RNA-dependent integrin α3 protein localization regulated by the Muscleblind-like protein MLP1

Abstract

We show that localized expression of the integrin α 3 protein is regulated at the level of RNA localization by the human homologue of Drosophila Muscleblind, MLP1/MBLL/MBNL2, a unique Cys 3 His zinc-finger protein. This is supported by the following observations: MLP1 knockdown abolishes localization of integrin α 3 to the adhesion complexes; MLP1 is localized in adhesion plaques that contain phospho-focal adhesion kinase; this localization is microtubule-dependent; integrin α 3 transcripts colocalize with MLP1 in distinct cytoplasmic loci; integrin α 3 transcripts are physically associated with MLP1 in cells and MLP1 binds to a specific ACACCC motif in the integrin α 3 3′ untranslated region (UTR) in vitro; and a green fluorescent protein (GFP) open reading frameintegrin α 3 3′ UTR chimeric gene directs GFP protein localization to distinct cytoplasmic loci near the cell periphery, which is dependent on MLP1 and is mediated by the ACACCC motif but is independent of the integrin α 3 signal peptide.We have recently identified a gene that is differentially expressed in many tumours. The cognate protein was named MLP1 for Muscleblind-like protein 1 (GenBank accession #AF491866) because of its high degree of homology with the Drosophila Muscleblind (Mbl) protein. The Drosophila mbl gene is involved in regulatory processes that are associated with the terminal differentiation of photoreceptors and muscles 1,2 . The Mbl protein is characterized by novel Cys 3 His zinc-finger motifs, CX 7 CX 6 CX 3 H-CX 7 CX 4 CX 3 H. Highly conserved homologues of mbl are found from worms and insects to mammals. Three human Mbl-like proteins -named EXP/MBNL1, MBLL/MBNL2 and MBXL/CHCR/MBNL3 -have been partially characterized 3-7 . MLP1 is the same as MBLL/MBNL2 and, as the former name was first used in the GenBank database, we will continue to use it throughout this report for brevity. These three proteins display RNA-binding activity towards transcripts that contain expanded trinucleotide repeats in myotonic dystrophy muscle cells, presumably sequestering the target RNAs 3-5 . These observations, however, did not shed light on the normal cellular functions of this family of proteins, although a role in mRNA processing has been implicated 7 . The developmental functions of Drosophila Mbl do not include early proliferation or differentiation of the muscle precursors 2 . In mbl mutants, muscles detach from the epidermis, indicating a defect in integrin-mediated adhesion. In addition, Mbl protein is found associated with the Zband that is rich in actin. Therefore, these mbl homologues may represent a unique group of regulatory genes that control the cellular functions that are related to cell-matrix interactions and cytoskeletal organization.

Developmental regulation of muscleblind‐like (MBNL) gene expression in the chicken embryo retina

“…The intensity of the MBNL1 bands in the retina appeared highest on ED15. Similar analysis with the MBNL2 antibody detected a single protein band of ca 40 kDa in both retina and muscle extracts, resembling the apparent molecular mass of human MBNL2 (Adereth et al, 2005). Based on our data, the expected size range of MBNL2 in chick is 22 kDa to 43 kDa, suggesting that the band at ϳ40 kDa is the most abundant one.…”

“…Given that it is now well established that protein synthesis does occur in axons and synaptic terminals (Hirokawa, 2006;Martin and Zukin, 2006;Schuman et al, 2006;Wells, 2006), it is conceivable that MBNL1 may participate in the transport of mRNAs for some photoreceptor synaptic proteins toward their terminals, much as MBNL2 does in the case of the transport of integrin-␣3 mRNA toward the areas of focal adhesion complexes (Adereth et al, 2005). We are currently investigating this possibility.…”

Muscleblind-Like Proteins

“…An unexpected function for MBNL2 in cytoplasmic mRNA transport has been reported, 39 but it is not yet known whether MBNL1 can also perform this function. In embryonic chick retina development, MBNL1 and MBNL2 become distributed in different regions of the photoreceptor, 52 but it is not known whether they perform similar or different functions there.…”

“…37,38 Endogenous MBNL2 has been little studied, but it does colocalize with ribonuclear foci in sections of cortical neurons 29 and heart 30 from DM1 patients and is able to regulate alternative splicing. 35 MBNL2 has also been reported to colocalize with integrin alpha3 mRNA at integrin-containing adhesion plaques, and it was suggested that MBNL2 may transport integrin alpha3 mRNA from the nucleus to the cytoplasm, 39 but the question of whether MBNL1 has a similar role was not examined. MBNL3 mRNA was present in placenta but was absent from all other tissue tested including skeletal muscle and heart.…”

Ribonuclear foci at the neuromuscular junction in myotonic dystrophy type 1

“…Muscleblind proteins are nuclear factors that participate in the developmental regulation of alternative splicing [6,23], and they also may regulate the transport and decay of mRNA [24]. However, except for microtubuleassociated protein tau, it is not known which transcripts expressed in subsynaptic nuclei or motor neurons are subject to CUG exp -induced spliceopathy.…”