Abstract Ribosomes are among the most ancient, intricate, and essential cellular machines whose function is to process genetic information to generate cellular proteins. Human ribosomes are incredibly complex, consisting of 80 ribosomal proteins and four heavily modified ribosomal RNAs (rRNA), which must be precisely assembled. Further, the largest pre-rRNA is transcribed from a polycistronic gene repeated more than 300 times in the genome. This pre-rRNA must be post-transcriptionally processed to remove external and internal spacers to release mature rRNAs. The majority of these assembly events occur in a specialized nuclear body called the nucleolus. Owing, in part, to their complex assembly process, it is now appreciated that not all ribosomes are the same, and it has been suggested that heterogenous ribosomes may have specialized functions. We have discovered that an undetermined stress response pathway regulates early events in rRNA processing. We have shown that this pathway is activated by oxidative stress, select viral infections, and chemotherapeutic drugs. Activation results in unprocessed pre-rRNA being stored in the nucleolus, which re-enters the maturation process when stress resolves. We are undertaking a multi-omics approach to uncover this pathway and determine how this prolonged stalling of processing and storage of pre-rRNA in the nucleolus affects the assembly and activity of ribosomes generated in this time window. We have developed a novel technique that allows for the time- resolved capture of ribosomes to address this question. Further, analysis of processing sites demonstrates profound sequence heterogeneity across the different repeated genes through the genome, raising the possibility that processing events are differentially regulated between rRNA genes. We expect to clarify the sequence motifs required for early rRNA processing and determine how naturally occurring variants contribute to the differential expression of sub-types of ribosomes. We seek to understand better the basic principles governing ribosome biogenesis in light of diverse sequence variation and how various stimuli alter the process of ribosome assembly.