The DEAD-box protein Drs1 facilitates compaction of ribosomal RNA domain III during nucleolar stages of large ribosomal subunit assembly

The ribosome translates the genetic code in mRNA and catalyzes protein synthesis in all living organisms. This complex macromolecular machine is composed of two subunits that in eukaryotes contain four ribosomal RNAs (rRNAs) and 79 ribosomal proteins (RPs). The functional centers of ribosomes, such as the peptidyl transferase center (PTC), the nascent polypeptide exit tunnel (NPET), and GTPase activating center (GAC) in the large subunit (LSU), are made primarily of rRNA. Consequently, a critical aspect of ribosome assembly is the correct folding of rRNA into the precise structures required for ribosome function. 

In eukaryotes such as Saccharomyces cerevisiae (baker’s yeast), ribosome assembly occurs in three subcellular compartments- it begins in the nucleolus with transcription of rRNA, followed by folding, compaction, modification, and processing of the rRNA and initial stages of incorporation of many RPs. Assembly continues in the nucleoplasm and concludes in the cytoplasm. Efficient and accurate assembly is enabled by more than 200 assembly factors (AFs), which transiently associate with pre-ribosomes. The functions of these AFs include RNA processing (by endo- and exonucleases), fine-tuning RNA structure via covalent modification (methylation and pseudo-uridylation), chaperoning rRNA folding ( e.g., stabilizing helical junctions), remodeling protein-RNA subcomplexes and powering incorporation of RPs. 

The largest rRNA of the LSU (i.e., the 25S rRNA) folds into six distinct, phylogenetically conserved “domains” (domains I-VI). These domains of rRNA gather and around a cluster of “root helices”, much like the petals of a flower, in both two and three dimensions. Previous work shows that these domains of rRNA come together or “compact” around the root helices in a hierarchical manner- beginning with the two ends of the rRNA (domains I, II, and VI) followed by the middle domains (domains III, IV, and V). The large-scale rearrangements of rRNA necessary to compact these individual domains during assembly is facilitated by various AFs. 

Nineteen DEAD-box proteins (DBPs), a class of non-processive helicases, contribute to ribosome assembly in yeast. These DBPs contain two globular RecA domains connected by a non-descript “linker”. At the interface of the RecA domains are 12 conserved motifs involved in RNA-binding and ATP binding/hydrolysis. The coordination of ATP hydrolysis and RNA binding by DBPs results in a melting of ~12-15 bases in double-stranded RNA. Whether and how unwinding of such a short rRNA duplex can facilitate large-scale rearrangements of rRNA structure remains an important question to be addressed. It also remains unclear how DBPs are targeted to their specific rRNA substrates. The RNA binding domain of DBPs is not sequence specific; it binds to the phosphodiester backbone of the RNA. However, each different DBP contains specific auxiliary amino- and/or carboxy-termini (referred to as “extensions” or “tails”). To date, the role that these extensions play in DBP function are understudied, but they have been hypothesized to contribute to substrate specificity and auto-regulation of DBPs. 

Exactly how these RNA helicases facilitate ribosome assembly via unwinding activity is a long and under-studied question in our field. 

In this dissertation, I demonstrate that the DBP “Drs1” contributes to compaction of rRNA domain III during nucleolar stages of LSU assembly. The Introduction of this dissertation aims to give the reader a background on ribosome assembly and DBPs and provides four examples of the best studied DBPs in ribosome assembly. 

In Chapter 2, I demonstrate that: (1) Drs1 is present on assembling LSUs in the nucleolus, up to the stage where compaction of rRNA domain III occurs, (2) Drs1 interacts with proteins in and around rRNA domain III, (3) Mutations in conserved motifs of Drs1 predicted to disrupt its ATPase activity result in a block in compaction of rRNA domain III during nucleolar stages of LSU assembly, and (4) Mechanisms exist at the root helices of rRNA domain III to facilitate large-scale rearrangements of the large rRNA structure. 

In Chapter 3, I present and discuss preliminary data that show: (1) The N-terminal extension of Drs1 plays little to no role in the activity of Drs1, and (2) The C-terminal extension of Drs1 may contribute to the RNA-specificity/unwinding activity of Drs1. 

Altogether, this dissertation contributes an important context for the role of Drs1 in essential, large-scale rearrangements involved in assembly of the LSU in S. cerevisiae.