RESUMO
The RNA-editing protein ADAR is essential for early development in the mouse. Genetic evidence suggests that A to I editing marks endogenous RNAs as 'self'. Today, different Adar knockout alleles have been generated that show a common phenotype of apoptosis, liver disintegration, elevated immune response and lethality at E12.5. All the Adar knockout alleles can be rescued by a concomitant deletion of the innate immunity genes Mavs or Ifih1 (MDA5), albeit to different extents. This suggests multiple functions of ADAR. We analyze AdarΔ7-9 mice that show a unique growth defect phenotype when rescued by Mavs. We show that AdarΔ7-9 can form a truncated, unstable, editing deficient protein that is mislocalized. Histological and hematologic analysis of these mice indicate multiple tissue- and hematopoietic defects. Gene expression profiling shows dysregulation of Rps3a1 and Rps3a3 in rescued AdarΔ7-9. Consistently, a distortion in 40S and 60S ribosome ratios is observed in liver cells. This dysregulation is also seen in AdarΔ2-13; Mavs-/- but not in AdarE861A/E861A; Ifih1-/- mice, suggesting editing-independent functions of ADAR in regulating expression levels of Rps3a1 and Rps3a3. In conclusion, our study demonstrates the importance of ADAR in post-natal development which cannot be compensated by ADARB1.
Assuntos
Adenosina Desaminase/genética , Imunidade Inata/genética , Helicase IFIH1 Induzida por Interferon/genética , Proteínas Ribossômicas/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Alelos , Animais , Regulação da Expressão Gênica/genética , Fígado/metabolismo , Camundongos , Camundongos Knockout , Edição de RNA/genética , Proteínas de Ligação a RNA/genética , Subunidades Ribossômicas Maiores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/genética , Deleção de Sequência/genéticaRESUMO
RNA modifications are present in all classes of RNAs. They control the fate of mRNAs by affecting their processing, translation, or stability. Inosine is a particularly widespread modification in metazoan mRNA arising from deamination of adenosine catalyzed by the RNA-targeting adenosine deaminases ADAR1 or ADAR2. Inosine is commonly thought to be interpreted as guanosine by cellular machines and during translation. Here, we systematically test ribosomal decoding using mass spectrometry. We show that while inosine is primarily interpreted as guanosine it can also be decoded as adenosine, and rarely even as uracil. Decoding of inosine as adenosine and uracil is context-dependent. In addition, mass spectrometry analysis indicates that inosine causes ribosome stalling especially when multiple inosines are present in the codon. Indeed, ribosome profiling data from human tissues confirm inosine-dependent ribosome stalling in vivo. To our knowledge this is the first study where decoding of inosine is tested in a comprehensive and unbiased way. Thus, our study shows novel, unanticipated functions for inosines in mRNAs, further expanding coding potential and affecting translational efficiency.