolding proteins found at the nuclear envelope.
They form the LInker of Nucleoskeleton-and-Cytoskeleton (LINC) complex, connecting the nucleus to the cytoskeleton.
Nesprin genes produce diverse tissue-specific variants, complicating research.

Purpose of the Study:

To present methods for identifying and validating nesprin isoforms.
To address the complexity arising from alternative splicing and variant generation.
To facilitate a clearer understanding of nesprin functions.

Main Methods:

Utilizing 5' and 3' Rapid Amplification of cDNA Ends (RACE).
Employing RT-PCR in conjunction with EST database searching.
Developing strategies to distinguish true nesprin variants from degradation products.

Main Results:

Established protocols for identifying and validating novel nesprin isoforms.
Provided a framework for differentiating specific nesprin variants.
Aided in resolving ambiguities in nesprin research.

Conclusions:

Accurate identification of nesprin variants is crucial for understanding their roles.
The discussed methods enable precise characterization of nesprin isoforms.
This work advances the study of nesprin biology in various cell types.