RESUMEN
Proper base-pairing of a miRNA with its target mRNA is a key step in miRNA-mediated mRNA repression. RNA remodelling by RNA-binding proteins (RBPs) can improve access of miRNAs to their target mRNAs. The largest isoform p45 of the RBP AUF1 has previously been shown to remodel viral or AU-rich RNA elements. Here, we show that AUF1 is capable of directly promoting the binding of the miRNA let-7b to its target site within the 3'UTR of the POLR2D mRNA. Our data suggest this occurs in two ways. First, the helix-destabilizing RNA chaperone activity of AUF1 disrupts a stem-loop structure of the target mRNA and thus exposes the miRNA target site. Second, the RNA annealing activity of AUF1 drives hybridization of the miRNA and its target site within the mRNA. Interestingly, the RNA remodelling activities of AUF1 were found to be isoform-specific. AUF1 isoforms containing a YGG motif are competent RNA chaperones, whereas isoforms lacking the YGG motif are not. Overall, our study demonstrates that AUF1 has the ability to modulate a miRNA-target site interaction, thus revealing a new regulatory function for AUF1 proteins during post-transcriptional control of gene expression. Moreover, tests with other RBPs suggest the YGG motif acts as a key element of RNA chaperone activity.
Asunto(s)
Empalme Alternativo , Ribonucleoproteína Nuclear Heterogénea D0/genética , MicroARNs/genética , ARN Mensajero/genética , Motivos de Unión al ARN/genética , Regiones no Traducidas 3'/genética , Algoritmos , Secuencia de Aminoácidos , Regulación de la Expresión Génica , Ribonucleoproteína Nuclear Heterogénea D0/metabolismo , Humanos , Cinética , MicroARNs/metabolismo , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , ARN Mensajero/metabolismoRESUMEN
The 3'-terminal stem-loop (3'SL) of the RNA genome of the flavivirus West Nile (WNV) harbors, in its stem, one of the sequence elements that are required for genome cyclization. As cyclization is a prerequisite for the initiation of viral replication, the 3'SL was proposed to act as a replication silencer. The lower part of the 3'SL is metastable and confers a structural flexibility that may regulate the switch from the linear to the circular conformation of the viral RNA. In the human system, we previously demonstrated that a cellular RNA-binding protein, AUF1 p45, destabilizes the 3'SL, exposes the cyclization sequence, and thus promotes flaviviral genome cyclization and RNA replication. By investigating mutant RNAs with increased 3'SL stabilities, we showed the specific conformation of the metastable element to be a critical determinant of the helix-destabilizing RNA chaperone activity of AUF1 p45 and of the precision and efficiency of the AUF1 p45-supported initiation of RNA replication. Studies of stability-increasing mutant WNV replicons in human and mosquito cells revealed that the cultivation temperature considerably affected the replication efficiencies of the viral RNA variants and demonstrated the silencing effect of the 3'SL to be temperature dependent. Furthermore, we identified and characterized mosquito proteins displaying similar activities as AUF1 p45. However, as the RNA remodeling activities of the mosquito proteins were found to be considerably lower than those of the human protein, a potential cell protein-mediated destabilization of the 3'SL was suggested to be less efficient in mosquito cells. In summary, our data support a model in which the 3'SL acts as an RNA thermometer that modulates flavivirus replication during host switching.