ABSTRACT
The importance of translational regulation in tumour biology is increasingly appreciated. Here, we leverage polyribosomal profiling to prospectively define translational regulatory programs underlying epithelial-to-mesenchymal transition (EMT) in breast epithelial cells. We identify a group of ten translationally regulated drivers of EMT sharing a common GU-rich cis-element within the 3'-untranslated region (3'-UTR) of their mRNA. These cis-elements, necessary for the regulatory activity imparted by these 3'-UTRs, are directly bound by the CELF1 protein, which itself is regulated post-translationally during the EMT program. CELF1 is necessary and sufficient for both mesenchymal transition and metastatic colonization, and CELF1 protein, but not mRNA, is significantly overexpressed in human breast cancer tissues. Our data present an 11-component genetic pathway, invisible to transcriptional profiling approaches, in which the CELF1 protein functions as a central node controlling translational activation of genes driving EMT and ultimately tumour progression.
Subject(s)
CELF1 Protein/metabolism , Epithelial-Mesenchymal Transition/physiology , Gene Expression Regulation/physiology , Animals , Breast Neoplasms , CELF1 Protein/genetics , Cell Line, Tumor , Cell Movement , Cell Proliferation , Cell Survival , Epithelial Cells , Female , Gene Regulatory Networks , Humans , Mice , Neoplasms, Experimental , Prospective Studies , Protein Array Analysis , Protein Biosynthesis , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
T lymphocytes stimulated through their antigen receptor (TCR) preferentially express mRNA isoforms with shorter 3´ untranslated regions (3´-UTRs) derived from alternative pre-mRNA cleavage and polyadenylation (APA). However, the physiological relevance of APA programs remains poorly understood. CD5 is a T-cell surface glycoprotein that negatively regulates TCR signaling from the onset of T-cell activation. CD5 plays a pivotal role in mediating outcomes of cell survival or apoptosis, and may prevent both autoimmunity and cancer. In human primary T lymphocytes and Jurkat cells we found three distinct mRNA isoforms encoding CD5, each derived from distinct poly(A) signals (PASs). Upon T-cell activation, there is an overall increase in CD5 mRNAs with a specific increase in the relative expression of the shorter isoforms. 3´-UTRs derived from these shorter isoforms confer higher reporter expression in activated T cells relative to the longer isoform. We further show that polypyrimidine tract binding protein (PTB/PTBP1) directly binds to the proximal PAS and PTB siRNA depletion causes a decrease in mRNA derived from this PAS, suggesting an effect on stability or poly(A) site selection to circumvent targeting of the longer CD5 mRNA isoform by miR-204. These mechanisms fine-tune CD5 expression levels and thus ultimately T-cell responses.
Subject(s)
CD5 Antigens/genetics , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , Lymphocyte Activation/genetics , Lymphocyte Activation/immunology , MicroRNAs/genetics , Polyadenylation , Polypyrimidine Tract-Binding Protein/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , 3' Untranslated Regions , Base Sequence , CD5 Antigens/metabolism , Gene Expression Regulation , Humans , Jurkat Cells , Models, Biological , Poly A , RNA Interference , RNA Isoforms , RNA, Messenger/geneticsABSTRACT
Regulation of messenger ribonucleic acid (mRNA) subcellular localization, stability and translation is a central aspect of gene expression. Much of this control is mediated via recognition of mRNA 3' untranslated regions (UTRs) by microRNAs (miRNAs) and RNA-binding proteins. The gold standard approach to assess the regulation imparted by a transcript's 3' UTR is to fuse the UTR to a reporter coding sequence and assess the relative expression of this reporter as compared to a control. Yet, transient transfection approaches or the use of highly active viral promoter elements may overwhelm a cell's post-transcriptional regulatory machinery in this context. To circumvent this issue, we have developed and validated a novel, scalable piggyBac-based vector for analysis of 3' UTR-mediated regulation in vitro and in vivo. The vector delivers three independent transcription units to the target genome--a selection cassette, a turboGFP control reporter and an experimental reporter expressed under the control of a 3' UTR of interest. The pBUTR (piggyBac-based 3' UnTranslated Region reporter) vector performs robustly as a siRNA/miRNA sensor, in established in vitro models of post-transcriptional regulation, and in both arrayed and pooled screening approaches. The vector is robustly expressed as a transgene during murine embryogenesis, highlighting its potential usefulness for revealing post-transcriptional regulation in an in vivo setting.
