title: Dissecting domains necessary for activation and repression of splicing by Muscleblind-like protein 1.
abstract: BACKGROUND
  Alternative splicing contributes to the diversity of the proteome, and provides the cell with an important additional layer of regulation of gene expression. Among the many RNA binding proteins that regulate alternative splicing pathways are the Muscleblind-like (MBNL) proteins. MBNL proteins bind YGCY motifs in RNA via four CCCH zinc fingers arranged in two tandem arrays, and play a crucial role in the transition from embryonic to adult muscle splicing patterns, deregulation of which leads to Myotonic Dystrophy. Like many other RNA binding proteins, MBNL proteins can act as both activators or repressors of different splicing events.
  
  
  RESULTS
  We used targeted point mutations to interfere with the RNA binding of MBNL1 zinc fingers individually and in combination. The effects of the mutations were tested in assays for splicing repression and activation, including overexpression, complementation of siRNA-mediated knockdown, and artificial tethering using MS2 coat protein. Mutations were tested in the context of both full length MBNL1 as well as a series of truncation mutants. Individual mutations within full length MBNL1 had little effect, but mutations in ZF1 and 2 combined were more detrimental than those in ZF 3 and 4, upon splicing activation, repression and RNA binding. Activation and repression both required linker sequences between ZF2 and 3, but activation was more sensitive to loss of linker sequences.
  
  
  CONCLUSIONS
  Our results highlight the importance of RNA binding by MBNL ZF domains 1 and 2 for splicing regulatory activity, even when the protein is artificially recruited to its regulatory location on target RNAs. However, RNA binding is not sufficient for activity; additional regions between ZF 2 and 3 are also essential. Activation and repression show differential sensitivity to truncation of this linker region, suggesting interactions with different sets of cofactors for the two types of activity.
authors.forename: Christopher
authors.initials: C
authors.lastname: Edge
authors.forename: Clare
authors.initials: C
authors.lastname: Gooding
authors.affiliation: Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, UK. cwjs1@cam.ac.uk.
authors.forename: Christopher W J
authors.initials: CW
authors.lastname: Smith
mesh_terms: D001665:Binding Sites / Q000235:genetics; D005091:Exons; D015972:Gene Expression Regulation, Neoplastic; D006367:HeLa Cells; D006801:Humans; D009132:Muscles / Q000378:metabolism*; D017354:Point Mutation; D011487:Protein Conformation; D055503:Protein Multimerization; D012313:RNA / Q000378:metabolism; D022821:RNA Splice Sites; D012326:RNA Splicing; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011973:Receptors, LDL / Q000235:genetics / Q000378:metabolism; D014335:Tropomyosin / Q000235:genetics / Q000378:metabolism; D016335:Zinc Fingers
chemical_list: C493663:MBNL1 protein, human; D022821:RNA Splice Sites; D016601:RNA-Binding Proteins; D011973:Receptors, LDL; C490775:TPM1 protein, human; D014335:Tropomyosin; C036053:VLDL receptor; D012313:RNA