Significance Ribosome biogenesis requires a number of assembly factors. However, the exact mechanism of this process remains largely unknown. We could successfully reconstitute the 50S subunit from the 45S precursor by a 2′- O -methylation of U2552 (Um2552) mediated by rRNA methyltransferase RlmE, in the presence of the wash fraction from crude ribosomes. To our knowledge, this experiment is the first demonstration of enzymatic formation of a ribosomal subunit in vitro from its precursor via the action of an assembly factor. Mechanistic studies revealed that RlmE-mediated Um2552 formation promotes interdomain interactions of 23S rRNA, in concert with the incorporation of L36, triggering late steps of 50S subunit assembly. RlmE and Um2552 are conserved in other organisms, including human, indicating the functional importance of this process in ribosome biogenesis.