During eukaryotic ribosome biogenesis, nascent ribosomal RNA (rRNA) forms pre-ribosomal particles containing ribosomal proteins and assembly factors. Subsequently, these immature rRNAs are processed and remodelled. Little is known about the premature assembly states of rRNAs and their structural rearrangement during ribosome biogenesis. Using cryo-EM we characterize a pre-60S particle, where the 5S rRNA and its associated ribosomal proteins L18 and L5 (5S ribonucleoprotein (RNP)) are rotated by almost 180°when compared with the mature subunit. Consequently, neighbouring 25S rRNA helices that protrude from the base of the central protuberance are deformed. This altered topology is stabilized by nearby assembly factors (Rsa4 and Nog1), which were identified by fitting their three-dimensional structures into the cryo-EM density. We suggest that the 5S RNP performs a semicircular movement during 60S biogenesis to adopt its final position, fulfilling a chaperone-like function in guiding the flanking 25S rRNA helices of the central protuberance to their final topology.

The yeast Mex67-Mtr2 complex and its homologous metazoan counterpart TAP-p15 operate as nuclear export receptors by binding and translocating mRNA through the nuclear pore complexes. Here, we show how Mex67-Mtr2 can also function in the nuclear export of the ribosomal 60S subunit. Biochemical and genetic studies reveal a previously unrecognized interaction surface on the NTF2-like scaffold of the Mex67-Mtr2 heterodimer, which in vivo binds to pre-60S particles and in vitro can interact with 5S rRNA. Crucial structural requirements for this binding platform are loop insertions in the middle domain of Mex67 and Mtr2, which are absent from human TAP-p15. Notably, when the positively charged amino acids in the Mex67 loop are mutated, interaction of Mex67-Mtr2 with pre-60S particles and 5S rRNA is inhibited, and 60S subunits, but not mRNA, accumulate in the nucleus. Thus, the general mRNA exporter Mex67-Mtr2 contains a distinct electrostatic interaction surface for transporting 60S preribosomal cargo.

Ribosomal proteins are synthesized in the cytoplasm, before nuclear import and assembly with ribosomal RNA (rRNA). Little is known about coordination of nucleocytoplasmic transport with ribosome assembly. Here, we identify a transport adaptor, symportin 1 (Syo1), that facilitates synchronized coimport of the two 5S-rRNA binding proteins Rpl5 and Rpl11. In vitro studies revealed that Syo1 concomitantly binds Rpl5-Rpl11 and furthermore recruits the import receptor Kap104. The Syo1-Rpl5-Rpl11 import complex is released from Kap104 by RanGTP and can be directly transferred onto the 5S rRNA. Syo1 can shuttle back to the cytoplasm by interaction with phenylalanine-glycine nucleoporins. X-ray crystallography uncovered how the a-solenoid symportin accommodates the Rpl5 amino terminus, normally bound to 5S rRNA, in an extended groove. Symportin-mediated coimport of Rpl5-Rpl11 could ensure coordinated and stoichiometric incorporation of these proteins into pre-60S ribosomes.R ibosomes perform their role in translation in the cytoplasm, but ribosome assembly occurs predominantly in a specialized nuclear compartment, the nucleolus (1-4). The construction of ribosomes follows an ordered assembly of~80 ribosomal proteins (r-proteins) and four ribosomal RNAs (rRNAs) into a small (40S) and large (60S) ribosomal subunit. This process is spatially and temporally coordinated, starting with cotranscriptional assembly of a first preribosomal particle (90S) in the nucleolus that is subsequently separated into pre-40S and pre-60S ribosomes, which follow independent processing and maturation steps before export into the cytoplasm (5-8). R-proteins are synthesized in the cytoplasm and are imported into the nucleus by nuclear import receptors of the importinb/karyopherin family (9, 10). These transport receptors recognize different types of nuclear localization sequences (NLSs), and hence a number of import receptors have been implicated in decoding NLSs of r-proteins in a redundant way (9, 11). After nuclear import and before incorporation into nascent ribosomes, r-proteins are released from the transport receptor by its interaction with RanGTP (12). At present, it is thought that each r-protein is individually transported into the nucleus by its import receptor. However, a number of r-proteins form functional clusters on the ribosomal surface or assemble at distinct temporal or spatial entry points during ribosome formation (13-15), thus raising the possibility of coordinated nuclear import and assembly of r-proteins.One such pair of functionally related r-proteins is Rpl5 and Rpl11, which are close to each other on the mature 60S subunit and bind to opposite sites on the 5S rRNA ( Fig.

Pre-ribosomal particles evolve in the nucleus through transient interaction with biogenesis factors, before export to the cytoplasm. Here, we report the architecture of the late pre-60S particle purified from Saccharomyces cerevisiae through Arx1, a nuclear export factor with structural homology to methionine aminopeptidases, or its binding partner Alb1. Cryo-electron microscopy reconstruction of the Arx1-particle at 11.9 Å resolution reveals regions of extra densities on the pre-60S particle attributed to associated biogenesis factors, confirming the immature state of the nascent subunit. One of these densities could be unambiguously assigned to Arx1. Immuno-electron microscopy and UV cross-linking localize Arx1 close to the ribosomal exit tunnel in direct contact with ES27, a highly dynamic eukaryotic rRNA expansion segment. The binding of Arx1 at the exit tunnel may position this export factor to prevent premature recruitment of ribosome-associated factors active during translation.

Shuttling transport receptors carry cargo through nuclear pore complexes (NPCs) via transient interactions with Phe-Gly (FG)-rich nucleoporins. Here, we identify Arx1, a factor associated with a late 60S preribosomal particle in the nucleus, as an unconventional export receptor. Arx1 binds directly to FG nucleoporins and exhibits facilitated translocation through NPCs. Moreover, Arx1 functionally overlaps with the other 60S export receptors, Xpo1 and Mex67-Mtr2, and is genetically linked to nucleoporins. Unexpectedly, Arx1 is structurally unrelated to known shuttling transport receptors but homologous to methionine aminopeptidases (MetAPs), however, without enzymatic activity. Typically, the MetAP fold creates a central cavity that binds the methionine. In contrast, the predicted central cavity of Arx1 is involved in the interaction with FG repeat nucleoporins and 60S subunit export. Thus, an ancient enzyme fold has been adopted by Arx1 to function as a nuclear export receptor.