<span class="paragraphSection"><div class="boxTitle">Abstract</div>Production of the eukaryotic ribosomal subunits (40S and 60S) is a highly dynamic process in which numerous assembly factors (AFs) coordinate structural rearrangements of pre-ribosomal complexes to achieve their mature, functional architectures. Across the domains of life, GTPases leverage their functions as molecular switches to induce conformational changes that drive key steps in subunit maturation. Three GTPases, GTPBP4, GNL2, and GNL3, have been detected in nucleolar/nucleoplasmic human pre-60S complexes. Here, we compositionally analyze the pre-ribosomal particles associated with each of these GTPases and demonstrate the requirement of these enzymes, and their abilities to bind and hydrolyze GTP, for distinct steps in pre-ribosomal RNA processing. We further reveal that the GNL3 paralog, GNL3L, also associates with pre-ribosomes, and we map GNL3L binding sites on pre-rRNAs as well as identifying RNA contact sites on GNL3L. Lack of GNL3L impairs synthesis of the 60S rRNAs and expression of GTPase-inactive GNL3L causes defects in early steps of pre-rRNA processing. Impaired GTP hydrolysis by GNL3L leads to its accumulation on pre-60S particles, together with other AFs with proximal binding sites. Our data further demonstrate that the GTPase activity of GNL3L is required for maintaining 60S subunit levels, protein synthesis, and cellular proliferation.</span>