Author(s): Hudak KA, Bauman JD, Tumer NE
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Abstract Several cap-binding proteins from both the nucleus and cytosol have been identified that mediate processes such as pre-mRNA splicing, translation initiation, and mRNA turnover. Here we describe a novel cap-binding protein, pokeweed antiviral protein (PAP), a 29-kDa type I ribosome-inactivating protein (RIP) isolated from Phytolacca americana. In addition to depurinating the sarcin/ricin loop of the large rRNA, an activity common to all RIPs, we have reported recently that PAP depurinates capped, but not uncapped RNAs in vitro. Here we characterize this activity further and, using affinity chromatography, show that PAP binds to the m7Gppp cap structure. PAP UV-crosslinks to m7GpppG-capped luciferase mRNA more efficiently than GpppG-capped luciferase mRNA, indicating specificity for the methylated guanosine. We present evidence that PAP does not remove the cap structure or depurinate the m7Gppp as shown by primer extension of capped and uncapped luciferase transcripts incubated with PAP. Modeling studies of cap interaction with PAP predict that the cap structure would bind to the active site of PAP in a similar manner to guanine. We map the depurination sites on the capped luciferase RNA and illustrate that depurination occurs at specific adenine and guanine residues throughout the RNA sequence. Incubation of isolated ribosomes with PAP and increasing molar concentrations of m7GpppG relative to PAP resulted in a decrease in the level of rRNA depurination. Therefore, at elevated concentrations, the methylated cap structure competes with the adenine or guanine for binding to PAP, even though the affinity of PAP for capped message is almost fourfold lower than for rRNA. These results demonstrate that the activity of PAP is not limited to rRNA depurination, but that PAP binds to the cap structure and depurinates mRNAs downstream of the cap in vitro. These findings may have implications for understanding PAP activity in vivo.
This article was published in RNA
and referenced in Biochemistry & Pharmacology: Open Access