The Effects of HDV Ribozymes on Both the Initiation and Postpolyadenylation of HDV RNS Transcription (II)

  • Hsieh, Sen-Yung (PI)

Project: National Science and Technology CouncilNational Science and Technology Council Academic Grants

Project Details

Abstract

To investigate the roles of hepatitis delta virus (HDV) ribozyme on both the initiation of transcription and the transcription after polyadenylation, we adopted HDV cDNA transfection system to assay the effects of various ribozyme mutations on HDV RNA replication and the transcripts after polyadenylation. In terms of the effect on HDV RNA replication, we found that 1) mutation on either the genomic or the antigenomic HDV ribozyme, or mutation on either the ribozyme targeting site (self-cleavage site) or the ribozyme domain (enzyme domain), the genome replication capacity was lost; 2) the primary transcripts which generated from the cDNA and would be used as template for RNA to RNA synthesis was equal in amount, whether the cDNA contained wild type or mutant ribozyme sequences. Clearly, both the ribozymes on genome and antigenome are essential for HDV replication and the roles of ribozymes are more than stabilization of the transcripts. To further dissect the molecular mechanism of the ribozymes on HDV RNA replication, we used in vitro synthesized unit length HDV RNA with linear or circular conformation and containing either wide type or mutant ribozyme sequences for transfection. We found that 1) the circular RNAs were much better template for RNA synthesis than the linear RNAs; 2) there was no difference for RNA synthesis either the RNA with wild type or the template with mutant ribozyme sequences. Our findings suggested that one of the most significant roles of ribozyme in supporting HDV RNA replication is to process the HDV RNA as a circular template which is required for efficient transcription initiation. Previously, we have demonstrated that the ribozyme 3' juxtaposition to the polyadenylation site endowed stabilization and antitermination effect of the transcription after Polyadenylation. By using mutations on either the ribozyme targeting site or enzyme domain, we found the stabilization and antitermination effect were lost, suggesting that ribozyme activity was directly related. We further utilized the in vitro synthesized HDV RNA, to examine the unusual 5'-OH end of the downstream cleaved fragment and found the 5'-OH RNA had a longer half life than the 5'-phosphorylated RNA (8 minutes vs. 4 minutes) in a cellular lysate assay. However, since the difference was small, ribozyme function per se or cellular factors that interact with ribozyme might be involved.

Project IDs

Project ID:PB8407-1047
External Project ID:NSC84-2331-B182-045-MH
StatusFinished
Effective start/end date01/08/9431/10/95

Keywords

  • Hepatitis delta vitus
  • Ribozyme
  • Transcription
  • Polyadenylation

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