Figure 5.

hnRNP Q specifically interacts with an adenosine-rich region in the 5′ end of the AANAT IRES. (A) Schematic diagram of the full-length rat AANAT IRES and its various mutant RNAs used for UV cross-linking experiments and monitoring efficiency of IRES-dependent translation in vivo. The conserved hnRNP Q-binding region among vertebrate species is represented as a closed box. Their locations in the AANAT 5′UTR are indicated as numbers from the 5′ end. (B) Three adenosine residues, within the conserved hnRNP Q-binding element, were replaced with cytosine residues. The asterisk denotes the nucleotide changes in the hnRNP R-binding region. Accession numbers for the AANAT 5′UTRs shown are as follows: rat, DQ075321; mouse, NM_009591 and U83462; human, U40347 and U40391; monkey, XM_523725; and sheep, DQ839412. (C) Radiolabeled riboprobes and purified hnRNP Q (hnQ437) were incubated with or without competitor RNAs [homopolymer(A) or cold r46], and subjected to UV cross-linking. (D) Rat pinealocyte cells were transfected with bicistronic reporter plasmids and incubated for 48 h before harvest. Cell lysates were prepared and subjected to luciferase assays. The ratio for the empty vector pRF was set to 1.