CNR - Institute of Neuroscience CNR
Institute of Neuroscience
 

Project

Molecular enzymology of DM-related MBLN proteins

Background

MBLN are alternative splicing regulators fundamental for mRNA processing. Myotonic Distrophy (DM) is an autosomal dominant multisystemic disorder and the most common form of muscular dystrophy in adults that affects 1 in 8000 individuals worldwide. One form of the disorder (DM1) is caused by an expanded (CTG)n, in the 3' untranslated region of the Dystrophia Myotonica Protein Kinase (DMPK) gene. In 1998, a second form of myotonic dystrophy (DM2) was described that closely mimic the DM1 phenotype. The DM2 mutation consists in the expansion of a tetranucleotidic repetition (CCTG)n in the first intron of the ZNF9 (Zinc Finger Protein) gene. Commom to DM1 and DM2 is the expression of expanded mutant CUG- and CCUG-containing RNA transcripts, which accumulate in the nuclei of diseased cells to form nuclear foci (ribonuclear inclusions) altering the regulation and localization of the alternative splicing regulators including, among others, MBLN proteins. Human Muscleblind paralogs MBNL1, MBLN2 and MBLN3 promote inclusion or exclusion of specific exons on different pre-mRNA by antagonizing the activity of CUG-BP and ETR-3-like factors (CELF proteins), with different functional specializations. MBNL1 seems to promote muscle differentiation, MBLN3 appears to function in opposite manner, and MBLN2 participates in integrin α3 subcellular localization. MBNL1 and MBNL2 are found in numerous tissues, with maximal MBNL1 expression in muscle and heart.

Objectives

While there is a good experimental evidence supporting the correlation between the altered function of muscleblind proteins (MBNL) and the symptoms of myotonic dystrophy (DM), the molecular mechanism(s) underlying their sequestration by the RNA repeat extensions from the normal alternative splicing machinery is still poorly understood. This project is aimed at undertaking a biochemical investigation on the regulatory residues and/or domains of the isoform MBNL1 potentially implicated in its altered function. Since a number of highly conserved putative phosphorylation sites and a classical type II consensus for SH3 domains are located in the MBNL1 primary structure, two aspects are on focus in our research: i) the regulation of MBNL1 by phosphorylation of specific residues and ii) the MBNL1 interaction potential mediated by proline-rich domain(s).

Methods

These goals are addressed firstly with in vitro experiments by using the recombinant MBNL1 protein aiming, on one side, at the identification of the site(s) phosphorylated in MBNL1 by purified protein kinase(s) and, on the other, at the characterization of the MBNL1 interaction with purified tyrosine kinases of the Src family (SFKs). Then the search will be transferred to a cellular model, taking advantage of the myogenic C2C12 cell line and of the availability of primary human myoblasts from controls or DM1 and DM2 patients, to possibly correlate by using phospho-specific antibodies the phosphorylation state of MBNL1 with its altered function. Moreover, since Src family tyrosine kinases, besides MBNL1, are directly involved in myogenesis and neural differentiation, we will assess, by co-IP and MS analyses, whether these proteins interact also in the cell with MBNL1.

Grants

Progetto di Eccellenza CARIPARO 2008 (Resp. Prof. Corrado Angelini)

Collaborations

  • Prof. Corrado Angelini, Dipartimento di Neuroscienze, Università di Padova, Italy.
  • Prof. Giuseppe Novelli, Dipartimento di Biopatologia e Diagnostica per Immagini, Università di Roma Tor Vergata, Italy.
  • Prof. Elisabetta Gianazza, Dipartimento di Scienze Farmacologiche, Università di Milano, Italy.
  • Prof. Hugo Monaco, Dipartimento di Biotecnologie, Università di Verona, Italy.

 

PI photo

Lorenzo Pinna

Contact information

email  E-mail

email  049 8276108

Participating staff

Flavio Meggio
Giorgio Cozza
Mario A. Pagano
Oriano Marin
Anna Brunati
Elena Tibaldi