Biased Pol II Fidelity Contributes to Conservation of Functional Domains in the Potato Spindle Tuber Viroid Genome

C.E. Credits: P.A.C.E. CE Florida CE
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Abstract

Polymerase errors are the major source of variation in virus and viroid genomes, and thus polymerase error rates are critical determinants of adaptation potential.  Potato spindle tuber viroid (PSTVd) has a circular non-coding RNA genome of 359 nucleotides that replicates and spreads systemically in host plants.  Consequently, all functions needed to establish a productive infection are mediated by sequence and structure elements within the genomic RNA.  During replication, PSTVd hijacks host transcription machinery, and specifically DNA-dependent RNA polymerase II (Pol II), to copy its genome.  We sought to determine the Pol II error rate when transcribing PSTVd RNA, a non-native template.  However, estimation of in vivo error/mutation rates is complicated by selection, which leads to loss or proliferation of certain mutations.  To circumvent this issue, we sequenced minus-strand PSTVd dimers from concatemeric replication intermediates.  The underlying rationale is that mutations found in only one of the monomers were likely generated de novo during Pol II transcription of the circular plus-strand RNA genome.  This approach yielded an apparent error rate ~100-fold greater than Pol II transcription on DNA templates.  Nevertheless, the error spectrum was similar to that observed during mRNA transcription by Pol II.  Remarkably, however, de novo mutations were rare in the most highly conserved, replication-critical genome regions, suggesting these sequences are selected for both important function and enhanced transcription fidelity.  Such biased fidelity reveals a novel strategy to ensure population survival while maximizing adaptation potential.  Comparison of mutations identified by minus-strand dimer sequencing with mutations observed in progeny variants derived from wild type PSTVd showed that most de novo mutations are lost through selection.


Learning Objectives: 

1.  Identify viroids as infectious non-coding RNAs

2.  Understand polymerase errors as drivers of virus/viroid evolution


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