Small RNAs of Nuclear Viroids Figure 2. Size distribution of HSVd- and GYSVd1-sRNAs reveals prominent peaks of 21-, 22- and 24-nt species. Histograms comparing the size distribution of 20–24-nt HSVd-sRNAs (A) and GYSVd1-sRNAs (B) in the different grapevine tissues. doi:10.1371/journal.pone.0007686.g002 analyzed the nucleotide at this position in the grapevine vd- sRNAs. In HSVd, we observed the prevalence of C in all size groups and tissues (Fig. 3A and Fig. S4). U was the second most abundant residue at the 59 terminal position in most cases except in 24-nt sRNAs, in which A was present with a frequency comparable to or higher than U (Fig. 3A and Fig. S4). When polarity of sRNAs was taken into consideration, C was confirmed as the most frequent residue at the 59 terminal position of both (+) and (2) HSVd-sRNAs in all tissues and in most size classes (Fig. 3B and 3C, and Fig. S5), indicating that this feature is not dependent on the viroid RNA polarity. This analysis also showed that 24-nt HSVd-sRNAs with A residue at the 59 terminal position were mostly of (2) polarity in all tissues including tendril, where they were prevalent among the (2) 24-nt HSVd-sRNAs (Fig. 3C and Fig. S5). Interestingly, similar results were obtained when GYSVd1-sRNAs were analyzed (Fig. 3D and Fig. S4) except that G was the second most frequent residue at the 59 terminus of several (+) GYSVd1-sRNAs, including those from tendril (Fig. 3E and 3F, and Fig. S6). Altogether these data show that the frequency of specific residues at the 59 terminal position of both (+) and (2) vd-sRNAs does not reflect the nucleotide frequency within their respective (+) and (2) viroid genomic RNAs and support the conclusion that the nucleotide at the 59 terminal position of vd- sRNAs from both viroids is unevenly distributed. The high coincidence of the distribution profiles in all tissues and for both viroids is noteworthy, and corroborates the reproducibility of the results here obtained. Most HSVd- and GYSVd1-sRNAs Derive from Specific Regions of the Genomic (+) and (2) RNAs Non-redundant HSVd-sRNAs from grapevine tissues almost covered the whole viroid genome (data not shown). However, when the cloning frequency of each sRNAs was considered, their distribution profile showed that they mostly derive from specific and very restricted regions (hotspots) of the HSVd genomic (+) and (2) RNAs. Figure 4A illustrates HSVd-sRNA mapping from tendrils, but similar distribution profiles were obtained with HSVd-sRNAs from the other tissues (Fig. S7 and S8). Approx- imately 85% and 76% (+) and (2) HSVd-sRNAs mapped at only two and one major hotspots, respectively, hereafter denoted HS1, HS2 and HS3. The hotspots ranged in size between 30 and 50 nt and in total covered only 20% of the viroid genome. In the rod- like secondary structure proposed for HSVd, (+) sRNAs hotspots (HS1 and HS2) mapped to the upper and lower strands, partially covering the central and the variable domains (Fig. 5A). Although these two hotspots partially overlap in the viroid rod-like secondary structure, complementary vd-sRNAs with two 39- protruding nucleotides in each strand were not found, suggesting PLoS ONE | www.plosone.org 4 November 2009 | Volume 4 | Issue 11 | e7686