Lineage-specific roles of the cytoplasmic polyadenylation factor CPEB4 in the regulation of melanoma drivers

Abstract

Nuclear 3'-end-polyadenylation is essential for the transport, stability and translation of virtually all eukaryotic mRNAs. Poly(A) tail extension can also occur in the cytoplasm, but the transcripts involved are incompletely understood, particularly in cancer. Here we identify a lineage-specific requirement of the cytoplasmic polyadenylation binding protein 4 (CPEB4) in malignant melanoma. CPEB4 is upregulated early in melanoma progression, as defined by computational and histological analyses. Melanoma cells are distinct from other tumour cell types in their dependency on CPEB4, not only to prevent mitotic aberrations, but to progress through G1/S cell cycle checkpoints. RNA immunoprecipitation, sequencing of bound transcripts and poly(A) length tests link the melanoma-specific functions of CPEB4 to signalling hubs specifically enriched in this disease. Essential in these CPEB4-controlled networks are the melanoma drivers MITF and RAB7A, a feature validated in clinical biopsies. These results provide new mechanistic links between cytoplasmic polyadenylation and lineage specification in melanoma.

MITF—the first 25 years

“…In addition to transcriptional regulation, as might be expected for a key transcription factor, MITF is also subject to posttranscriptional regulation via both control of its mRNA polyadenylation by CPEB4 (Pérez-Guijarro et al 2016) and the action of microRNAs that play a key role in melanoma biology (Bell and Levy 2011;Kunz 2013). To date, several microRNAs have been described as inhibiting MITF expression, including miR-26a in melanoma (Qian et al 2017), miR-340 in osteoclasts (Zhao et al 2017), and miR-137 (Bemis et al 2008), miR-148 (Haflidadóttir et al 2010, miR-155 (Arts et al 2015), and miR-182 (Yan et al 2012).…”

Systems analysis identifies melanoma-enriched pro-oncogenic networks controlled by the RNA binding protein CELF1

“…Therefore, it will be interesting to address to which extent this melanoma RBP landscape is shared with other tumor types. In particular, genes modulating 3′ UTR-associated functions may provide insight into the control of lineage specification as we recently reported for the cytoplasmic polyadenylation factor CPEB4 20 . In this context, unbiased transcriptomic and proteomic data sets generated here may serve as a tractable platform for the identification of new roles of RBPs controlling the still elusive mechanisms underlying lineage specificity or tumor-type identity.…”

“…Moreover, X-ray 43 and nuclear magnetic resonance studies 44 revealed that CELF1 is organized in complex 3D oligomeric structures with open and close conformations that could result in ambiguous readings of iCLIP. Therefore, we selected to perform RNA immunoprecipitations (RIP) using protocols we have proven efficient for RBPs such as the cytoplasmic polyadenylation factor CPEB4 in melanoma cells 20 . RIP was then followed by RNA sequencing (RIP-Seq), resulting in at least 10 million reads/sample aligned to the human genome (GRCh37/hg19).…”

“…Cell lines have been authenticated using GenePrint 10 Loci Service. Primary human melanocytes, keratinocytes, and fibroblasts were isolated from foreskins of healthy donors by differential tripsinization 20 . Melanocytes were cultured in Medium 254 (Invitrogen) supplemented with 1% melanocyte growth factors (HMGS, Invitrogen), 0.2 mM CaCl 2 , and 100 μg/mL penicillin/streptomycin; keratinocytes were cultured in Epilife Medium (Invitrogen) supplemented with 1% keratinocyte growth factors (HKGS, Invitrogen), 0.2 mM CaCl 2 and 100 μg/mL penicillin/streptomycin and fibroblasts were cultured in DMEM, supplemented with 10% FBS, and 100 μg/mL penicillin/streptomycin.…”

“…Of those, the least understood are 3′ UTR-related events. We have recently identified two 3′ end-interacting factors (the cytoplasmic polyadenylation protein CPEB4, and the translation modulator UNR) with key roles in melanoma progression 19 , 20 . Nevertheless, genome-wide analyses of the genomic landscape of RBPs in melanoma are still pending.…”