Fig. 1
E. histolytica has two sRNA populations (27 nt and 31 nt) and 31 nt sRNA is oligo-A modified at 3′-end. a Size distribution for two size-fractionated sRNA libraries (15-30 nt and 30-45 nt). Both libraries were cloned using 5′-P independent cloning method (TAP). Total reads (dashed lines) and mapped reads (solid lines) are shown. The 15-30 nt size-fractionated library shows a single peak at 27 nt for both total and mapped reads. The 30-45 nt size-fractionated library has two sRNA peaks (27 nt and 31 nt) for the total reads, and only the 27 nt sRNAs, but not 31 nt sRNAs, can be mapped to genome. b Nucleotide distribution analysis for the mapped and non-mapped reads. There is a 5′-G bias for the first nucleotide in all populations. The 30–45 non-mapped reads show a 5′-G bias for the first nucleotide, and a string of 3 or 4 As are identified at 3′-end. After trimming of the 3′-end As, these reads can be remapped to the genome (Table 1) indicating non-templated oligo-A tailing event to the 27 nt sRNAs. c Northern blot detects both 27 nt and 31 nt sRNA populations. Three sRNAs (probes called A, B and C) were cloned in both size sRNA populations; Northern blot analysis detected signals at both sRNA sizes, indicating that the two sRNA species co-exist in the cell. A sRNA enriched RNA from E. histolytica trophozoites (20 μg) was used for each sample and probed with end-labeled [32P] oligonucleotide probes corresponding to the cloned sRNAs, see Suppl. Table 7 and Suppl. original blot for Fig. 1c. d Both 27 nt and 31 nt sRNA populations are resistant to cleavage by Terminator enzyme, they are shifted for one nucleotide distance via capping assay, indicating a 5′-polyP structure for both sRNA populations. A “spike-in” control of 22 nt RNA with a 5′-monoP is readily degraded by Terminator enzyme. An increase in size following treatment with capping enzyme indicates that RNAs have 5′-di- or tri-phosphate structure. The probe “A” and a probe specific to EhLINE were used (Suppl. Table 7 and Suppl. original blot for Fig. 1d.) e E. invadens 30-45 nt size-fractionated library has two sRNA peaks. Similar to E. histolytica, the 27 nt peak can be mapped to the genome but not 31 nt peak. The plot shows the trophozoite dataset (similar plots are observed for two other time point datasets, data not shown)