histolytica both endogenous and exogenously triggered antisense small RNAs have 50 polyP termini. Since our analysis to date had focused on the antisense small RNAs, we wanted to determine whether the small RNAs that map sense to genes also have 50 polyP directly termini. For this purpose, we analyzed small RNAs that mapped sense to the EHI 020920 locus. This gene is highly enriched for both antisense and sense small RNAs with small RNA co verage extending into the apparent 50 upstream region. Northern blot analysis detected signal at 27nt with probes that detect antisense small RNAs and with probes that detect sense small RNAs. All the blots were performed using the same membrane, and therefore the relative band intensity reflects the abundance of small RNAs.
We observed a correlation of 5 enriched distribution of Inhibitors,Modulators,Libraries small RNAs with the intensity of probe 1 probe 2 probe 3. We then performed a Terminator exonuclease assay and a 50 end Inhibitors,Modulators,Libraries Capping assay on the total RNA sample. The signal for sense small RNAs were resistant to Terminator treatment and shifted up after capping assay, indicating that these small RNAs that map sense to the EHI 020920 locus have 50 polyP termini. Probe 1, which would detect antisense small RNAs had the expected biochemical features consistent with 50 polyP termini. The control was, as expected, degraded by Terminator en zyme and unaffected by treatment with capping enzyme. We further examined a second example the locus that contains both antisense and sense small RNAs to EHI 130480 and EHI 130490 as well as one potential unannotated gene.
Probes for detecting antisense and sense small RNAs were chosen for EHI 130480. Northern Inhibitors,Modulators,Libraries blot analysis showed signal for both probes at 27nt. the sense small RNA was resistant to Terminator exonuclease and thus has 50 polyphosphate termini. Thus, for the two loci tested we determined that the sense small RNAs also have Inhibitors,Modulators,Libraries 50 polyP termini. The biochemical features of sense small RNAs having 50 polyP termini was unexpected. Analysis for pairing between antisense and sense small RNAs showed no enriched pairs for these gene loci. In a typical RNAi pathway, dsRNA is chop ped into a siRNA duplex, where enriched pairing be tween antisense and sense small RNAs can be found. The lack of pairing between antisense and sense small RNAs in group II genes and the 50 polyP termini for both antisense and sense small RNAs indicates that these small RNAs could be individually processed from bidirectional transcripts at these loci.
To Inhibitors,Modulators,Libraries determine if this may have occurred, we performed strand specific RT PCR for EHI 020920, EHI 130480 and EHI 130490. At all three loci, both sense and antisense transcripts can be detected, PF-2341066 albeit with antisense transcripts at much lower level than the sense transcript. Overall transcript levels as assayed by RT PCR correlated with the abundance of antisense and sense small RNAs mapped to these loci.