A nuclear import pathway exploited by pathogenic noncoding RNAs

Figure 1. IMPa-4 and VIRP1 in the same pathway for PSTVd infection. A, RNA immunoprecipitation. IMPa genes were fused with a TAP-tag, which contains 9X cMyc, 2X IgG binding domain, and 1X His6. IMPa genes were transiently expressed in PSTVd-infected N. benthamiana plants via agroinfiltration and then harvested for immunoprecipitation using magnetic IgG beads. RNAs in the immunoprecipitation were subject to RT-PCR followed by electrophoresis using native PAGE gels. Histone H2A serves as a negative control. NC, infiltration with agrobacterium harboring no construct. IP, immunoprecipitated fraction. B, Virus-induced gene silencing of IMPa-4 inhibiting PSTVd systemic infection in tomato. RNA gel blots showing PSTVd and TRV accumulation in infiltrated leaves. RT-PCR shows the specific downregulation of IMPa-4 expression by the TRVIMPa-4 construct. “c” depicts circular genomic PSTVd. G, sg1, and sg2 indicate the genomic RNA1, subgenomic1 from RNA1, and subgenomic2 from RNA1, respectively. NC, tomato without PSTVd or TRV inoculation. C, Virus-induced gene silencing inhibiting PSTVd nuclear accumulation in systemic leaves. Whole-mount in situ hybridization showed that PSTVd-infected nuclei were…

Figure 2. IMPa-4 and VIRP1 interaction in plants. A, Co-immunoprecipitation. Agroinfiltration-based transient expression of FLAG-tagged VIRP1 in N. benthamiana serves a bait to pull down co-expressed IMPa proteins with a TAP-tag. IP, immunoprecipitated fraction. B, BiFC. Nicotiana benthamiana seedlings were used for transient expression of various combinations of constructs via agroinfiltration. 35S:RFP-Histone2B serves as a marker for the nucleus. Scale bar, 8 μm. White dashed lines outline the positions of nuclei.

Figure 3. Characterizing PSTVd C-loop. A, Rationale for PSTVd C-loop mutagenesis. As explained in the Results, the critical trans-WC–Hoogsteen base pairing between A171 and U187 is subject to mutagenesis. Based on the RNA basepair catalog, structure-maintaining and structure-disruptive mutants were designed and highlighted in green and magenta, respectively. B, EMSA illustrating the interaction between C-loop mutants and VIRP1. Arrows and arrow heads indicate shifted RNA (in RNA–protein complex) and free RNA, respectively. C, Box plot showing quantification of EMSA results. The percent of RNA shifted in total RNA used for each reaction was calculated. The WT RNA shifted percentage was set as 100% in each replicate, and the mutant RNA shifted percentage was normalized to that of WT RNA. All C-loop mutants have a significant reduction in VIRP1 binding as compared with WT, based on two-tailed t test. D, RNA gel bots detecting PSTVd systemic infection in N. benthamiana. WT PSTVd serves as positive control. Ethidium bromide staining of rRNAs serves as loading control. c, circular PSTVd. E, Whole-mount in situ hybridization showing the presence of viroid RNAs in the nuclei (signal appears as…

Figure 4. Characterizing a C-loop variant in HSVd. A, Rationale for HSVd C-loop mutagenesis. The critical trans-WC–Hoogsteen base pairing between C125 and G172 is subject to mutagenesis. Based on the RNA basepair catalog, C125–G172, C125–C172, C125–A172 but not C125–U172 can form the critical tWH pairing. The magenta cross depicts the structure-destructive design. B, EMSA illustrating the interaction between C-loop mutants and VIRP1. Arrowheads indicate the position of free probe, while the arrow indicates the position of RNA–protein complex. Multiple bands in “RNA only” lanes are likely caused by different confirmations of RNAs. C, Box plot showing quantification of EMSA results. Normalization method was the same as described in Figure 3. All C-loop mutants have a significant reduction in VIRP1 binding as compared with WT and G157C RNA, based on two-tailed t test. **P < 0.1. ***P < 0.001. D, RNA gel bots detecting HSVd systemic infection in N. benthamiana. WT HSVd serves as positive controls. Ethidium bromide staining of rRNAs serves as loading control. c, circular genomic HSVd. Green and magenta colors depict structure-maintaining and structure-disruptive mutants, respectively.

Figure 5. A working model illustrating the IMPa-4/VIRP1/C-loop-based RNA nuclear import. VIRP1 recognizes viroid C-loop to form an RNP complex, which is transported into the nucleus by IMPa-4. The IMPb responsible for viroid nuclear import remains to be identified.