title: Functional studies on the ATM intronic splicing processing element.
abstract: In disease-associated genes, the understanding of the functional significance of deep intronic nucleotide variants may represent a difficult challenge. We have previously reported a new disease-causing mechanism that involves an intronic splicing processing element (ISPE) in ATM, composed of adjacent consensus 5' and 3' splice sites. A GTAA deletion within ISPE maintains potential adjacent splice sites, disrupts a non-canonical U1 snRNP interaction and activates an aberrant exon. In this paper, we demonstrate that binding of U1 snRNA through complementarity within a approximately 40 nt window downstream of the ISPE prevents aberrant splicing. By selective mutagenesis at the adjacent consensus ISPE splice sites, we show that this effect is not due to a resplicing process occurring at the ISPE. Functional comparison of the ATM mouse counterpart and evaluation of the pre-mRNA splicing intermediates derived from affected cell lines and hybrid minigene assays indicate that U1 snRNP binding at the ISPE interferes with the cryptic acceptor site. Activation of this site results in a stringent 5'-3' order of intron sequence removal around the cryptic exon. Artificial U1 snRNA loading by complementarity to heterologous exonic sequences represents a potential therapeutic method to prevent the usage of an aberrant CFTR cryptic exon. Our results suggest that ISPE-like intronic elements binding U1 snRNPs may regulate correct intron processing.
authors: International Centre for Genetic Engineering and Biotechnology Padriciano 99, 34012 Trieste, Italy.
authors: Marzena A
authors: MA
authors: Lewandowska
authors: Cristiana
authors: C
authors: Stuani
authors: Alireza
authors: A
authors: Parvizpur
authors: Francisco E
authors: FE
authors: Baralle
authors: Franco
authors: F
authors: Pagani
mesh_terms: D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism; D016384:Consensus Sequence; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005091:Exons; D006801:Humans; D007438:Introns; D051379:Mice; D008969:Molecular Sequence Data; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D012322:RNA Precursors / Q000378:metabolism; D022821:RNA Splice Sites; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D012322:RNA Precursors; D022821:RNA Splice Sites; D012333:RNA, Messenger; D017412:Ribonucleoprotein, U1 Small Nuclear; D025521:Tumor Suppressor Proteins; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C576325:Atm protein, mouse; D017346:Protein Serine-Threonine Kinases