@article{hu_pillai-nair_hemenway_2007, title={Long-distance RNA-RNA interactions between terminal elements and the same subset of internal elements on the potato virus X genome mediate minus- and plus-strand RNA synthesis}, volume={13}, ISSN={["1469-9001"]}, DOI={10.1261/rna.243607}, abstractNote={Potexvirus genomes contain conserved terminal elements that are complementary to multiple internal octanucleotide elements. Both local sequences and structures at the 5′ terminus and long-distance interactions between this region and internal elements are important for accumulation of potato virus X (PVX) plus-strand RNA in vivo. In this study, the role of the conserved hexanucleotide motif within SL3 of the 3′ NTR and internal conserved octanucleotide elements in minus-strand RNA synthesis was analyzed using both a template-dependent, PVX RNA-dependent RNA polymerase (RdRp) extract and a protoplast replication system. Template analyses in vitro indicated that 3′ terminal templates of 850 nucleotides (nt), but not 200 nt, supported efficient, minus-strand RNA synthesis. Mutational analyses of the longer templates indicated that optimal transcription requires the hexanucleotide motif in SL3 within the 3′ NTR and the complementary CP octanucleotide element 747 nt upstream. Additional experiments to disrupt interactions between one or more internal conserved elements and the 3′ hexanucleotide element showed that long-distance interactions were necessary for minus-strand RNA synthesis both in vitro and in vivo. Additionally, multiple internal octanucleotide elements could serve as pairing partners with the hexanucleotide element in vivo. These cis-acting elements and interactions correlate in several ways to those previously observed for plus-strand RNA accumulation in vivo, suggesting that dynamic interactions between elements at both termini and the same subset of internal octanucleotide elements are required for both minus- and plus-strand RNA synthesis and potentially other aspects of PVX replication.}, number={2}, journal={RNA}, author={Hu, Bin and Pillai-Nair, Neeta and Hemenway, Cynthia}, year={2007}, month={Feb}, pages={267–280} }
 @article{pillai-nair_kim_hemenway_2003, title={Cis-acting regulatory elements in the potato virus X 3 ' non-translated region differentially affect minus-strand and plus-strand RNA accumulation}, volume={326}, ISSN={["0022-2836"]}, DOI={10.1016/S0022-2836(02)01369-4}, abstractNote={The 72 nt 3′ non-translated region (NTR) of potato virus X (PVX) RNA is identical in all sequenced PVX strains and contains sequences that are conserved among all potexviruses. Computer folding of the 3′ NTR sequence predicted three stem-loop structures (SL1, SL2, and SL3 in the 3′ to 5′ direction), which generally were supported by solution structure analyses. The importance of these sequence and/or structural elements to PVX RNA accumulation was further analyzed by inoculation of Nicotiana tabacum (NT-1) protoplasts with PVX transcripts containing mutations in the 3′ NTR. Analyses of RNA accumulation by S1 nuclease protection indicated that multiple sequence elements throughout the 3′ NTR were important for minus-strand RNA accumulation. Formation of SL3 was required for accumulation of minus-strand RNA, whereas SL1 and SL2 formation were less important. However, sequences within all of these predicted structures were required for minus-strand RNA accumulation, including a conserved hexanucleotide sequence element in the loop of SL3, and the CU nucleotide in a U-rich sequence within SL2. In contrast, 13 nucleotides that were predicted to reside in SL1 could be deleted without any significant reduction in minus or plus-strand RNA levels. Potential polyadenylation signals (near upstream elements; NUEs) in the 3′ NTR of PVX RNA were more important for plus-strand RNA accumulation than for minus-strand RNA accumulation. In addition, one of these NUEs overlapped with other sequence required for optimal minus-strand RNA levels. These data indicate that the PVX 3′ NTR contains multiple, overlapping elements that influence accumulation of both minus and plus-strand RNA.}, number={3}, journal={JOURNAL OF MOLECULAR BIOLOGY}, author={Pillai-Nair, N and Kim, KH and Hemenway, C}, year={2003}, month={Feb}, pages={701–720} }