ESR 12 Project - Nucleoid associated proteins and their function
Work Package 5: Novel insights into maintenance of mitochondrial genome.
Partner: Hans Spelbrink
Institution: Radboud University, Nijmegen, The Netherlands
Duration: 36 months
Objectives: We have recently shown that different pools of nucleoids exist that can be separated biochemically. In particular we showed that replicating mtDNA is specifically found in a membrane associated insoluble digitonin fraction that can be separated by flotation on iodixanol gradients and based on Western blot analysis contains replication factors that are expected in an mtDNA replication platform, such as Twinkle and polymerase γ. Preliminary mass spectrometry analysis has confirmed this. I) By imposing selective cell culture and isolation conditions it is the aim of this project to identify novel nucleoid associated proteins that are typical for different aspects of mtDNA maintenance and gene expression. The dynamic nature of nucleoids raise questions of the protein composition of nucleoids in relation not only to replication and transcription but also questions of the in situ organization of these machineries and their link to mitochondrial biogenesis and dynamics such as fission and fusion. II) For this reason our aim is to chart how the processes of mtDNA replication, transcription, translation, and fusion/fission are organized relative to one another within the mitochondrial network. Marker proteins for these processes in combination with ClickIt chemistry and other in situ nucleic acid labeling methods will be used to determine the functional relationship of these interdependent aspects of mitochondrial gene expression.
Expected results: Several laboratories in recent years have applied nucleoid purification with some success and identified novel nucleoid protein candidates but also many proteins involved in for example mitochondrial translation. Current strategies that mostly use variations of affinity purification nevertheless have several drawbacks. One is a trade-off between stringency and confidence. For this reason we are currently optimizing very high-stringency purification strategies that use a combination of either mtDNA- or RNA-protein crosslinking, and purification under highly denaturing conditions. The rationale for using also RNA-protein crosslinking is that RNA molecules are intimately associated with the nucleoid and RNA intermediates are implicated in the replication process itself. A second drawback is the difficulty to identify low-abundant proteins. We will therefore combine our current knowledge of e.g. nucleoid dynamics with more targeted enrichment, such as flotation on iodixanol gradients, in combination with proteomics. Using these approaches we expect not only to identify minimal mitochondrial DNA- and RNA-binding proteomes but also to identify low-abundant proteins and assign preliminary function to some of the identified proteins by their association with a specific functional fraction. The sets of proteins are expected to overlap but a considerable proportion, in particular of the RNA-binding proteome will likely not be found in the DNA-binding proteome, such as many of the mitochondrial ribosomal proteins, proteins involved in RNA processing and mitochondrial tRNA synthetases. On the basis of the DNA- & RNA-binding and the more targeted proteomics we will analyze selected proteins by molecular biology methods and identify their relationship to the processes of DNA replication, transcription, RNA processing and translation. Using in situ cell biological methods we expect in particular to answer whether transcription of mtDNA is also confined to a subset of nucleoids (as is replication), where this process takes place, if replication and transcription are mutually exclusive and if transcription is coupled to translation.