Late-assembly of human ribosomal protein S20 in the cytoplasm is essential for the functioning of the small subunit ribosome

• The step of S20 assembled on 40S is happened in the cytoplasm.
• A small subunit assembled with a nuclear S20NLS is translational incompetence.
• Using energy depletion and recovery to manipulate the cellular compartment of S20NLS.
• Cytoplasm-retained S20NLS is crucial for creating a functional small subunit.

Using immuno-fluorescent probing and Western blotting analysis, we reveal the exclusive cytoplasm nature of the small subunit ribosomal protein S20. To illustrate the importance of the cellular compartmentation of S20 to the function of small subunit 40S, we created a nuclear resident S20NLS mutant gene and examined polysome profile of cells that had been transfected with the S20NLS gene. As a result, we observed the formation of recombinant 40S carried S20NLS but this recombinant 40S was never found in the polysome, suggesting such a recombinant 40S was translation incompetent. Moreover, by the tactic of the energy depletion and restoration, we were able to restrain the nuclear-resided S20NLS in the cytoplasm. Yet, along a progressive energy restoration, we observed the presence of recombinant 40S subunits carrying the S20NLS in the polysome. This proves that S20 needs to be cytoplasmic in order to make a functional 40S subunit. Furthermore, it also implies that the assembly order of ribosomal protein in eukaryote is orderly regulated.