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Analysis of the mechanism of action of the antisense RNA that controls the replication of the repABC plasmid p42d.

ABSTRACT: Replication and segregation of the Rhizobium etli symbiotic plasmid (pRetCFN42d) depend on the presence of a repABC operon, which carries all the plasmid-encoded elements required for these functions. All repABC operons share three protein-encoding genes (repA, repB, and repC), an antisense RNA (ctRNA) coding gene, and at least one centromere-like region (parS). The products of repA and repB, in conjunction with the parS region, make up the segregation system, and they negatively regulate operon transcription. The last gene of the operon, repC, encodes the initiator protein. The ctRNA is a negative posttranscriptional regulator of repC. In this work, we analyzed the secondary structures of the ctRNA and its target and mapped the motifs involved in the complex formed between them. Essential residues for the effective interaction localize at the unpaired 5' end of the antisense molecule and the loop of the target mRNA. In light of our results, we propose a model explaining the mechanism of action of this ctRNA in the regulation of plasmid replication in R. etli.

SUBMITTER: Cervantes-Rivera R

PROVIDER: S-EPMC2897686 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Analysis of the mechanism of action of the antisense RNA that controls the replication of the repABC plasmid p42d.

Cervantes-Rivera Ramón R Romero-López Cristina C Berzal-Herranz Alfredo A Cevallos Miguel A MA

Journal of bacteriology 20100430 13

Replication and segregation of the Rhizobium etli symbiotic plasmid (pRetCFN42d) depend on the presence of a repABC operon, which carries all the plasmid-encoded elements required for these functions. All repABC operons share three protein-encoding genes (repA, repB, and repC), an antisense RNA (ctRNA) coding gene, and at least one centromere-like region (parS). The products of repA and repB, in conjunction with the parS region, make up the segregation system, and they negatively regulate operon ...[more]

PMID: 20435728

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Project description:BACKGROUND: Recent evidence proposes a novel concept that mammalian natural antisense RNAs play important roles in cellular homeostasis by regulating the expression of several genes. Identification and characterization of retroviral antisense RNA would provide new insights into mechanisms of replication and pathogenesis. HIV-1 encoded-antisense RNAs have been reported, although their structures and functions remain to be studied. We have tried to identify and characterize antisense RNAs of HIV-1 and their function in viral infection. RESULTS: Characterization of transcripts of HEK293T cells that were transiently transfected with an expression plasmid with HIV-1NL4-3 DNA in the antisense orientation showed that various antisense transcripts can be expressed. By screening and characterizing antisense RNAs in HIV-1NL4-3-infected cells, we defined the primary structure of a major form of HIV-1 antisense RNAs, which corresponds to a variant of previously reported ASP mRNA. This 2.6 kb RNA was transcribed from the U3 region of the 3' LTR and terminated at the env region in acutely or chronically infected cell lines and acutely infected human peripheral blood mononuclear cells. Reporter assays clearly demonstrated that the HIV-1 LTR harbours promoter activity in the reverse orientation. Mutation analyses suggested the involvement of NF-?? binding sites in the regulation of antisense transcription. The antisense RNA was localized in the nuclei of the infected cells. The expression of this antisense RNA suppressed HIV-1 replication for more than one month. Furthermore, the specific knockdown of this antisense RNA enhanced HIV-1 gene expression and replication. CONCLUSIONS: The results of the present study identified an accurate structure of the major form of antisense RNAs expressed from the HIV-1NL4-3 provirus and demonstrated its nuclear localization. Functional studies collectively demonstrated a new role of the antisense RNA in viral replication. Thus, we suggest a novel viral mechanism that self-limits HIV-1 replication and provides new insight into the viral life cycle.

Dataset Information

Analysis of the mechanism of action of the antisense RNA that controls the replication of the repABC plasmid p42d.

Publications

Analysis of the mechanism of action of the antisense RNA that controls the replication of the repABC plasmid p42d.

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