RNAs are integral components of molecular machines that carry out all essential processes in gene expression. To expand the functional landscape of these molecular machines, RNAs synergize with proteins to form large complexes called ribonucleoproteins (RNPs). RNPs are formed initially during transcription, where synthesis of the RNA is coupled to RNA folding and association of proteins. A possible consequence of this coupling is that improper co-transcriptional folding may delay protein association, thereby hindering RNP assembly. Yet, RNPs like the ribosome form within minutes in the cell suggesting that there are mechanisms to prevent misfolding or slow assembly. The ribosome represents an ideal model system for studying co-transcriptional RNP assembly, because it contains a highly structured RNA that must be properly folded and assembled to function. Decades of studies on bacterial ribosome assembly have supported a model for assembly in which ribosomal protein association is strictly hierarchical; however, recent evidence from my work and others suggests that while stable incorporation may be hierarchical, underlying transient protein binding nonetheless influences the RNA folding path. The mechanism for how proteins chaperone the RNA during transcription to accelerate assembly is currently unclear. Furthermore, while a similar ordered assembly mechanism has been proposed for eukaryotic ribosome assembly, it is likely that underlying protein binding dynamics also plays a role in guiding folding of the RNA during transcription. This proposal aims to understand the molecular consequences that arise from coupling between transcription, RNA folding, and ribosome assembly by measuring RNA folding directly during co- transcriptional assembly (Aim 1) and visualizing protein association on nascent eukaryotic RNAs (Aim 2). To examine RNA folding during transcription-coupled ribosome assembly in Aim 1, I will probe the RNA structure in real time in vitro during transcription using dimethyl sulfate (DMS) mutational profiling with sequencing (DMS- MaPseq). This method will provide a complete picture of the folding pathway while the RNA is being synthesized in the presence and absence of proteins, thereby allowing for dissection of the individual contributions to the assembly mechanism. Studying RNA folding directly will be complemented by single-molecule experiments in Aim 2 that directly examine protein/RNP binding kinetics. Specifically, I will examine binding of UtpA and U3 snoRNP in real time to nascent yeast ribosomal RNA. Transitioning to studying transcription-coupled ribosome biogenesis in eukaryotes will provide new insight into how transient binding may be a common theme in RNP assembly. Results from the K99 phase will be expanded upon in the independent phase to examine folding and assembly of larger yeast assembly intermediates, such as the 5? external transcribed spacer particle. In total, these aims will advance our understanding of the mechanistic underpinnings of how transcription-coupled RNP assembly occurs normally and shed light on how RNP assembly can be altered in disease.
PROJECT NARATIVE RNAs and proteins come together to form molecular machines that carry out essential processes in gene expression. This proposal aims to characterize assembly of the ribosome, which is an RNA-protein complex that synthesizes new proteins. Since defective ribosome assembly leads to genetic diseases and is associated with many cancers, it is important to elucidate the underlying molecular details which dictate assembly.
