RESUMO
Cytidine to uridine editing of apolipoprotein B (apoB) mRNA requires the cytidine deaminase APOBEC-1 as well as a tripartite sequence motif flanking a target cytidine in apoB mRNA and an undefined number of auxiliary proteins that mediate RNA recognition and determine site-specific editing. Yeast engineered to express APOBEC-1 and apoB mRNA supported editing under conditions of late log phase growth and stationary phase. The cis -acting sequence requirements and the intracellular distribution of APOBEC-1 in yeast were similar to those described in mammalian cells. These findings suggest that auxiliary protein functions necessary for the assembly of editing complexes, or 'editosomes', are expressed in yeast and that the distribution of editing activity is to the cell nucleus.
Assuntos
Apolipoproteínas B/genética , Citidina Desaminase/genética , Citidina/genética , Edição de RNA , Saccharomyces cerevisiae/genética , Uridina/genética , Desaminase APOBEC-1 , Animais , Sequência de Bases , Citidina Desaminase/metabolismo , Primers do DNA , Ratos , Frações Subcelulares/metabolismoRESUMO
Yeast co-expressing rat APOBEC-1 and a fragment of human apolipoprotein B (apoB) mRNA assembled functional editosomes and deaminated C6666 to U in a mooring sequence-dependent fashion. The occurrence of APOBEC-1-complementing proteins suggested a naturally occurring mRNA editing mechanism in yeast. Previously, a hidden Markov model identified seven yeast genes encoding proteins possessing putative zinc-dependent deaminase motifs. Here, only CDD1, a cytidine deaminase, is shown to have the capacity to carry out C-->U editing on a reporter mRNA. This is only the second report of a cytidine deaminase that can use mRNA as a substrate. CDD1-dependent editing was growth phase regulated and demonstrated mooring sequence-dependent editing activity. Candidate yeast mRNA substrates were identified based on their homology with the mooring sequence-containing tripartite motif at the editing site of apoB mRNA and their ability to be edited by ectopically expressed APOBEC-1. Naturally occurring yeast mRNAs edited to a significant extent by CDD1 were, however, not detected. We propose that CDD1 be designated an orphan C-->U editase until its native RNA substrate, if any, can be identified and that it be added to the CDAR (cytidine deaminase acting on RNA) family of editing enzymes.
Assuntos
Citidina Desaminase/metabolismo , Edição de RNA , Leveduras/enzimologia , Desaminase APOBEC-1 , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Citidina Desaminase/análise , Citidina Desaminase/química , Citidina Desaminase/genética , Imunofluorescência , Teste de Complementação Genética , Humanos , Cinética , Cadeias de Markov , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Estrutura Terciária de Proteína , Edição de RNA/genética , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Leveduras/genéticaRESUMO
Two 3'-5' exoribonucleases, polynucleotide phosphorylase and ribonuclease II play a central role in the degradation of bacterial mRNA to ribonucleotides. Sequences with the potential to form stem-loop structures can stabilize upstream mRNA against 3'-5' exoribonucleolytic attack in vivo by blocking the processive activities of these enzymes. For many mRNA species stem-loop structures appear to provide a very efficient block to decay from the 3' end, such that the rate-determining step for mRNA decay occurs elsewhere in the transcript. We have examined the stalling of 3'-5' exoribonucleases at stem-loop structures in vitro. Although stem-loop structures alone can impede the progress of both enzymes, the duration of stalling at these structures in vitro is insufficient to account for the increased half-lives that they confer on mRNA in vivo. These data suggest that an additional factor, such as a stem-loop binding protein, is required for stabilization of mRNA by stem-loop structures in vivo. The implications for the regulation of mRNA stability are discussed.