Abstract
Insertional RNA editing has been observed and characterized in mitochondria of myxomycetes. The single subunit mitochondrial RNA polymerase adds nontemplated nucleotides co-transcriptionally to produce functional tRNA, rRNA and mRNAs with full genetic information. Addition of nontemplated nucleotides to the 3′ ends of RNAs have been observed in polymerases related to the mitochondrial RNA polymerase. This activity has been observed with T7 RNA polymerase (T7 RNAP), the well characterized prototype of the single subunit polymerases, as a nonspecific addition of nucleotides to the 3′ end of T7 RNAP transcripts in vitro. Here we show that this novel activity is an editing activity that can add specific ribonucleotides to 3′ ends of RNA or DNA when oligonucleotides, able to form intramolecular or intermolecular hairpin loops with recessed 3′ ends, are added to T7 RNA polymerase in the presence of at least one ribonucleotide triphosphate. Specific ribonucleotides are added to the recessed 3′ ends through Watson-Crick base pairing with the non-base paired nucleotide adjacent to the 3′ end. Optimization of this activity is obtained through alteration of the lengths of the 5′-extension, hairpin loop, and hairpin duplex. These properties define a T7 RNAP activity different from either transcriptional elongation or initiation.
Highlights
RNA editing is an alteration of the genetic information in RNA relative to its DNA template
Several laboratories have reported that single subunit RNAPs could add radiolabeled nucleotide triphosphates to the 3′ end of RNA in the absence of a DNA template[20,48,52] and that T7 RNA polymerase (T7 RNAP) could add ribonucleotides to the 3′ end of DNA and RNA44,46,47,53–55. We hypothesized that these observations indicated that these RNAPs had a novel activity similar to the RNA editing observed in mitochondria of Physarum polycephalum[7] and other myxomycetes[56]
We have developed an in vitro assay for labeling the 3′ ends of RNA and DNA oligonucleotides using T7 RNAP and a single radiolabeled ribonucleotide triphosphate
Summary
RNA editing is an alteration of the genetic information in RNA relative to its DNA template It is an additional step in gene expression, where specific nucleotides at specific positions are substituted for, added to, or deleted from the RNA in order to produce functional RNAs, especially mRNAs with a functional open reading frame. Specificity of the nucleotide insertion or deletion site and the number of nucleotides inserted or deleted at a site is determined by an antisense RNA, termed a guide RNA [gRNA], which has the genetic information to produce functional mRNAs significantly different from the template DNA13. With complex RNA populations without DNA templates in vitro any of the four ribonucleotides can be added Both aspects of the specificity of this co-transcriptional RNA editing involve the mtRNAP. The conservation of these structural and functional similarities extend to other single subunit polymerases, such as the bacteriophage RNAPs22–24, DNA polymerases (DNAPs) such as the Klenow fragment of DNAP I in E. coli[25,26,27], and reverse transcriptases (RNA-directed DNA polymerases) such as the HIV-1 reverse transcriptase[28,29,30], constituting a super-family of single subunit polymerases with structural and functional homology
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