In eukaryotic cells, the synthesis of ribosomes is a complex process involving several hundred genes whose functions span transcription of precursor ribosomal ribonucleic acids (pre-rRNAs), processing of pre-rRNAs, assembly of ribosomal proteins (r-proteins) with pre-rRNAs, and nuclear export of the ribosomal particles 
. Ribosome biogenesis is an essential process, with mutations of ribosome biogenesis genes either causing lethality or increasing susceptibility to cancer—e.g., bone marrow failure and leukemia 
or breast cancer 
. This pathway has been extensively studied over the past 30–40 y, and a broad picture of the major events is known for the yeast Saccharomyces cerevisiae
. First, 35S polycistronic pre-rRNA is transcribed from the ribosomal deoxyribonucleic acid (rDNA) repeat by RNA polymerase I in the nucleolus. During transcription, the small-subunit processome and some small-subunit r-proteins assemble onto the 35S pre-rRNA to form a 90S particle. The 35S pre-rRNA is cleaved to release the pre-40S particle, which contains a 20S pre-rRNA. The pre-60S complex assembles on the rest of the transcript, and both subunits are further processed in the nucleus and independently exported through the nuclear pore complex (NPC) to the cytoplasm, where they undergo further maturation—e.g., cleavage of 20S pre-rRNA to 18S rRNA. The mature small subunit contains 32 proteins and 18S rRNA, while the large subunit contains 46 proteins and three rRNAs: 5.8S, 25S, both derived from the 35S precursor, and 5S, which is transcribed separately by RNA polymerase III.
Ribosome biogenesis is a temporally and spatially dynamic process requiring coordination of many trans-acting factors at different stages along the pathway, including at least 170 protein factors that act to modify and cleave pre-rRNAs and help to assemble and export ribosomal particles 
. Many of these protein factors were first identified by yeast genetics. Later, biochemical purifications coupled with mass spectrometric analysis greatly expanded the number of known factors 
. In addition, a large-scale effort using oligonucleotide microarrays identified 115 mutants that exhibited pre-rRNA processing defects, and 10 new genes were confirmed to affect pre-rRNA processing 
. Despite these intensive studies, new ribosome biogenesis genes are still emerging, and recent computational analysis suggests that over 200 genes constitute the ribosome biogenesis regulon 
, indicating that the genes in this fundamental cellular pathway have not been completely identified.
We asked if recent functional genomic and proteomic studies could be applied in a predictive fashion to identify additional ribosomal biogenesis genes. In particular, functional networks of genes have been reconstructed, incorporating literally millions of experimental observations into probabilistic networks indicating genes likely to work together in cells. The emerging technique of network-guided genetics (e.g., 
) leverages such networks to computationally associate candidate genes with a biological process of interest, much as a genetic screen might do. We used such a probabilistic gene network 
to predict the genes most likely to participate in yeast ribosome biogenesis based on connectivity to known ribosomal biogenesis genes, and we present here experimental confirmation of at least 15 new genes affecting ribosome biogenesis. Beyond providing new insights into ribosome biogenesis, this study therefore also represents one of the most extensive experimental studies to date of the principle of network-guided genetics, which we demonstrate to be a powerful approach for rational discovery of candidate genes, applicable to diverse biological processes.