Prostate cancer resistance leads to a global deregulation of translation factors and unconventional translation.

Emerging evidence associates translation factors and regulators to tumorigenesis. However, our understanding of translational changes in cancer resistance is still limited. Here, we generated an enzalutamide-resistant prostate cancer (PCa) model, which recapitulated key features of clinical enzalutamide-resistant PCa. Using this model and poly(ribo)some profiling, we investigated global translation changes that occur during acquisition of PCa resistance. We found that enzalutamide-resistant cells exhibit an overall decrease in mRNA translation with a specific deregulation in the abundance of proteins involved in mitochondrial processes and in translational regulation. However, several mRNAs escape this translational downregulation and are nonetheless bound to heavy polysomes in enzalutamide-resistant cells suggesting active translation. Moreover, expressing these corresponding genes in enzalutamide-sensitive cells promotes resistance to enzalutamide treatment. We also found increased association of long non-coding RNAs (lncRNAs) with heavy polysomes in enzalutamide-resistant cells, suggesting that some lncRNAs are actively translated during enzalutamide resistance. Consistent with these findings, expressing the predicted coding sequences of known lncRNAs JPX, CRNDE and LINC00467 in enzalutamide-sensitive cells drove resistance to enzalutamide. Taken together, this suggests that aberrant translation of specific mRNAs and lncRNAs is a strong indicator of PCa enzalutamide resistance, which points towards novel therapeutic avenues that may target enzalutamide-resistant PCa.

DDX3 regulates endoplasmic reticulum stress-induced ATF4 expression.

Accumulation of unfolded and potentially toxic proteins in the endoplasmic reticulum (ER) activates a cell stress adaptive response, which involves a reprogramming of general gene expression. ATF4 is a master stress-induced transcription factor that orchestrates gene expression in cells treated with various ER stress inducers including those used to treat cancers. ER stress-induced ATF4 expression occurs mainly at the translational level involving the activity of the phosphorylated (P) translation initiation factor (eIF) eIF2α. While it is well established that under ER stress PeIF2α drives ATF4 expression through a specialised mode of translation re-initiation, factors (e.g. RNA-binding proteins and specific eIFs) involved in PeIF2α-mediated ATF4 translation remain unknown. Here we identified the RNA-binding protein named DDX3 as a promotor of ATF4 expression in cancer cells treated with sorafenib, an ER stress inducer used as a chemotherapeutic. Depletion experiments showed that DDX3 is required for PeIF2α-mediated ATF4 expression. Luciferase and polyribosomes assays showed that DDX3 drives ER stress-induced ATF4 mRNA expression at the translational level. Protein-interaction assays showed that DDX3 binds the eIF4F complex, which we found to be required for ER stress-induced ATF4 expression. This study thus showed that PeIF2α-mediated ATF4 mRNA translation requires DDX3 as a part of the eIF4F complex.

Analysis of the translatome in solid tumors using polyribosome profiling/RNA-Seq.

Gene expression involves multiple steps from the transcription of a mRNA in the nucleus to the production of the encoded protein in the cytoplasm. This final step occurs through a highly regulated process of mRNA translation on ribosomes that is required to maintain cell homeostasis. Alterations in the control of mRNA translation may lead to cell's transformation, a hallmark of cancer development. Indeed, recent advances indicated that increased translation of mRNAs encoding tumor-promoting proteins may be a key mechanism of tumor resistance in several cancers. Moreover, it was found that proteins whose encoding mRNAs are translated at higher efficiencies may be effective biomarkers. Evaluation of global changes in translation efficiency in human tumors has thus the potential of better understanding what can be used as biomarkers and therapeutic targets. Investigating changes in translation efficiency in human cancer cells has been made possible through the development and use of the polyribosome profiling combined with DNA microarray or deep RNA sequencing (RNA-Seq). While helpful, the use of cancer cell lines has many limitations and it is essential to define translational changes in human tumor samples in order to properly prioritize genes implicated in cancer phenotype. We present an optimized polyribosome RNA-Seq protocol suitable for quantitative analysis of mRNA translation that occurs in human tumor samples and murine xenografts. Applying this innovative approach to human tumors, which requires a complementary bioinformatics analysis, unlocks the potential to identify key mRNA which are preferentially translated in tumor tissue compared to benign tissue as well as translational changes which occur following treatment. These technical advances will be of interest to those researching all solid tumors, opening possibilities for understanding what may be therapeutic Achilles heels' or relevant biomarkers.

A Polyadenylation-Dependent 3' End Maturation Pathway Is Required for the Synthesis of the Human Telomerase RNA.

Telomere maintenance by the telomerase reverse transcriptase requires a noncoding RNA subunit that acts as a template for the synthesis of telomeric repeats. In humans, the telomerase RNA (hTR) is a non-polyadenylated transcript produced from an independent transcriptional unit. As yet, the mechanism and factors responsible for hTR 3' end processing have remained largely unknown. Here, we show that hTR is matured via a polyadenylation-dependent pathway that relies on the nuclear poly(A)-binding protein PABPN1 and the poly(A)-specific RNase PARN. Depletion of PABPN1 and PARN results in telomerase RNA deficiency and the accumulation of polyadenylated precursors. Accordingly, a deficiency in PABPN1 leads to impaired telomerase activity and telomere shortening. In contrast, we find that hTRAMP-dependent polyadenylation and exosome-mediated degradation function antagonistically to hTR maturation, thereby limiting telomerase RNA accumulation. Our findings unveil a critical requirement for RNA polyadenylation in telomerase RNA biogenesis, providing alternative approaches for telomerase inhibition in cancer.

Poly(A) tail-mediated gene regulation by opposing roles of Nab2 and Pab2 nuclear poly(A)-binding proteins in pre-mRNA decay.

The 3' end of most eukaryotic transcripts is decorated by poly(A)-binding proteins (PABPs), which influence the fate of mRNAs throughout gene expression. However, despite the fact that multiple PABPs coexist in the nuclei of most eukaryotes, how functional interplay between these nuclear PABPs controls gene expression remains unclear. By characterizing the ortholog of the Nab2/ZC3H14 zinc finger PABP in Schizosaccharomyces pombe, we show here that the two major fission yeast nuclear PABPs, Pab2 and Nab2, have opposing roles in posttranscriptional gene regulation. Notably, we find that Nab2 functions in gene-specific regulation in a manner opposite to that of Pab2. By studying the ribosomal-protein-coding gene rpl30-2, which is negatively regulated by Pab2 via a nuclear pre-mRNA decay pathway that depends on the nuclear exosome subunit Rrp6, we show that Nab2 promotes rpl30-2 expression by acting at the level of the unspliced pre-mRNA. Our data support a model in which Nab2 impedes Pab2/Rrp6-mediated decay by competing with Pab2 for polyadenylated transcripts in the nucleus. The opposing roles of Pab2 and Nab2 reveal that interplay between nuclear PABPs can influence gene regulation.