The Removal of RNA Primers from DNA Synthesized by the Reverse Transcriptase of the Retrotransposon Tf 1 Is Stimulated by Tf 1 Integrase

Abstract

The Tf 1 retrotransposon represents a group of long terminal repeat retroelements that use an RNA self-primer for initiating reverse transcription while synthesizing the minus-sense DNA strand. Tf 1 reverse transcriptase (RT) was found earlier to generate the self-primer in vitro . Here, we show that this RT can remove from the synthesized cDNA the entire self-primer as well as the complete polypurine tract (PPT) sequence (serving as a second primer for cDNA synthesis). However, these primer removals, mediated by the RNase H activity of Tf 1 RT, are quite inefficient. Interestingly, the integrase of Tf 1 stimulated the specific Tf 1 RT-directed cleavage of both the self-primer and PPT, although there was no general enhancement of the RT's RNase H activity (and the integrase by itself is devoid of any primer cleavage). The RTs of two prototype retroviruses, murine leukemia virus and human immunodeficiency virus, showed only a partial and nonspecific cleavage of both Tf 1 -associated primers with no stimulation by Tf 1 integrase. Mutagenesis of Tf 1 integrase revealed that the complete Tf 1 integrase protein (excluding its chromodomain) is required for stimulating the Tf 1 RT primer removal activity. Nonetheless, a double mutant integrase that has lost its integration functions can still stimulate the RT's activity, though heat-inactivated integrase cannot enhance primer removals. These findings suggest that the enzymatic activity of Tf 1 integrase is not essential for stimulating the RT-mediated primer removal, while the proper folding of this protein is obligatory for this function. These results highlight possible new functions of Tf 1 integrase in the retrotransposon's reverse transcription process.

New methods as alternative or corrective measures for the pitfalls and artifacts of reverse transcription and polymerase chain reactions (RT-PCR) in cloning chimeric or antisense-accompanied RNA

“…Although retrovirus uses cellular tRNA to prime mRNA for reverse transcriptases to synthesize the 1st DNA strand, 31 , 32 endogenous small RNAs such as mRNA fragments can efficiently prime cDNA synthesis by reverse transcriptases 12 , 20 , 22 . RNA samples contain a huge number of short RNA fragments, such as degraded RNAs, excised introns and other processed mRNAs that are known to us recently, 1 which can serve as ERP for RT.…”

The dUTPase-related gene of bovine immunodeficiency virus is critical for viral replication, despite the lack of dUTPase activity of the encoded protein

“…The columns were then washed with 50 ml of this washing buffer. Finally, elution was performed with 20–500 mM imidazole linear gradients in the same buffer [ 37 , 48 , 49 , 51 ]. The purity of the column-eluted dUTPase enzymes were assessed by SDS-PAGE and western analysis with monoclonal anti-6HIS HRP- conjugated antibodies (from Sigma).…”

RNA Chimeras as a Gene Signature of Breast Cancer

“…No matter which one goes into which, it indicates a previously unknown mechanism for RNA fusion, besides the transcriptional-slippage and trans-splicing, because transportation of nRNA to the mitochondria or fusion of the mtRNA to the nRNA should occur after the nuclear transcription has been terminated and splicing has been completed. The identification of those ESTs in which the downstream partner is fused to the poly-A tail of the upstream partner also supports a ''post-transcription and post-splicing'' mechanism, because polyadenylation occurs after both RNA transcript cleavage and splicing completion, although the polyadenylation may continue in the cytoplasm [21]. Moreover, this new mechanism may occur outside the nucleus, whereas both transcriptional-slippage and trans-splicing of nRNA can only occur in the nucleus.…”

“…This SHS will serve as the primer to initiate a second RT reaction that elongates the cDNA, thus creating a chimera. This is possible since a retrovirus uses cellular tRNA to prime mRNA for reverse transcriptases to synthesize the first DNA strand (17,18) and reverse transcriptases are known to be able to utilize endogenous small RNAs to efficiently prime cDNA synthesis in vitro (19)(20)(21). Since pentamers are often used in PCR, we assume that the random annealing only requires a SHS as short as five nucleotides, resulting in a chimeric cDNA in which the two partners share this SHS.…”