Periodic Reporting for period 1 - ItohRibo (Structural study on mitochondrial ribosome assembly in human cells)
Reporting period: 2018-06-21 to 2020-06-20
The protein synthesis machinery, the ribosome, is not constructed of freely diffusing macromolecules; rather, its formation is an intricate and well-defined hierarchical process involving hundreds of proteins and a few RNA molecules working in cooperation and under tight regulation. In mitochondria, this fundamental process has an additional level of complexity, as it requires cooperative effort involving the precise regulation of two genomes. Although mitochondrial rRNAs are encoded in the mitochondrial genome, all 82 proteins of mitochondrial ribosome (mitoribosome) and numerous assembly factors are encoded in the nuclear genome and are therefore imported from cytosol. The high complexity of mitoribosomal assembly, and the unique association of a tRNA as a structural component of mitoribosome imply the involvement of as-yet-unknown mitochondria-specific auxiliary factors. Because many of the features of this system are unique to mitochondria, which have traditionally been difficult to investigate, little is known about the process of mitoribosome assembly.
I expected to reveal mechanistic insights into how mitoribosomal proteins are assembled in a cascade while nascent mitochondrial rRNA molecules are processed and folded. Since high resolution cryo-EM allows now a unique ability to investigate heterogeneous ribosomal populations and built de novo models, the proposed work will not only reveal maturation states, but also new factors in the mitoribosome assembly process.
Overall goal was to describe the full molecular sequence of the maturation steps along the ribosomal assembly pathway time line in human mitochondria and to put that sequence in the mirochondrial-cellular context. The objectives aimed to act on the fundamental question of the mitochondrial protein synthesis machinery assembly and uncover first insights into how unprecedented structural occurrences, unique to the ribosomes from human mitochondria, are formed.
During the period, in total of 6 assembly intermediate states from human mitochondria were identified by extensive 3D classifications of cryo-EM data and the model fitting is going on. In additon, two pre-initiation complexes of translation were also identified and published.
I expected to reveal mechanistic insights into how mitoribosomal proteins are assembled in a cascade while nascent mitochondrial rRNA molecules are processed and folded. Since high resolution cryo-EM allows now a unique ability to investigate heterogeneous ribosomal populations and built de novo models, the proposed work will not only reveal maturation states, but also new factors in the mitoribosome assembly process.
Overall goal was to describe the full molecular sequence of the maturation steps along the ribosomal assembly pathway time line in human mitochondria and to put that sequence in the mirochondrial-cellular context. The objectives aimed to act on the fundamental question of the mitochondrial protein synthesis machinery assembly and uncover first insights into how unprecedented structural occurrences, unique to the ribosomes from human mitochondria, are formed.
During the period, in total of 6 assembly intermediate states from human mitochondria were identified by extensive 3D classifications of cryo-EM data and the model fitting is going on. In additon, two pre-initiation complexes of translation were also identified and published.
I targeted on the rRNA modification enzyme MRM3, based on the assumption that the modification enzymes bind to the specific assemble intermediates of mitoribosome. Nucleotide modifications of rRNA are important steps for ribosome assemble.
I cultured the FLAG-tagged overexpressing MRM3 cells, generated from human culture cell line. I isolated mitochondria and MRM3-bound mitoribosome was fished by immunoprecipitation targeting FLAG tag. I prepare grids for cryo-EM and collected particle images on an electron microscope Titan Krios. I processed the data, picked the particles of mtLSU and reconstructed the cryo-EM map in 3 Å resolution. Large parts of rRNA near from the subunit interface were disordered. Three additional proteins were bound. The mtLSU map is similar to those reported as native assembly intermediates from wild-type cells. Although there are some extra densities, they could not be resolved due to the high flexibility.
I also cultured the MRM3-KO cells, isolated mitochondria and purified mitoribosome by a sucrose gradient. I prepare grids and collected particle images on a Titan Krios. I processed data, picked particles, classified mtLSU and mtSSU particles and reconstructed cryo-EM maps in 2.6-3.2 angstrom resolution. The mtLSU is almost identical for the reported native assembly intermediate, except that an additional one rRNA helix is disordered. In contrast, mtSSU particles are highly heterogeneity and with some extra protein densities. I performed focused 3D classifications on the tRNA and mRNA binding sits, resulted in seven obvious states. One is canonical mtSSU and two are translation-initiation complexes, while the rests seem the intermediates in the late assembly process. Since MRM3 is a modification enzyme working for mtLSU, it can be speculated that MRM3-KO prevent association of mtLSU and mtSSU and accumulated mtSSU assemble intermediates. The mitoribosome-specific protein mS37 is absent from all of the intermediates, suggesting that mS37 comes in the very end of the assembly process.
In addition, mitoriosome fished by FLAG-tagged IF3 was also prepared and cryo-EM data was collected and processed. Two pre-initiation complexes were identified and important of mS37 during translation initiation was suggested.
I cultured the FLAG-tagged overexpressing MRM3 cells, generated from human culture cell line. I isolated mitochondria and MRM3-bound mitoribosome was fished by immunoprecipitation targeting FLAG tag. I prepare grids for cryo-EM and collected particle images on an electron microscope Titan Krios. I processed the data, picked the particles of mtLSU and reconstructed the cryo-EM map in 3 Å resolution. Large parts of rRNA near from the subunit interface were disordered. Three additional proteins were bound. The mtLSU map is similar to those reported as native assembly intermediates from wild-type cells. Although there are some extra densities, they could not be resolved due to the high flexibility.
I also cultured the MRM3-KO cells, isolated mitochondria and purified mitoribosome by a sucrose gradient. I prepare grids and collected particle images on a Titan Krios. I processed data, picked particles, classified mtLSU and mtSSU particles and reconstructed cryo-EM maps in 2.6-3.2 angstrom resolution. The mtLSU is almost identical for the reported native assembly intermediate, except that an additional one rRNA helix is disordered. In contrast, mtSSU particles are highly heterogeneity and with some extra protein densities. I performed focused 3D classifications on the tRNA and mRNA binding sits, resulted in seven obvious states. One is canonical mtSSU and two are translation-initiation complexes, while the rests seem the intermediates in the late assembly process. Since MRM3 is a modification enzyme working for mtLSU, it can be speculated that MRM3-KO prevent association of mtLSU and mtSSU and accumulated mtSSU assemble intermediates. The mitoribosome-specific protein mS37 is absent from all of the intermediates, suggesting that mS37 comes in the very end of the assembly process.
In addition, mitoriosome fished by FLAG-tagged IF3 was also prepared and cryo-EM data was collected and processed. Two pre-initiation complexes were identified and important of mS37 during translation initiation was suggested.
On the organelle level, the mitochondrion is compartmentalized into sub-organelle sections such as nucleoids, RNA granules and membrane-related milieus. These sub-organelle compartments co-localize with various stages of the mitoribosome assembly. As the earliest stages of mitoribosome assembly are initiated co-transcriptionally, the early assembly is likely to take place near or at the nucleoids, later moving to the RNA granules and finally on to the inner mitochondrial membrane where translation takes place. By assigning the assembly intermediates obtained by singly particle cryo-EM into the electron cryo-tomography (cryo-ET) data, we will obtain the full picture of the mitoribosome assembly and maturation, which will help us to understand the mitochondria-related health and diseases.