Research Article: Novel splicing in IGFN1 intron 15 and role of stable G-quadruplex in the regulation of splicing in renal cell carcinoma

Date Published: October 18, 2018

Publisher: Public Library of Science

Author(s): Shiv Prakash Verma, Parimal Das, Ravindra N. Singh.

http://doi.org/10.1371/journal.pone.0205660

Abstract

The IGFN1 (Immunoglobulin-Like And Fibronectin Type III Domain Containing 1) gene has a role in skeletal muscle function and is also involved in metastatic breast cancer, and the isoforms with three N-terminal globular domains are sufficient for its function in skeletal muscle. Two novel splicing isoforms of IGFN1 have been identified in renal cell carcinoma (RCC), one with 5’exon extension and an isoform with a novel exon. The role of G-quadruplex, a non-B DNA, was explored for the splicing alteration of IGFN1 in RCC. G-quadruplexes are the secondary structures acquired by stacking of G-quartets by Hoogsteen hydrogen bonding in DNA and RNA. IGFN1 has intronic potential G-quadruplex forming sequence (PQS) folding into G-quadruplex and is studied for its involvement in aberrant splicing. A PQS in the intron 15 of IGFN1 gene was observed in our in silico analysis by QGRS mapper and non BdB web servers. We observed PQS folds into stable G-quadruplex structure in gel shift assay and circular dichroism (CD) spectroscopy in the presence of G-quadruplex stabilizing agents Pyridostatin (PDS) and KCl, respectively. G-quadruplex formation site with single base resolution was mapped by Sanger sequencing of the plasmid constructs harbouring the cloned PQS and its mutant. This stable G-quadruplex inhibits reverse transcriptase and taq polymerase in reverse transcriptase & PCR stop assays. PDS changes the different splicing isoforms of IGFN1 in UOK146 cell line, displaying involvement of intronic G-quadruplex in IGFN1 splicing. These results lead us to propose that a stable G-quadruplex structure is formed in IGFN1 intron and a reason behind IGFN1 aberrant splicing which could be targeted for therapeutic intervention.

Partial Text

IGFN1 is specifically expressed in skeletal muscle and has sequence and structural homology to myosin binding protein-C fast and slow-type skeletal muscle isoforms. During muscle denervation IGFN1 is substantially upregulated leading to the down-regulation of protein synthesis via eEF1A interaction [1]. In Chinese population, IGFN1 is associated with susceptibility to Primary retroperitoneal liposarcoma [2].

Many diseases, particularly some neurodegenerative disorders are proven to be caused by G-quadruplex sequences [26,12,13]. This non-B DNA is also studied for its involvement in many types of cancers e.g. stomach, liver T-cell leukemia and follicular lymphoma etc. [10,18,19,27]. Intronic G-quadruplex affect the splicing of many genes such as hTERT, FMRP, TP53, PAX9, BACE2 [21, 28–31]. Theses splicing events lead to the enrichment of pathogenic form or increases the activity of particular isoform which is the reason behind the disease pathogenesis. In RCC novel splicing variants were identified in IGFN1 transcripts and validated by Sanger sequencing. Two novel variants one with 5’ exon extension and other with a novel exon were identified. Novel exon and the 5’exon extension in the IGFN1 has significant role in the relative expression of different isoforms of IGFN1. Identification of the factors and features which could be used or targeted to change the relative expression could be of therapeutic importance. Since two novel isoforms have been discovered in the present study, the role of these novel isoform towards cancer more particularly RCC could be of interest for biomarker discovery or therapeutics intervention.

 

Source:

http://doi.org/10.1371/journal.pone.0205660