Pokeweed antiviral protein binds to the cap structure of eukaryotic mRNA and depurinates the mRNA downstream of the cap
Bibliographic record
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.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".