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
The Ten-a gene of Drosophila melanogaster encodes several alternative variants of a full length member of the Odz/Tenm protein family. A number of Ten-a mutants created by inexact excisions of a resident P-element insertion are embryonic lethal, but show no pair-rule phenotype. In contrast, these mutants, and deficiencies removing Ten-a, do enhance the segmentation phenotype of a weak allele of the paralog gene odz (or Ten-m) to the odz amorphic phenotype. Germ line clone derived Ten-a(-) embryos display a pair-rule phenotype which phenocopies that of odz. Post segmentation eye patterning phenotypes of Ten-a mutants establish it as a pleiotropic patterning co-partner of odz.