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. 2013 May 10;41(13):6577–6594. doi: 10.1093/nar/gkt369

Figure 6.

Figure 6.

hnRNP F/H binds to the 3′ UTR of AATP11 and regulates its level at the PCF stage. (A) Quantitative real-time PCR analysis of the AATP11 transcript on RNAi silencing of hnRNP F/H in the PC stage. cDNA was prepared from total RNA (1 µg) derived from uninduced cells (−Tet) or cells after 2.5 days of silencing (+Tet), as described in ‘Materials and Methods’ section. Real-time PCR was performed as described in ‘Materials and Methods’ section using cDNA (diluted 1:100), and concentration curves were used to determine the amount of PCR product amplified in uninduced cells (−Tet) or cells after 2.5 days of silencing (+Tet). The results shown are the average of three independent experiments. (B) Schematic representation of the mini-genes containing different sized 3′UTRs of AATP11 cloned downstream to the luciferase gene. Four different sized (1186, 975, 365 and 109 nt) fragments of 3′UTR of AATP11 were amplified as described in ‘Materials and Methods’ section and cloned downstream to the luciferase gene in the pNS21b expression vector. The constructs were then transfected into a cell line silenced for hnRNP F/H using RNAi. The sizes of the 3′UTRs and AAGAA hnRNP F/H-binding motifs are indicated. (C) Expression of the mini-gene transcripts in cells expressing the hnRNP F/H silencing construct. RNA was prepared from transgenic parasites expressing the luciferase AATP11 minigenes shown in panel B. Expression was monitored in hnRNP F/H cells after 2.5 days of silencing. Total RNA (30 μg) was separated on a 1.2% agarose, 2.2 M formaldehyde gel. The RNA was blotted and hybridized with a randomly labelled probe specific for the luciferase gene. The 7SL RNA was used as a control for equal loading. (D) Schematic representation of the luciferase minigenes carrying the 365 nt long 3′UTR of the AATP11 model gene with two AAGAA motifs. The ‘wild 365 luciferase’ minigene consists of a 365 nt long 3′UTR of AATP11 cloned downstream to the luciferase gene. The ‘mut 365 luciferase’ minigene carries 3′UTR of AATP11 with a second AAGAA mutated motif. The sequence of the domain carrying the mutations is boxed, and the base substitutions used to generate the mutation are depicted. (E) Expression of the wild and mutated minigenes. RNA was prepared from the transgenic parasites expressing the above luciferase minigenes as described in (D), and northern analysis was performed as described in (C).