RESUMO
The design of proteins that can bind any RNA sequence of interest has many potential biological and medical applications. Here we have expanded the recognition of Pumilio and FBF homology protein (PUF) repeats beyond adenine, guanine and uracil and evolved them to specifically bind cytosine. These repeat sequences can be used to create PUF domains capable of selectively binding RNA targets of diverse sequence and structure.
Assuntos
Citosina/metabolismo , Engenharia de Proteínas , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Mutação , RNA/química , RNA/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Sequências Repetitivas de Aminoácidos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Mammalian mitochondrial DNA is transcribed as precursor polycistronic transcripts containing 13 mRNAs, 2 rRNAs, punctuated by 22 tRNAs. The mechanisms involved in the excision of mitochondrial tRNAs from these polycistronic transcripts have remained largely unknown. We have investigated the roles of ELAC2, mitochondrial RNase P proteins 1 and 3, and pentatricopeptide repeat domain protein 1 in the processing of mitochondrial polycistronic transcripts. We used a deep sequencing approach to characterize the 5' and 3' ends of processed mitochondrial transcripts and provide a detailed map of mitochondrial tRNA processing sites affected by these proteins. We show that MRPP1 and MRPP3 process the 5' ends of tRNAs and the 5' unconventional, non tRNA containing site of the CO1 transcript. By contrast, we find that ELAC2 and PTCD1 affect the 3' end processing of tRNAs. Finally, we found that MRPP1 is essential for transcript processing, RNA modification, translation and mitochondrial respiration.