RNAi screening of subtracted transcriptomes reveals tumor suppression by taurine-activated GABAA receptors involved in volume regulation.

Camiel L C Wielders,Hein Te Riele,Floris Foijer,Huibert D Mansvelder,Joanne Verheij,Pim Van Nierop,Tinke L Vormer,Jesper B Andersen,Johannes C Lodder

doi:10.1371/journal.pone.0196979

Abstract

To identify coding and non-coding suppressor genes of anchorage-independent proliferation by efficient loss-of-function screening, we have developed a method for enzymatic production of low complexity shRNA libraries from subtracted transcriptomes. We produced and screened two LEGO (Low-complexity by Enrichment for Genes shut Off) shRNA libraries that were enriched for shRNA vectors targeting coding and non-coding polyadenylated transcripts that were reduced in transformed Mouse Embryonic Fibroblasts (MEFs). The LEGO shRNA libraries included ~25 shRNA vectors per transcript which limited off-target artifacts. Our method identified 79 coding and non-coding suppressor transcripts. We found that taurine-responsive GABAA receptor subunits, including GABRA5 and GABRB3, were induced during the arrest of non-transformed anchor-deprived MEFs and prevented anchorless proliferation. We show that taurine activates chloride currents through GABAA receptors on MEFs, causing seclusion of cell volume in large membrane protrusions. Volume seclusion from cells by taurine correlated with reduced proliferation and, conversely, suppression of this pathway allowed anchorage-independent proliferation. In human cholangiocarcinomas, we found that several proteins involved in taurine signaling via GABAA receptors were repressed. Low GABRA5 expression typified hyperproliferative tumors, and loss of taurine signaling correlated with reduced patient survival, suggesting this tumor suppressive mechanism operates in vivo.

Highlights

Anchorage-independent proliferation, a hallmark of transformation, involves deregulation of oncogenes and tumor suppressor genes [1]
To produce Low-complexity by Enrichment for Genes shut Off (LEGO) libraries, we designed a set of oligonucleotide adapters A, B, and C (Figures A, B in S1 Fig) that allow (1) highly optimized PCR-based Suppression Subtractive Hybridization (SSH) of two transcriptomes [7] to selectively amplify cDNA sequences from repressed polyadenylated transcripts, and (2) subsequent enzymatic processing of selected cDNA sequences into a library of pRETRO Super shRNA vectors for screening (Figure I in S1 Fig)
Production of LEGO libraries enriched for suppressors of anchorless growth To identify novel tumor suppressor pathways, we produced and screened two LEGO (Lowcomplexity by Enrichment for Genes shut Off) shRNA libraries that focus on putative suppressors of anchorage-independent growth

Summary

Introduction

Anchorage-independent proliferation, a hallmark of transformation, involves deregulation of oncogenes and tumor suppressor genes [1]. To identify additional tumour suppressor genes that are repressed when DKO RASV12 cells fully transform, we enzymatically produced low-complexity shRNA libraries that were subsequently used for loss-of-function screening.

Results

Conclusion