Potato Spindle Tuber Viroid RNA-Templated Transcription: Factors and Regulation

Viroids are circular noncoding RNAs that infect plants. Without encoding any protein, these noncoding RNAs contain the necessary genetic information for propagation in hosts. Nuclear-replicating viroids employ DNA-dependent RNA polymerase II (Pol II) for replication, a process that makes a DNA-dependent enzyme recognize RNA templates. Recently, a splicing variant of transcription factor IIIA (TFIIIA-7ZF) was identified as essential for Pol II to replicate potato spindle tuber viroid (PSTVd). The expression of TFIIIA-7ZF, particularly the splicing event, is regulated by a ribosomal protein (RPL5). PSTVd modulates its expression through a direct interaction with RPL5 resulting in optimized expression of TFIIIA-7ZF. This review summarizes the recent discoveries of host factors and regulatory mechanisms underlying PSTVd-templated transcription processes and raises new questions that may help future exploration in this direction. In addition, it briefly compares the machinery and the regulatory mechanism for PSTVd with the replication/transcription system of human hepatitis delta virus.

Potato Spindle Tuber Viroid Modulates Its Replication through a Direct Interaction with a Splicing Regulator

ABSTRACT Viroids are circular noncoding RNAs (ncRNAs) that infect plants. Despite differences in the genetic makeup and biogenesis, viroids and various long ncRNAs all rely on RNA structure-based interactions with cellular factors for function. Viroids replicating in the nucleus utilize DNA-dependent RNA polymerase II for transcription, a process that involves a unique splicing form of transcription factor IIIA (TFIIIA-7ZF). Here, we provide evidence showing that potato spindle tuber viroid (PSTVd) interacts with a TFIIIA splicing regulator (ribosomal protein L5 [RPL5]) in vitro and in vivo. PSTVd infection compromises the regulatory role of RPL5 over splicing of TFIIIA transcripts, while ectopic expression of RPL5 reduces TFIIIA-7ZF expression and attenuates PSTVd accumulation. Furthermore, we illustrate that the RPL5 binding site on the PSTVd genome resides in the central conserved region critical for replication. Together, our data suggest that viroids can regulate their own replication and modulate specific regulatory factors leading to splicing changes in only one or a few genes. This study also has implications for understanding the functional mechanisms of ncRNAs and elucidating the global splicing changes in various host-pathogen interactions. IMPORTANCE Viroids are the smallest replicons among all living entities. As circular noncoding RNAs, viroids can replicate and spread in plants, often resulting in disease symptoms. Potato spindle tuber viroid (PSTVd), the type species of nuclear-replicating viroids, requires a unique splicing form of transcription factor IIIA (TFIIIA-7ZF) for its propagation. Here, we provide evidence showing that PSTVd directly interacts with a splicing regulator, RPL5, to favor the expression of TFIIIA-7ZF, thereby promoting viroid replication. This finding provides new insights to better understand viroid biology and sheds light on the noncoding RNA-based regulation of splicing. Our discovery also establishes RPL5 as a novel negative factor regulating viroid replication in the nucleus and highlights a potential means for viroid control.

Gamma Radiation-Induced Damage in the Zinc Finger of the Transcription Factor IIIA

A zinc finger motif is an element of proteins that can specifically recognize and bind to DNA. Because they contain multiple cysteine residues, zinc finger motifs possess redox properties. Ionizing radiation generates a variety of free radicals in organisms. Zinc finger motifs, therefore, may be a target of ionizing radiation. The effect of gamma radiation on the zinc finger motifs in transcription factor IIIA (TFIIIA), a zinc finger protein, was investigated. TFIIIA was exposed to different gamma doses from 60Co sources. The dose rates were 0.20 Gy/min and 800 Gy/h, respectively. The binding capacity of zinc finger motifs in TFIIIA was determined using an electrophoretic mobility shift assay. We found that 1000 Gy of gamma radiation impaired the function of the zinc finger motifs in TFIIIA. The sites of radiation-induced damage in the zinc finger were the thiol groups of cysteine residues and zinc (II) ions. The thiol groups were oxidized to form disulfide bonds and the zinc (II) ions were indicated to be reduced to zinc atoms. These results indicate that the zinc finger motif is a target domain for gamma radiation, which may decrease 5S rRNA expression via impairment of the zinc finger motifs in TFIIIA.