ABSTRACT
Ribonucleotidyl transferases (rNTases) add untemplated ribonucleotides to diverse RNAs. We have developed TRAID-seq, a screening strategy in Saccharomyces cerevisiae to identify sequences added to a reporter RNA at single-nucleotide resolution by overexpressed candidate enzymes from different organisms. The rNTase activities of 22 previously unexplored enzymes were determined. In addition to poly(A)- and poly(U)-adding enzymes, we identified a cytidine-adding enzyme that is likely to be part of a two-enzyme system that adds CCA to tRNAs in a eukaryote; a nucleotidyl transferase that adds nucleotides to RNA without apparent nucleotide preference; and a poly(UG) polymerase, Caenorhabditis elegans MUT-2, that adds alternating uridine and guanosine nucleotides to form poly(UG) tails. MUT-2 is known to be required for certain forms of RNA silencing, and mutants of the enzyme that result in defective silencing did not add poly(UG) tails in our assay. We propose that MUT-2 poly(UG) polymerase activity is required to promote genome integrity and RNA silencing.
Subject(s)
Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans/genetics , Nucleotidyltransferases/genetics , RNA Interference , RNA Nucleotidyltransferases/genetics , Saccharomyces cerevisiae/genetics , Animals , Caenorhabditis elegans/enzymology , Mutation , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
Alterations in regulatory networks contribute to evolutionary change. Transcriptional networks are reconfigured by changes in the binding specificity of transcription factors and their cognate sites. The evolution of RNA-protein regulatory networks is far less understood. The PUF (Pumilio and FBF) family of RNA regulatory proteins controls the translation, stability, and movements of hundreds of mRNAs in a single species. We probe the evolution of PUF-RNA networks by direct identification of the mRNAs bound to PUF proteins in budding and filamentous fungi and by computational analyses of orthologous RNAs from 62 fungal species. Our findings reveal that PUF proteins gain and lose mRNAs with related and emergent biological functions during evolution. We demonstrate at least two independent rewiring events for PUF3 orthologs, independent but convergent evolution of PUF4/5 binding specificity and the rewiring of the PUF4/5 regulons in different fungal lineages. These findings demonstrate plasticity in RNA regulatory networks and suggest ways in which their rewiring occurs.
Subject(s)
Fungal Proteins/genetics , Gene Regulatory Networks , RNA, Messenger/genetics , RNA-Binding Proteins/genetics , 3' Untranslated Regions , Aspergillus nidulans/genetics , Binding Sites , Evolution, Molecular , Fungal Proteins/metabolism , Gene Expression Regulation, Fungal , Neurospora crassa/genetics , Phylogeny , RNA-Binding Proteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Cytoplasmic polyadenylation is important in the control of mRNA stability and translation, and for early animal development and synaptic plasticity. Here, we focus on vertebrate poly(A) polymerases that are members of the recently described GLD2 family. We identify and characterize two closely related GLD2 proteins in Xenopus oocytes, and show that they possess PAP activity in vivo and in vitro and that they bind known polyadenylation factors and mRNAs known to receive poly(A) during development. We propose that at least two distinct polyadenylation complexes exist in Xenopus oocytes, one of which contains GLD2; the other, maskin and Pumilio. GLD2 protein interacts with the polyadenylation factor, CPEB, in a conserved manner. mRNAs that encode GLD2 in mammals are expressed in many tissues. In the brain, mouse, and human GLD2 mRNAs are abundant in anatomical regions necessary for long-term cognitive and emotional learning. In the hippocampus, mouse GLD2 mRNA colocalizes with CPEB1 and Pumilio1 mRNAs, both of which are likely involved in synaptic plasticity. We suggest that mammalian GLD2 poly(A) polymerases are important in synaptic translation, and in polyadenylation throughout the soma.
Subject(s)
Brain/enzymology , Oocytes/enzymology , Polynucleotide Adenylyltransferase/metabolism , RNA-Binding Proteins/metabolism , Xenopus Proteins/metabolism , 3T3 Cells , Amino Acid Sequence , Animals , Brain/metabolism , Catalytic Domain , Female , Glutathione Transferase/metabolism , Humans , Mice , Microinjections , Molecular Sequence Data , Oocytes/metabolism , Polynucleotide Adenylyltransferase/chemistry , Polynucleotide Adenylyltransferase/genetics , Protein Biosynthesis , Protein Structure, Secondary , Protein Structure, Tertiary , RNA, Messenger/analysis , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid , Spleen/cytology , Xenopus , Xenopus Proteins/chemistry , Xenopus Proteins/genetics , mRNA Cleavage and Polyadenylation Factors/metabolismABSTRACT
RNA-protein interactions are essential for the proper execution and regulation of every step in the life of a eukaryotic mRNA. Here we describe a three-hybrid system in which RNA-protein interactions can be analyzed using simple phenotypic or enzymatic assays in Saccharomyces cerevisiae. The system can be used to detect or confirm an RNA-protein interaction, to analyze RNA-protein interactions genetically, and to discover new protein or RNA partners when only one is known. Multicomponent complexes containing more than one protein can be detected, identified, and analyzed. We describe the method and how to use it, and discuss applications that bear particularly on eukaryotic mRNAs.
