Targeted Long-Read Sequencing Decodes the Transcriptional Atlas of the Founding RAS Gene Family Members.

Panagiotis G Adamopoulos,Panagiotis Tsiakanikas,Andreas Scorilas,Michaela A Boti

doi:10.3390/ijms222413298

Panagiotis G Adamopoulos, Panagiotis Tsiakanikas + Show 2 more

Open Access

https://doi.org/10.3390/ijms222413298

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Abstract

The complicity of human RAS proteins in cancer is a well-documented fact, both due to the mutational hyperactivation of these GTPases and the overexpression of the genes encoding these proteins. Thus, it can be easily assumed that the study of RAS genes at the transcriptional and post-transcriptional level is of the utmost importance. Although previous research has shed some light on the basic mechanisms by which GTPases are involved in tumorigenesis, limited information is known regarding the transcriptional profile of the genes encoding these proteins. The present study highlights for the first time the wide spectrum of the mRNAs generated by the three most significant RAS genes (KRAS, NRAS and HRAS), providing an in-depth analysis of the splicing events and exon/intron boundaries. The implementation of a versatile, targeted nanopore-sequencing approach led to the identification of 39 novel RAS mRNA transcript variants and to the elucidation of their expression profiles in a broad panel of human cell lines. Although the present work unveiled multiple hidden aspects of the RAS gene family, further study is required to unravel the biological function of all the novel alternative transcript variants, as well as the putative protein isoforms.

Highlights

The human RAS superfamily of small guanosine triphosphatases (GTPases) consists of more than 150 members and is subdivided into five distinct categories (RAS, RHO, RAB, ARF and RAN) on the basis of the protein-members’ sequences, structures and functional characteristics [1]
The RAS subfamily consists of GTPases that are anchored to the membrane via post-transcriptional modifications that occurred at their C-terminal region, guiding their localization into distinct membrane compartments [2,3,4] where they are capable of regulating signal-transduction pathways [5]
We developed and employed a versatile targeted nanopore sequencing approach to the in-depth detection of new alternative splicing events of the founding RAS family gene members (KRAS, NRAS and HRAS), and the identification of novel mRNA transcripts

Summary

Introduction

The human RAS superfamily of small guanosine triphosphatases (GTPases) consists of more than 150 members and is subdivided into five distinct categories (RAS, RHO, RAB, ARF and RAN) on the basis of the protein-members’ sequences, structures and functional characteristics [1]. According to the GenBank® database, the human genes that encode the founding Ras GTPases (KRAS, NRAS and HRAS) produce 9 protein-coding alternative splice variants that lead to the generation of 6 distinct protein isoforms. These isoforms are characterized by high sequence similarities, sharing major structural and functional features. The G-domain consists of the residues 1–166 and propagates signaling when bound to GTP, whereas it is inactive when bound to GDP. It consists of two lobes and its active site resides within the first half of the domain, referred as the effector lobe (residues 1–86). The specific part of the catalytic domain contains three of the five highly conserved motifs that are observed in small GTPases [8,9], and is 100% identical in sequence among the RAS isoforms [10,11]

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