Periodic Reporting for period 3 - Human-Ribogenesis (Structural Biology of Human Ribosome Biogenesis)
Okres sprawozdawczy: 2023-10-01 do 2025-03-31
Ribosomes are large molecular machines consisting of several RNAs (rRNAs) and dozens of proteins (ribosomal proteins). In every cell the ribosomes are essential for the translation of the genetically encoded information into polypeptides or proteins which are required to execute most of the biochemical activity required for life. In human cells the assembly of the two ribosomal subunits, small 40S and large 60S subunit, is a vital process of daunting complexity, which requires several hundreds of assembly factors (AFs). Together with small nucleolar RNAs (snoRNAs) these factors facilitate a cascade of modification, processing and folding events of the ribosomal RNA (rRNA) that are tightly coordinated with the incorporation of a large set of ribosomal proteins between the nucleolus and the cytoplasm. This process has been mostly studied in fungal model organisms such as S. cerevisiae, however, assembly and rRNA processing pathways may differ between yeast and humans, and it has not been sufficiently analyzed in humans.
The emergence of numerous diseases caused by defective assembly of ribosomes, so-called ribosomopathies, calls for a deeper understanding of human ribosome biogenesis. Many unique AFs have been identified in humans and several ribosomopathies have been assigned to ribosome biogenesis defects. Yet, at the beginning of this project only very little information has been published (i.e. Ameismeier et al., 2018) that provides direct molecular information on the structural basis and architectural transitions of human ribosome maturation.
Therefore, it is planned in this project to provide a near complete structural inventory of human ribosome biogenesis by using state-of-the-art cryo-electron microscopy (cryo-EM) on purified native pre-ribosomal particles. Corresponding analysis by biochemical methods and functional assays is planned to gain complementary functional information. Finally, shot-gun cryo-EM of total pre-40S, pre-60S and 90S intermediates will be established in order to quantitatively characterize the equilibrium flow of ribosome assembly in normal and challenged human cells. Together, these insights will provide the basis for a mechanistic understanding of human ribosome biogenesis and will thereby lay the foundation for better relating this process to regulatory pathways and disease.
The emergence of numerous diseases caused by defective assembly of ribosomes, so-called ribosomopathies, calls for a deeper understanding of human ribosome biogenesis. Many unique AFs have been identified in humans and several ribosomopathies have been assigned to ribosome biogenesis defects. Yet, at the beginning of this project only very little information has been published (i.e. Ameismeier et al., 2018) that provides direct molecular information on the structural basis and architectural transitions of human ribosome maturation.
Therefore, it is planned in this project to provide a near complete structural inventory of human ribosome biogenesis by using state-of-the-art cryo-electron microscopy (cryo-EM) on purified native pre-ribosomal particles. Corresponding analysis by biochemical methods and functional assays is planned to gain complementary functional information. Finally, shot-gun cryo-EM of total pre-40S, pre-60S and 90S intermediates will be established in order to quantitatively characterize the equilibrium flow of ribosome assembly in normal and challenged human cells. Together, these insights will provide the basis for a mechanistic understanding of human ribosome biogenesis and will thereby lay the foundation for better relating this process to regulatory pathways and disease.
The project’s first task aimed at the structural basis of the assembly of the human 40S small ribosomal subunit by single particle cryo-EM. Here, we could provide information of the last cytoplasmic steps of human pre-40S assembly (Ameismeier et al., Nature, 2020) which includes the decisive ultimate cleavage of the rRNA to its mature 18S state by a dedicated nuclease. In addition, we succeeded in visualizing the complete nucleoplasmic phase of 40S subunit maturation (see Figure, Cheng et al., NAR, 2022) which is mainly characterized by the formation of the so-called head domain of this subunit. Since we visualized a large collection of human and of yeast intermediates, we were in the position to compare and noticed to our surprise that there are actually rather small differences between the assembly pathways of the species during this phase which might be a general evolutionary feature.
The next task aims at the structural elucidation of human 60S large ribosomal subunit assembly. We succeeded to isolate another set of nuclear intermediates on the basis of a mutation in the assembly factor NLE1 (Rsa4 in yeast). These intermediates represented rather late nuclear particles, which showed again that there is large a degree of conservation when compared to the known yeast intermediates (manuscript in poreparation). Another aspect of 60S maturation, the incorporation of the so-called 5S RNP and its signalling to the P53 stress surveillance system has been successfully analysed and provided first insights into how the 5S RNP is prepared for 60S incorporation and, in case of accumulation, sequesters MDM2 in order to stabilize P53 (Estrada et al., Nat. Struct. Mol. Biol., 2023). The unexpectedly high degree of conservation between human and yeast maturation justified to address special aspects of 60S subunit maturation again in the easy-to-handle fungal model with the high probability to gain insights which both systems have in common. We could thereby visualize the so far earliest 5S RNP incorporation in the fungal system (Lau et al., EMBO Rep., 2023) and te remodelling of the pre-60S subunit by the ATPases Rea1 Spb4 (Mitterer et al., eLife, 2023).
The next task aims at the structural elucidation of human 60S large ribosomal subunit assembly. We succeeded to isolate another set of nuclear intermediates on the basis of a mutation in the assembly factor NLE1 (Rsa4 in yeast). These intermediates represented rather late nuclear particles, which showed again that there is large a degree of conservation when compared to the known yeast intermediates (manuscript in poreparation). Another aspect of 60S maturation, the incorporation of the so-called 5S RNP and its signalling to the P53 stress surveillance system has been successfully analysed and provided first insights into how the 5S RNP is prepared for 60S incorporation and, in case of accumulation, sequesters MDM2 in order to stabilize P53 (Estrada et al., Nat. Struct. Mol. Biol., 2023). The unexpectedly high degree of conservation between human and yeast maturation justified to address special aspects of 60S subunit maturation again in the easy-to-handle fungal model with the high probability to gain insights which both systems have in common. We could thereby visualize the so far earliest 5S RNP incorporation in the fungal system (Lau et al., EMBO Rep., 2023) and te remodelling of the pre-60S subunit by the ATPases Rea1 Spb4 (Mitterer et al., eLife, 2023).
Taken together, we were successful in tackling the pre-40S assembly pathway by having contributed to an almost complete picture of its maturation pathway from the nucleolus to the final stages in the cytoplasm both for humans and yeast. For the pre-60S aim, we have developed mutations which led to the accumulation and successful characterization of nuclear pre-60S particles, and we succeeded in characterization of the long-sought 5S RNP before and immediately after incorporation into the early pre-60S. Importantly, we could conclude from our studies that the maturation pathways appear to be very well conserved between the better studied fungal systems and humans. This allows for the most difficult questions to still be addressed in the fungal systems with a high probability to essentially also reflect the human situation. We will therefore decide on a case-by-case basis what system to use for which questions and will aim at having a closer look at pathological contexts in the human system.