Ribosomal RNAs are synthesized in the nucleolus and are then processed and assembled with imported cytosolic proteins to form the mature ribosomes. In this grant application, we propose to investigate the structures of the RNA and complexes involved in two different aspects of ribosome biogenesis, using multi-dimensional multi-nuclear NMR spectroscopy. The first project concerns the specific interactions with nucleolin with pre-ribosomal RNA. Nucleolin, an abundant nucleolar protein, interacts with multiple sites on pre-ribosomal RNA and plays an important though still incompletely characterized role in the ribosome biogenesis. A consensus nucleolin RNA binding site has been identified by in vitro selection, and is comprised of a stem-loop structure. with 6 conserved nucleotides in the loop. The RNA is specifically recognized by two of the four RBD domains found in nucleolin; (2) Determine the structure of RNA oligonucleotides that bind specifically to nucleolin. We propose to: (1) Determine the structures of RNA oligonucleotides that bind specifically to nucleolin; (2) Determine the structures of RNA oligonucleotides that bind specifically to nucleolin; (2) Determine the structure of the RNA-binding domains of nucleolin; and (3) Determine the structure of a nucleolin RBD12-NRE RNA complex. Since no structures of RNA binding protein-RNA complexes that require two RBD domains have yet been determined, the structure determination of this complex should provide important new insights into RNA-protein recognition. The second project focuses on the class of snoRNAs involved in pseudouridylation. The snoRNPs are small nucleolar ribonucleic acid particles that are important for modification of ribosomal RNA nucleotides. One of these modifications is pseudouridylation, in which the glycosidic bond is moved from N1 to C5 of uracil. This modification is mediated by a class of snoRNPs which contain snoRNA with a conserved secondary structure and consensus H/ACA boxes. We propose to: (4) Investigate the structure of snoRNAs involved in pseudouridylation and their interaction with the rRNA substrate. The goal of this part of the project is to determine the structure of these """"""""guide"""""""" snoRNAs, both free in solution and in complex with the unmodified and modified target rRNA. The long term objectives of the work described in this grant are to gain a better understanding of RNA folding, RNA-protein, and RNA-RNA interactions. The specific projects proposed will also lead to new insights into the structural mechanisms involved in pre-ribosomal RNA processing.
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