Dual Role of G-runs and hnRNP F in the Regulation of a Mutation-Activated Pseudoexon in the Fibrinogen Gamma-Chain Transcript

Valeria Rimoldi, Giulia Soldà, Rosanna Asselta, Silvia Spena, Cristiana Stuani, Emanuele Buratti, Stefano Duga

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Most pathological pseudoexon inclusion events originate from single activating mutations, suggesting that many intronic sequences are on the verge of becoming exons. However, the precise mechanisms controlling pseudoexon definition are still largely unexplored. Here, we investigated the cis-acting elements and trans-acting regulatory factors contributing to the regulation of a previously described fibrinogen gamma-chain (FGG) pseudoexon, which is activated by a deep-intronic mutation (IVS6-320A>T). This pseudoexon contains several G-run elements, which may be bound by heterogeneous nuclear ribonucleoproteins (hnRNPs) F and H. To explore the effect of these proteins on FGG pseudoexon inclusion, both silencing and overexpression experiments were performed in eukaryotic cells. While hnRNP H did not significantly affect pseudoexon splicing, hnRNP F promoted pseudoexon inclusion, indicating that these two proteins have only partially redundant functions. To verify the binding of hnRNP F and the possible involvement of other trans-acting splicing modulators, pulldown experiments were performed on the region of the pseudoexon characterized by both a G-run and enrichment for exonic splicing enhancers. This 25-bp-long region strongly binds hnRNP F/H and weakly interacts with Serine/Arginine-rich protein 40, which however was demonstrated to be dispensable for FGG pseudoexon inclusion in overexpression experiments. Deletion analysis, besides confirming the splicing-promoting role of the G-run within this 25-bp region, demonstrated that two additional hnRNP F binding sites might instead function as silencer elements. Taken together, our results indicate a major role of hnRNP F in regulating FGG pseudoexon inclusion, and strengthen the notion that G-runs may function either as splicing enhancers or silencers of the same exon.

Original languageEnglish
Article numbere59333
JournalPLoS One
Volume8
Issue number3
DOIs
Publication statusPublished - Mar 22 2013

Fingerprint

Heterogeneous-Nuclear Ribonucleoprotein Group F-H
fibrinogen
mutation
Mutation
exons
ribonucleoproteins
proteins
Exons
Transcriptional Silencer Elements
Trans-Splicing
Heterogeneous-Nuclear Ribonucleoproteins
serine
arginine
eukaryotic cells
binding sites
Proteins
Trans-Activators
Experiments
Eukaryotic Cells
Serine

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Dual Role of G-runs and hnRNP F in the Regulation of a Mutation-Activated Pseudoexon in the Fibrinogen Gamma-Chain Transcript. / Rimoldi, Valeria; Soldà, Giulia; Asselta, Rosanna; Spena, Silvia; Stuani, Cristiana; Buratti, Emanuele; Duga, Stefano.

In: PLoS One, Vol. 8, No. 3, e59333, 22.03.2013.

Research output: Contribution to journalArticle

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