Thus, we conclude that al though bidirectional transcription has not been previ ously documented in E. histolytica, both antisense and sense small screening library RNAs are likely templated from bidirectional transcripts and generated by an RdRP dependent mech anism resulting in small RNAs with 50 polyphosphate termini. Natural antisense transcripts have been shown to be a major source of siRNA generation in Drosophila melanogaster Inhibitors,Modulators,Libraries and in plants. In the parasite, Giardia lamblia, bidirectional transcription produces abundant sterile antisense transcripts. Further studies to characterize the extent of natural antisense transcripts in E. histolytica and the mechanism for generation of 50 polyP small RNAs are needed.

Inhibitors,Modulators,Libraries Our demonstration that small RNAs to group II genes have 50 polyP termini and strand specific RT PCR detecting transcripts in both directions indicates that these sense small RNAs are likely generated from antisense transcript, and indicates that some portion of sense small RNAs in our dataset although called sense, are truly antisense to the transcript Inhibitors,Modulators,Libraries derived from the opposite strand of the examined gene. Small RNAs are derived from both unspliced and spliced transcripts In order to identify whether small RNAs map to spliced or unspliced transcripts, we downloaded both genomic and mRNA sequences for all E. histolytica protein cod ing genes with at least one predicted intron. Small RNAs mapping to introns are defined as those that map to the genomic gene sequence but not the mRNA sequence. Small RNAs mapping to exon exon junctions are those that map to mRNA sequence but not the genomic gene sequence.

We found a total of 52 small RNA reads that spanned exon exon junctions and 1,187 small RNA reads that mapped to predicted introns. We further examined Inhibitors,Modulators,Libraries the protein coding genes with at least 50 small RNAs and which also have at least one in tron. For this list, we first checked for potential false introns caused by genome sequence error, and excluded EHI 018150, EHI 180820 and EHI 137120 as they have many Ns in their intron sequences. We then checked the predicted intronic se quence in the remaining genes for an in frame stop codon or frame disruption, as this strongly suggests that the intron is correctly predicted. Lastly, we checked for paralogs within these genes. With these criteria, we examined the three categories of genes to which small RNAs mapped to identify small RNAs that mapped to Inhibitors,Modulators,Libraries exons, introns and exon exon junctions.

For the group I genes, 18 unique genes had small RNAs that mapped to predicted introns and among them, 4 unique genes also had small RNAs that mapped to exon exon junctions. For Trichostatin A chemical structure the group II genes, 4 unique genes had antisense small RNAs that mapped to introns and 3 of these also had sense small RNAs that mapped to introns.