Unraveling TRIM71-mediated RNA regulatory mechanisms during developmental and oncogenic processes

Abstract

A dysregulation in the tight control of cell proliferation and differentiation severely affects developmental processes, and can lead to oncogenic transformation in the adult organism. The stem cell-specific protein TRIM71 is essential for embryonic development and has been associated with tumorigenesis in several cancer types. TRIM71 belongs to the highly-conserved family of TRIM-NHL proteins, whose structural domain organization enables their role as post-transcriptional regulators via mRNA repression, and as post-translational regulators via protein ubiquitylation. These distinct molecular functions assist TRIM71 in its role promoting proliferation and preventing premature differentiation of embryonic stem cells (ESCs), progenitor cells and cancer cells. However, a better characterization of TRIM71 molecular functions is mandatory in order to understand its essential functions in development, and eventually to design novel stem cell-/cancer-specific therapeutic strategies targeting TRIM71. <br /> TRIM71 interacts with several proteins involved in the miRNA pathway, but the functional outcome of such interactions remains unclear. In the first chapter of this thesis, we investigated the role of TRIM71 in the miRNA pathway. Our work revealed that TRIM71 regulates miRNA expression and activity via two independent mechanisms. On one hand, TRIM71 represses the expression of the pro-differentiation and tumor suppressor miRNA let-7 by enhancing the TUT4/LIN28-mediated degradation of its precursor miRNA pre-let-7. On the other hand, TRIM71 represses the activity of pro-differentiation miRNAs such as let-7 and the brain-specific miR-128, by a mechanism that depends on AGO2 binding. We found that TRIM71 mutations present in congenital hydrocephalus (CH) patients – R608H and R796H – abrogate the interaction between TRIM71 and AGO2, suggesting that an impaired ability of TRIM71 to repress miRNA activity may be involved in the pathophysiology of this brain developmental disease. <br /> Furthermore, TRIM71 is known to act as an mRNA repressor, but the mechanisms for mRNA target recognition and degradation remain largely elusive. The second chapter of this thesis describes the mechanistic unraveling of TRIM71-mediated recognition and repression of the CDKN1A mRNA, which encodes for the cell cycle inhibitor and tumor suppressor p21. A negative correlation between TRIM71 and CDKN1A expression has been observed in patients with advanced hepatocellular carcinoma (HCC), and we showed that TRIM71 represses CDKN1A mRNA and controls proliferation of HepG2 HCC cells. Our work revealed that CDKN1A specific recognition involves the direct interaction of TRIM71 NHL domain with a highly conserved structural RNA motif present in the 3’UTR of CDKN1A. We found TRIM71-mediated CDKN1A regulation to be AGO2- and miRNA-independent. Instead, the Nonsense-Mediated Decay (NMD) factors SMG1, UPF1 and SMG7 assist TRIM71-mediated degradation of CDKN1A mRNA, among other targets. The canonical NMD pathway controls the degradation of transcripts with a premature termination codon (PTC), which are recognized via the Exon Junction Complex (EJC). However, NMD also regulates functional transcripts lacking PTCs (non-canonical NMD), but how such transcripts are recognized by the NMD machinery remains unknown. We showed that TRIM71 is required for the NMD machinery to recognize several mRNA targets, including CDKN1A, while the NMD degradation of PTC-containing targets is TRIM71-independent. Thus, our work uncovered a role for TRIM71 in non-canonical NMD, revealing the existence of a novel target-specific and cell-specific RNA surveillance mechanism which we have termed the TRIM71/NMD axis. Last, we proved that the CH mutants R608H and R796H fail to bind mRNA, thereby having an impaired TRIM71/NMD axis. Indeed, TRIM71-deficient murine ESCs (mESCs) and TRIM71 CH mutant mESCs show a premature CDKN1A/p21 upregulation accompanied by proliferation defects in the course of neural differentiation. Thus, the regulation of CDKN1A via the TRIM71/NMD axis, not only may play a fundamental role in tumorigenic processes such as HCC, but also in developmental diseases such as CH. <br /> Altogether, our work reports several novel mechanisms of TRIM71-mediated miRNA and mRNA regulation, and will stimulate future studies to investigate the in vivo implications of these mechanisms in developmental and oncogenic processes, as well as to identify other TRIM71 miRNA and mRNA targets of pathophysiological relevance.