Interaction of hnRNPA1/A2 and DAZAP1 with an Alu-Derived Intronic Splicing Enhancer Regulates ATM Aberrant Splicing

Tibor Pastor,Franco Pagani,Sue Cotterill

doi:10.1371/journal.pone.0023349

Tibor Pastor, Franco Pagani + Show 1 more

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https://doi.org/10.1371/journal.pone.0023349

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Abstract

We have previously identified an Alu-derived Intronic Splicing enhancer (ISE) in the Ataxia Teleangectasia Mutated gene (ATM) that facilitates intron pre-mRNA processing and leads to the inclusion of a cryptic exon in the final mRNA transcript. By using an RNA pull-down assay, we show here that hnRNPA1/A2, HuR and DAZAP1 splicing factors and DHX36 RNA helicase bind to the ISE. By functional studies (overexpression and siRNA experiments), we demonstrate that hnRNPA1 and DAZAP1 are indeed involved in ISE-dependent ATM cryptic exon activation, with hnRNPA1 acting negatively and DAZAP1 positively on splicing selection. On the contrary, HuR and DHX36 have no effect on ATM splicing pattern. These data suggest that splicing factors with both negative and positive effect can assemble on the intronic Alu repeats and regulate pre-mRNA splicing.

Highlights

Accurate intron excision and exon joining during the process of pre-mRNA splicing, enable generation of mature mRNAs that contain continuous coding sequence for protein synthesis
This deep intronic GTAA deletion in the Ataxia Teleangectasia Mutated gene (ATM) gene differs from the majority of described intronic variants as it is not directly related to the changes at splice sites but instead affects an Intronic Splicing Processing Element (ISPE) whose disruption abolishes a non-canonical interaction with U1 snRNP and leads to the activation of two nearby cryptic splice sites [5]
HuR was originally reported to be implicated in stabilization of AREcontaining mRNAs but it has been recently shown that is involved in splicing regulation where it promotes Fas exon 6 skipping by binding to an exonic splicing silencer [13]

Summary

Introduction

Accurate intron excision and exon joining during the process of pre-mRNA splicing, enable generation of mature mRNAs that contain continuous coding sequence for protein synthesis. Precise pre-mRNA splicing depends on the presence of splicing consensus sequences at 59 and 39 exon splice sites and additional intronic and exonic regulatory elements [1]. These elements are defined as Splicing Enhancer or Silencers according to their effect on splice site selection and are involved in normal and aberrant splicing regulation [2]. RNA-binding factors are key splicing regulators as their interaction with intronic and/or exonic sequences contributes to the splicing outcome. Each splicing factor has a positive or a negative effect on splicing, for example SR proteins are considered enhancers whereas hnRNPA1/A2 are silencers, but frequently the final effect on splicing is based on their binding position [3], which reflects specific interactions with multiple spliceosomal components on the nascent transcripts

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