ABSTRACT
mRNAs carry two layers of information, the genetic code and the information that dictates their post-transcriptional fate. The latter function relies on a complex interplay between cis-elements and trans-regulators, and unbiased identification of these elements is still challenging. To identify cis-elements that control gene expression, we use dimethyl sulfate (DMS) mutational profiling with sequencing and map changes in mRNA secondary structure following viral infection. Our dynamic structural data reveal a major role for ribosomes in unwinding secondary structures, which is further supported by the relationship we uncover between structure and translation efficiency. Moreover, our analysis revealed dozens of regions in viral and cellular mRNAs that exhibit changes in secondary structure. In-depth analysis of these regions reveals cis-elements in 3' UTRs that regulate mRNA stability and elements within coding sequences that control translation. Overall, our study demonstrates how mapping dynamic changes in mRNA structure allows unbiased identification of functional regulatory elements.
Subject(s)
Cytomegalovirus/genetics , Gene Expression Regulation , Host-Pathogen Interactions/genetics , Phosphoproteins/genetics , Protein Biosynthesis , RNA, Messenger/chemistry , Viral Matrix Proteins/genetics , Cell Line , Cytomegalovirus/drug effects , Cytomegalovirus/metabolism , Fibroblasts/drug effects , Fibroblasts/metabolism , Fibroblasts/virology , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Mutagens/pharmacology , Nucleic Acid Conformation , Phosphoproteins/metabolism , RNA Stability , RNA, Messenger/genetics , RNA, Messenger/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Ribosomes/genetics , Ribosomes/metabolism , Signal Transduction , Sulfuric Acid Esters/pharmacology , Viral Matrix Proteins/metabolismABSTRACT
MicroRNAs (miRNAs) play critical roles in a broad variety of biological processes by inhibiting translation initiation and by destabilizing target mRNAs. The CCR4-NOT complex effects miRNA-mediated silencing, at least in part through interactions with 4E-T (eIF4E transporter) protein, but the precise mechanism is unknown. Here we show that the cap-binding eIF4E-homologous protein 4EHP is an integral component of the miRNA-mediated silencing machinery. We demonstrate that the cap-binding activity of 4EHP contributes to the translational silencing by miRNAs through the CCR4-NOT complex. Our results show that 4EHP competes with eIF4E for binding to 4E-T, and this interaction increases the affinity of 4EHP for the cap. We propose a model wherein the 4E-T/4EHP interaction engenders a closed-loop mRNA conformation that blocks translational initiation of miRNA targets.