RESUMO
Mitochondria contain dedicated ribosomes (mitoribosomes), which synthesize the mitochondrial-encoded core components of the oxidative phosphorylation complexes. The RNA and protein components of mitoribosomes are encoded on two different genomes (mitochondrial and nuclear) and are assembled into functional complexes with the help of dedicated factors inside the organelle. Defects in mitoribosome biogenesis are associated with severe human diseases, yet the molecular pathway of mitoribosome assembly remains poorly understood. Here, we applied a multidisciplinary approach combining biochemical isolation and analysis of native mitoribosomal assembly complexes with quantitative mass spectrometry and mathematical modeling to reconstitute the entire assembly pathway of the human mitoribosome. We show that, in contrast to its bacterial and cytosolic counterparts, human mitoribosome biogenesis involves the formation of ribosomal protein-only modules, which then assemble on the appropriate ribosomal RNA moiety in a coordinated fashion. The presence of excess protein-only modules primed for assembly rationalizes how mitochondria cope with the challenge of forming a protein-rich ribonucleoprotein complex of dual genetic origin. This study provides a comprehensive roadmap of mitoribosome biogenesis, from very early to late maturation steps, and highlights the evolutionary divergence from its bacterial ancestor.
RESUMO
Paper-based nucleic acid detection and diagnosis are currently gaining much interest in point-of-care (POC) applications. The major steps involved in any nucleic acid amplification testing (NAAT) based diagnostics are nucleic acid isolation, reverse transcription (RT) (in the case of RNA), amplification and detection. RT is an important step in quantifying the viral load in case of disease diagnosis as well as quantifying gene expression levels in other molecular studies. cDNA synthesis is routinely carried out using a thermal cycler, with the process requiring temperatures between 40ºC to 65ºC. Here we report for the first time an instrument-free RT, performed at room temperature on cellulose-based paper devices. cDNA synthesis on paper was confirmed by RT-PCR and Sanger sequencing of the PCR products. Purified RNA from varied sources such as cell lysate, tissue and blood were used to test the methodology. Synthetic hepatitis C virus (HCV) RNA and human blood RNA were used as proof-of-concept to demonstrate the use of these devices in diagnostic applications. Further, ready-to-use paper-based reverse transcription (PRT) devices have been developed, wherein only the RNA sample is added on the device and the cDNA can be eluted after 30 min of incubation at room temperature. The devices were found to be stable for 30 days at - 20ºC storage. The cellulose-based PRT devices are simple, time saving and user-friendly for a complete instrument-free cDNA synthesis at room temperature.
