Abstract
RNA cap binding proteins have evolved to specifically bind to the N7-methyl guanosine cap structure found at the 5’ ends of eukaryotic mRNAs. The specificity of RNA capping enzymes towards GTP for the synthesis of this structure is therefore crucial for mRNA metabolism. The fact that ribavirin triphosphate was described as a substrate of a viral RNA capping enzyme, raised the possibility that RNAs capped with nucleotide analogues could be generated in cellulo. Owing to the fact that this prospect potentially has wide pharmacological implications, we decided to investigate whether the active site of the model Paramecium bursaria Chlorella virus-1 RNA capping enzyme was flexible enough to accommodate various purine analogues. Using this approach, we identified several key structural determinants at each step of the RNA capping reaction and generated RNAs harboring various different cap analogues. Moreover, we monitored the binding affinity of these novel capped RNAs to the eIF4E protein and evaluated their translational properties in cellulo. Overall, this study establishes a molecular rationale for the specific selection of GTP over other NTPs by RNA capping enzyme It also demonstrates that RNAs can be enzymatically capped with certain purine nucleotide analogs, and it also describes the impacts of modified RNA caps on specific steps involved in mRNA metabolism. For instance, our results indicate that the N7-methyl group of the classical N7-methyl guanosine cap is not always indispensable for binding to eIF4E and subsequently for translation when compensatory modifications are present on the capped residue. Overall, these findings have important implications for our understanding of the molecular determinants involved in both RNA capping and RNA metabolism.
Highlights
The addition of a 5’ cap structure to RNA transcripts synthesized by RNA polymerase II is fundamental to eukaryotic gene expression [1]
In order to probe into the structural flexibility of the active site of a GTase, we tested the relative propensity of the Paramecium bursaria Chlorella virus-1 (PBCV-1) GTase to accommodate modified substrates through the use of nucleotide analogues
The fact that some nucleotide analogues led to inhibition of the PBCV-1 GTase activity at high concentrations (> 2.0 mM) raised the possibility that they might be acting as chelator for the essential divalent metal ion co-factor, and these analogues were not included in subsequent analyses
Summary
The addition of a 5’ cap structure to RNA transcripts synthesized by RNA polymerase II is fundamental to eukaryotic gene expression [1]. In the nucleus for instance, the cap structure of pre-mRNA is recognized by the cap binding proteins (CBP20 et CBP80) [2] This cap binding complex (CBC) protects mRNA from degradation and assists RNA transport from the nucleus to the cytoplasm. The eukaryotic translation initiation factor 4E (eIF4E) binds to the RNA cap structure [3]. This association is mediated by two aromatic residues of the eIF4E protein; the mRNA binding is further stabilized by specific hydrogen bonds between the positive charge of the 7-methylguanosine and an acidic residue (reviewed in [4]). EIF4E assembles with eIF4G (a scaffold protein) and eIF4A (an RNA helicase) into the eIF4F complex [5]. The roles fulfilled by the RNA cap structure are crucial for RNA stability and translation
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