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

Abstract

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.

Keywords: HDAg; Pol II; RPL5; TFIIIA; alternative splicing; hepatitis delta virus; viroid replication.

Figure 1

Co-immunoprecipitation of TFIIIA-7ZF/Pol II/TBP. Overexpressed and HA-tagged TFIIIA-7ZF served as a bait and successfully pulled down endogenous Pol II largest subunit and TBP. IgG- and α-HA-conjugated resins were used as negative and positive treatments, respectively. The experimental methods were described previously [21]. IP, immunoprecipated fractions.

Figure 2

Two possible left terminal conformations during replication. (A) Traditional rod-shape left terminal conformation; (B) Alternative bifurcated left terminal conformation. The mapped TFIIIA-7ZF binding region is highlighted in a blue box.

Figure 3

RNA-immunoprecipitation supporting that TFIIIA-7ZF and RPL5 bind to HSVd in vivo. HA-tagged proteins (GFP, RPL5, and TFIIIA-7ZF) were expressed in HSVd-infected N. benthamiana plants via agroinfiltration and served as baits. RPL5 and TFIIIA-7ZF as baits successfully pulled down HSVd RNA. No-infiltrated and GFP-expressing leaves served as negative controls. The experimental methods were described previously [21]. PC, monomeric HSVd cDNA served as a size marker.

Figure 4

Loss of infectivity of a potato spindle tuber viroid (PSTVd) variant with 5S rRNA loop E sequences. Left panel displays loop E structural arrangements in wildtype (WT) PSTVd, N. benthamiana 5S rRNA, and a loop E swapped PSTVd^{IntA101G/C259G}. The right panel shows northern blots detecting the infection of WT PSTVd and PSTVd^{IntA101G/C259G} in local and systemic leaves of N. benthamiana plants. PC, a verified RNA sample from PSTVd-infected leaves served as a positive control. IVT, the unit-length in vitro transcript served as a control. Note: only circular PSTVd (an indicator of replication) is shown in the northern blots for inoculated leaves. The “○, ⎕, ∆” symbols depict Watson-Crick, Hoogsteen, and sugar edges, respectively. The hollow and solid symbols represent trans and cis glycosidic orientations, respectively. These symbolic annotations of loop E 3-dimensional structures were previously explained in detail [63,65].

Figure 5

Possible regulatory mechanisms for the expression of TFIIIA-7ZF and HDAg-S. (A) PSTVd directly binds to RPL5 to regulate alternative splicing of TFIIIA, resulting in optimized TFIIIA-7ZF expression and PSTVd replication; (B) The expression of HDAg-S and HDAg-L is regulated through A-to-I RNA editing at a specific site. Hepatitis delta virus (HDV) RNA structure may affect the editing. In addition, HDAg-S itself may also impact the editing efficiency in group 1 HDV.