Expression of murine muscle-enriched A-type lamin-interacting protein (MLIP) is regulated by tissue-specific alternative transcription start sites

Abstract

Muscle-enriched lamin-interacting protein (Mlip) is an alternatively spliced gene whose splicing specificity is dictated by tissue type. MLIP is most abundantly expressed in brain, cardiac, and skeletal muscle. In the present study, we systematically mapped the transcriptional start and stop sites of murine Mlip Rapid amplification of cDNA ends (RACE) of Mlip transcripts from the brain, heart, and skeletal muscle revealed two transcriptional start sites (TSSs), exon 1a and exon 1b, and only one transcriptional termination site. RT-PCR analysis of the usage of the two identified TSSs revealed that the heart utilizes only exon 1a for MLIP expression, whereas the brain exclusively uses exon 1b TSS. Loss of Mlip exon 1a in mice resulted in a 7-fold increase in the prevalence of centralized nuclei in muscle fibers with the Mlip exon1a-deficient satellite cells on single fibers exhibiting a significant delay in commitment to a MYOD-positive phenotype. Furthermore, we demonstrate that the A-type lamin-binding domain in MLIP is encoded in exon 1a, indicating that MLIP isoforms generated with exon 1b TSS lack the A-type lamin-binding domain. Collectively these findings suggest that Mlip tissue-specific expression and alternative splicing play a critical role in determining MLIP's functions in mice.

Keywords: MLIP; alternative splicing; brain; cellular localization; gene expression; heart; intracellular trafficking; muscle-enriched lamin-interacting protein; protein isoform; protein-protein interaction; skeletal muscle; splice variant; tissue-specific expression; transcriptional regulation; transcriptional start site.

Figure 1.

MLIP expression in the heart and brain of Mlip^+/+ and Mlip^ΔE1/ΔE1 mice. A, generation of Mlip knockout mouse model (Mlip ΔE1) by deletion of exon 1 (E1) of the Mlip gene and its proximal promoter. Orange arrows indicate flox sites. Mlip Ab, polyclonal antibody targeting an epitope in exon 11 of the gene. B, Western blot with MLIP antibody of Mlip^+/+ and Mlip^ΔE1/ΔE1 brain and heart. The polyclonal MLIP antibodies are specific for an epitope encoded within exon 11. Lamin B was used as a loading control. C, PCR amplification of Mlip cDNA from brain and heart of Mlip^+/+ and Mlip^ΔE1/ΔE1 mice using forward primers specific for Mlip exon 1b or exon 1a, respectively, and exon 14 reverse primer. Ubc was used as a loading control.

Figure 2.

Identification of two Mlip alternative transcriptional start sites. A, 5′-RACE reactions targeting Mlip were performed on mRNA extracted from adult mouse heart, brain, and skeletal muscle and subsequently identified by DNA sequencing. B, tissue distribution of exon 1a and exon 1b identified from 96 clones from heart, brain, and skeletal muscle. C and D, display of UCSC Genome Browser (ENCODE project data) showing histone H3 epigenetic marks (H3K27Ac, H3K4Me1, and H3K4Me3; the height of the peak correlates with the strength of signal in a given cell line) centered on exon 1a (C) and exon 1b (D) of the Mlip gene in different cell lines.

Figure 3.

MLIP tissue expression profile in Mlip^+/+ mice. A and B, PCR amplification of Mlip cDNA on tissues of Mlip^+/+ mice using different combinations of primers (arrows; A, a–e) targeting exons of the Mlip gene. *, nonspecific amplification. Ubc was used as a loading control. C, Western blot with MLIP antibody of MLIP^+/+ brain, heart, and skeletal (Sk.) muscle. *, tissue-specific isoforms. Actin was used as a loading control.

Figure 4.

Cellular localization of MLIP isoforms. A, schematic representation of the different synthetic human Myc-tagged Mlip isoforms used to transfect 293 cells. B, Western blot with Myc tag antibody. GAPDH was used as a loading control. C, immunostaining of 293 cells with Myc tag antibody (red), wheat germ agglutinin (WGA; green), and 4′,6-diamidino-2-phenylindole (DAPI; blue). Scale bars, 20 μm. D, Western blot with MLIP antibody of nuclear and cytoplasmic fractions isolated from MLIP^+/+ heart. GAPDH and lamin B were used as cellular fractionation controls. *, cytoplasmic-specific isoform.

Figure 5.

Structure, expression and splicing of the Mlip gene. A, Mlip exon 1a encodes an A-type lamin interaction domain. Purified recombinant MLIP isoforms and lamin A fusion proteins were incubated together. Complexes were immunoprecipitated and resolved by SDS-PAGE. B, schematic summary of MLIP–LMNA interaction assay. C, structure of the Mlip gene. The size of the boxes, corresponding to the exons, is proportional to the exon's size in bp. The lines (representing the introns) are not representative of the actual size of the introns in the genome. D, using alternative start sites located in exon 1a or exon1b, the gene gives rise to different mRNA expressed in a tissue-specific manner. ✓, tissue in which the given mRNA is expressed; BD, known binding domain. E, inclusion of the exons in the mRNA based on sequencing data. The gray boxes represent the exons largely spliced out (versatile regions), exon 3 (light green box) is included in about 50% of all the mRNA (“semi”-ubiquitous region), and exons 9–11 and exon 14 (dark green) constitute a ubiquitous region of the gene.