Figure 5.

Probing for functional AS sRNAs to the amebic gene. (A) Northern blot (10 µg of total RNA) probed for EhROM1 mRNA reveals absence of EHROM1 mRNA in the 19-trigger-ROM cell line. Control (EHI_118270) indicates equivalent mRNA in both lanes. (B) High-resolution northern blot analysis of 19-trigger-ROM and untransfected cell lines probed for EhROM1 AS sRNA (20 µg of enriched sRNA). An abundant population of AS sRNAs to the EhROM1 gene were detected, which persisted 2 and 3 months after drug release. Control (EHI_118130) indicates equivalent sRNA in all lanes. (C) Biochemical analysis of the EhROM1 AS sRNAs reveals that they are resistant to treatment with terminator enzyme and increase in size when treated with capping enzyme; both features are indicative of a 5′-polyphosphate structure. As a control a radioactively labeled 5′-monophosphate 18-mer (included in each lane) is degraded by terminator enzyme and does not increase in size when treated with capping enzyme (10 µg of enriched sRNA).