Fig 2.

AU-rich 5′ UTRs harboring IRES are regulated by NF45. (A) IRES activity was tested in d5 (NF45 shRNA) stable cells relative to c (nontargeting shRNA) cells using a pβgal/IRES/CAT-based bicistronic assay (schematic) with the IRES listed in Fig. 1B, as described in Materials and Methods. Note that a ratio of 1.0 is indicative of no change in IRES activities between the two cell lines. The IRES are ranked in order of decreasing IRES activities and 5′ UTR AU content from left to right (bottom panel). P values are shown for the identified NF45-dependent IRES compared to the EMCV IRES used as a control. (B) Steady-state mRNA levels of the indicated mRNAs were determined by qPCR in c and d5 cells, as well as in d5 cells transfected with GFP (d5-GFP) or a GFP-NF45R (d5-GFP-NF45R). Their expression was normalized to that of β-actin and expressed as a ratio of d5 to c or d5-NF45R to d5-GFP for each transcript. (C) Polyribosome-associated mRNAs from the indicated cell lines were determined as described in Materials and Methods. General polysome profiles, as well as heavy-to-light polysome ratios (HP/LP), are shown for d5 relative to c (d5-to-c ratio) and for d5-GFP-NF45R relative to d5-GFP (d5-NF45R–to–d5 ratio). (D) Detection and enrichment of cIAP1, XIAP, and NRF mRNAs from FLAG-NF45 RNA immunoprecipitation compared to a FLAG control, showing that NF45 specifically associates with these transcripts in vivo.