RESUMO
The interferon-induced proteins with tetratricopeptide repeats (IFITs) are a family of RNA-binding proteins that are very highly expressed during antiviral response of immune system. IFIT proteins recognize and tightly bind foreign RNA particles. These are primarily viral RNAs ended with triphosphate at the 5' or lacking methylation of the first cap-proximal nucleotide but also in vitro transcribed RNA synthesized in the laboratory. Recognition of RNA by IFIT proteins leads to the formation of stable RNA/IFIT complexes and translational shut off of non-self transcripts. Here, we present a fluorescent-based assay to study the interaction between RNA molecules and IFIT family proteins. We have particularly focused on two representatives of this family: IFIT1 and IFIT5. We found a probe that competitively with RNA binds the positively charged tunnel in these IFIT proteins. The use of this probe for IFIT titration allowed us to evaluate the differences in binding affinities of mRNAs with different variants of 5' ends.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Naftalenossulfonato de Anilina/química , Bioensaio , Corantes Fluorescentes/química , Proteínas de Neoplasias/química , Proteínas de Ligação ao Cap de RNA/química , Capuzes de RNA/química , Proteínas de Ligação a RNA/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sítios de Ligação , Ligação Competitiva , Humanos , Ligação de Hidrogênio , Cinética , Simulação de Acoplamento Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Ligação Proteica , Conformação Proteica , Análogos de Capuz de RNA/química , Análogos de Capuz de RNA/metabolismo , Proteínas de Ligação ao Cap de RNA/genética , Proteínas de Ligação ao Cap de RNA/metabolismo , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Espectrometria de Fluorescência , Eletricidade Estática , TermodinâmicaRESUMO
In response to foreign RNA, cellular antiviral mechanisms stimulate high expression of interferon-induced proteins with tetratricopeptide repeats (IFITs). Two members of the IFIT protein family, IFIT1 and IFIT5, are capable of binding the very terminal 5' end of mRNA. In eukaryotes, these mRNA termini contain a cap structure (m7GpppN, cap 0) that is often subjected to further modifications. Here, we performed a thorough examination of IFIT1 and IFIT5 binding to a wide spectrum of differently capped as well as fully uncapped mRNAs. The kinetic analysis of IFIT1 and IFIT5 interactions with mRNA ligands indicates that the cap structure modifications considerably influence the stability of IFIT1/RNA complexes. The most stable complexes were formed between IFIT1 and GpppG/A- and m7GpppG/A-RNAs. Unexpectedly, we found that NAD+- and NADH-capped RNAs associate with IFIT5 with kinetic parameters comparable to pppG-RNA. Finally, we measured interactions of IFIT1 with mRNAs bearing modified synthetic cap analogs that start to become the important tools in biotechnological and medicinal research. We found that incorporation of modified cap analogs to the RNA protects the latter, to a certain degree, from the translational inhibition caused by IFIT1 protein.
