NOVA1 regulates hTERT splicing and cell growth in non-small cell lung cancer

Andrew T Ludlow,Woodring E Wright,Jerry W Shay,Lu Zhang,Kimberly Batten,Tsung-Po Lai,Ilgen Mender,Jerome D Robin,Laura Yuan,Nicole Dahlson,Mohammed E Sayed,Enzo Tedone,Mandy Sze Wong

doi:10.1038/s41467-018-05582-x

Abstract

Alternative splicing is dysregulated in cancer and the reactivation of telomerase involves the splicing of TERT transcripts to produce full-length (FL) TERT. Knowledge about the splicing factors that enhance or silence FL hTERT is lacking. We identified splicing factors that reduced telomerase activity and shortened telomeres using a siRNA minigene reporter screen and a lung cancer cell bioinformatics approach. A lead candidate, NOVA1, when knocked down resulted in a shift in hTERT splicing to non-catalytic isoforms, reduced telomerase activity, and progressive telomere shortening. NOVA1 knockdown also significantly altered cancer cell growth in vitro and in xenografts. Genome engineering experiments reveal that NOVA1 promotes the inclusion of exons in the reverse transcriptase domain of hTERT resulting in the production of FL hTERT transcripts. Utilizing hTERT splicing as a model splicing event in cancer may provide new insights into potentially targetable dysregulated splicing factors in cancer.

Highlights

Alternative splicing is dysregulated in cancer and the reactivation of telomerase involves the splicing of TERT transcripts to produce full-length (FL) TERT
How the transcribed messenger RNA is processed (i.e., RNA-processing events), which is critical for determining if active telomerase is produced or not, is less well understood. One such RNA-processing regulatory mechanism is alternative splicing, which contributes to protein diversity and transcript abundance6. hTERT produces a transcript containing 16 exons that can be spliced into multiple isoforms[7,8,9,10], including the fulllength (FL) reverse transcriptase (RT) competent form
In non-small cell lung cancer cells that express high levels of neuro-oncological ventral antigen 1 (NOVA1), we found that stable reduction in NOVA1 levels shifted hTERT splicing toward inactive transcripts, reduced telomerase activity, which led to progressively shortened telomeres

Summary

Introduction

Alternative splicing is dysregulated in cancer and the reactivation of telomerase involves the splicing of TERT transcripts to produce full-length (FL) TERT. A lead candidate, NOVA1, when knocked down resulted in a shift in hTERT splicing to non-catalytic isoforms, reduced telomerase activity, and progressive telomere shortening. How the transcribed messenger RNA is processed (i.e., RNA-processing events), which is critical for determining if active telomerase is produced or not, is less well understood One such RNA-processing regulatory mechanism is alternative splicing, which contributes to protein diversity and transcript abundance. Little is currently known about the transacting factors that bind these regulatory regions Identification of such proteins would close a significant gap in telomerase regulation knowledge and potentially identify protein targets to shift the splicing of hTERT message to inactive forms to reduce telomerase activity, progressively shorten telomeres, and leading to reduced tumor growth in vivo. There are more than 500 RNA-binding proteins encoded in the genome and splicing is the result of cellular context, RNA secondary structure, RNA editing, and competition for splice sites, much is left to be learned concerning hTERT splicing regulation[16,17,18]

Objectives

Methods

Results

Conclusion