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.
Highlights
IGFN1 is expressed in skeletal muscle and has sequence and structural homology to myosin binding protein-C fast and slow-type skeletal muscle isoforms
In UOK146 only amplicon A & C were present whereas amplicon B along with A & C were present in other renal cell carcinoma (RCC) cell lines e.g. UOK109 and ACHN
Intronic G-quadruplex affect the splicing of many genes such as hTERT, FMRP, TP53, PAX9, BACE2 [21, 28,29,30,31]
Summary
IGFN1 is expressed in skeletal muscle and has sequence and structural homology to myosin binding protein-C fast and slow-type skeletal muscle isoforms. IGFN1 along with KY and FLNC is the part of a Z-band associated protein complex providing structural support to the skeletal muscle [3]. Alternative splicing regulates the proteomic diversity and apart from different splicing factors, RNA sequences with Non-B DNA structures are involved in the isoform specific gene expression. Non-B DNA is the non canonical form of DNA involved in almost all basic biological process e.g. replication, transcription, translation etc. These types of structure include hairpins/cruciforms, triplexes (H-DNA), Z-DNA, tetraplex, sticky DNA, slipped-DNA and G-quadruplex [5,6,7]. At DNA level G-quadruplexes are involved in BCL2 major breakpoint region t(14;18) translocation follicular lymphoma [18], HOX11 gene in t(10;14) translocation in T-cell leukemia [19], cMyc translocation and hypermutation [20], telomere replication
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