Ribosome synthesis is a highly complex and coordinated process involving more than 200 assembly factors. The synthesis and processing of ribosomal components occurs not only in the nucleolus but also in the nucleoplasm and the cytoplasm of eukaryotic cells.

Ribosome biogenesis begins with the synthesis of 5S and 45S pre-rRNAs by distinct RNA polymerases. The primary transcripts are extensively processed and modified before they are bound and folded by ribosomal proteins and assembly factors, which are imported from the cytoplasm. The extensive modification of ribosomal RNAs by snoRNPs is another distinct feature of eukaryotic ribosomes. Individually the modified bases may not seem to have a specific function collectively, all the modifications stabilize particular conformations of the ribosomal RNAs. Additionally, these modified bases are more concentrated in functional regions of the rRNA and regulate ribosomal activity in translation.

Both the rRNA modifications and processing of pre-rRNAs occur in the nucleolus because both these steps require components that are only found in the nucleolus. While the snoRNPs carry out chemical modification of the rRNA, other “nucleolar proteins” hydrolyze the transcribed “spacer RNA” in the precursor RNA to form the cleaved 18S, 5.8S and 28S rRNA. With the generation of the matured rRNAs, the free nucleolar proteins return to a nucleolar pool and are reutilized.

Cations such as Magnesium ions (Mg²⁺) play an important role in maintaining the structure of the ribosome. During experiments, the ribosome dissociates into subunits when Mg²⁺ is removed. Although the precise role of Mg²⁺ remains uncertain, it is plausible that the cations interact with ionized phosphates of the RNA to bridge the two ribosomal subunits.

Once ribosomal assembly is complete, some of the ribosomes associate with intracellular membranes, primarily the endoplasmic reticulum, whereas free ribosomes are distributed through the cytoplasm.