The assembly of ribonucleoprotein (RNP) complexes is central to processes such as protein
translation, mRNA splicing, and telomere maintenance. ATPases of the DEAD-box family are
ubiquitous, highly conserved enzymes that play essential roles during RNP assembly in all
kingdoms of life. DEAD-box proteins play critical roles in human health and disease; defects in
DEAD-box proteins underlie the progression of specific cancers as well as developmental
disorders, and are co-opted by RNA viruses such as HIV and West Nile for viral RNA processing.
Though they are part of the SF2 family of helicases, DEAD-box proteins lack key domains
present in processive SF2 helicases, and rely instead on trans factors that regulate ATP
hydrolysis and substrate binding. During RNP assembly, ATPase activity has been proposed to
drive the remodeling of secondary and tertiary RNA structures, coordinating the ordered
addition of proteins to form functional RNP assemblies. The requirement of trans modulators
makes DEAD-box ATPases ideal regulators, integrating RNP biogenesis with cellular signaling.
However, due to the transient nature of their interactions, we have no molecular understanding
of the how DEAD-box proteins engage and remodel their RNP assembly substrates. This
proposal describes a hybrid approach to define the molecular details of four essential DEAD-box
proteins (Dbp10, Drs1, Spb4 and Mak5) during the assembly of a complex RNP, the large
ribosomal (60S) subunit. We genetically manipulated yeast strains to trap and enrich distinct,
transient DEAD-box·RNP intermediates. The structural characterization of these dynamic
complexes by cryo-electron microscopy, as part of an integrated approach that includes cross-
linking mass-spectrometry and targeted in vitro reconstitution experiments, will shed light on
the molecular interactions of DEAD-box proteins with substrate RNA and modulating co-
factors. Because DEAD-box modulation of 60S maturation is closely associated with the
regulation of nucleolar pre-60S release, we will use a color-switching yeast strain to probe the
effect the expression of Dbp10, Drs1, Mak5 and Spb4 trapping mutants have on the subcellular
distribution of 60s intermediates. Together, these studies represent a unique approach to
understand the function of DEAD-box proteins in the centrally important 60S biogenesis
pathway. These innovative reagents and their use within an integrative experimental
approach will uniquely inform how DEAD-box proteins engage transient, dynamic
intermediates to modulate RNP assembly.