The Characterization of the Molecular Structure and the Partners of MLIP

Abstract

A novel gene, Muscle-enriched A-type Lamin-Interacting Protein (MLIP), was identified in our lab through a yeast two-hybrid screen using Lamin A/C N-terminal sequence as bait (10-230 amino acids), in an attempt to shed light on the complex nature of laminopathies. MLIP is a non-homologous, single copy gene conserved in amniotes. MLIP’s expression differs between tissues and is subjected to extensive tissue-dependent alternative splicing. Some of MLIP’s isoforms, lacking specific exons, have been identified and referred to as MLIP Δ2, Δ3, Δ4, Δ6, Δ8 and Δ10. The putative Lamin binding domain was identified to be within the first 41 amino acids of MLIP, spanning across exon 1 and 2 of the MLIP gene. MLIP’s Molecular function remains largely unknown. The main objective of this study is to map the exact location of MLIP:Lamin A/C binding domain and the identification of MLIP isoform specific binding partners in different tissues. We hypothesized that the MLIP:Lamin binding domain is specific interaction and is located on the protein encoded by exon 1. We also hypothesized that the phenotypic observations in laminopathies may be in part due to the differential MLIP isoform expression in different tissue, whereas MLIP isoforms have different binding partners in different tissues resulting in variable functions. Our results demonstrate that MLIP is a spliced protein that contains alternative promoters that are tissue specific, which we annotated as MLIP-1a and 1b. The MLIP alternative promoters are located on the 5’ end of the MLIP gene and contain an alternative start site situated on exon 3. We also found that MLIP-1a splice variants are predominantly expressed in the heart and skeletal muscle, whereas MLIP-1b splice variants are expressed in almost all tissue types studied. In addition, hMLIP binds to Lamin A/C and the hMLIP-Lamin binding domain is exclusively specific to the protein encoded in exon-1a of the MLIP gene. Moreover, MLIP and it’s splice variants (1a and 1b) show high levels of expression in the early post-natal heart which decrease with age, mimicking the expression of Lamin A/C. This gives us a good indication that MLIP:Lamin A/C interaction plays a vital role in heart development and mutations in that part of the gene might contribute to laminopathies pathology.