Identification and Validation of Putative Nesprin Variants

Nesprins are a family of multi-isomeric scaffolding proteins that were originally identified at the nuclear envelope (NE), where they bind to lamin A/C, emerin, and SUN-domain containing proteins, to form the LInker of Nucleoskeleton-and-Cytoskeleton (LINC) complex that connects the NE to the actin cytoskeleton. However, nesprin genes also give rise to a variety of tissue-specific variants of different sizes with potential roles beyond the NE. These variants are generated through alternative initiation, termination, and splicing, which makes nesprin biology very complex to study due to the difficulty in generating specific antibodies and/or short interfering RNAs (siRNA) to particular isoforms. In order to distinguish genuine nesprin variants and eliminate confusion with degradation products of larger nesprin isoforms, in this chapter we discuss methods including 5' and 3' Rapid Amplification of cDNA Ends (RACE) and RT-PCR in combination with EST database searching, for identifying and validating putative nesprin isoforms. This information is essential to allow a better understanding of nesprin functions in different cell types.