The long term goal of our research is to understand regulation of coordinate expression of ribosomal protein genes, and roles of non- ribosomal nucleolar proteins in rRNA processing and ribosome assembly. The mechanisms of equimolar synthesis of ribosomal proteins and rRNAs or of coordinate changes in their synthesis in response to altered demands for protein synthesis are not understood. We wt utilizing the yeast ribosomal protein gene CRY2 as a model system to study the feedback regulation of ribosomal protein gene synthesis in eukaryotes. Expression of CRY2 is repressed by rp59 encoded by CRY2, via conserved sequences in the 5' exon and intron of the CRY2 pre-mRNA that can fold into a stem- loop structure. Repression apparently results in unspliced CRY2 pre-mRNA being degraded in the cytoplasm. (1) We will test whether rp59 specifically blocks splicing of CRY2 pre-mRNA, thus indirectly diverting it to the cytoplasm where it is degraded, or whether rp59 directly promotes nuclear export of CRY2 pre-mRNA, leading to its decay in the cytoplasm. (2) We will further define primary sequence or secondary structural elements of CRY2 pre-mRNA necessary for regulation by probing the structure of the CRY2 RNA and by constructing site-directed mutations. (3) We will test whether rp59 binds CRY2 pre-mRNA, and use genetic selections and screens to identify sites in rp59 or CRY2 pre-mRNA necessary for binding. (4) Trans-acting factors other than rp59 involved in regulation of CRY2 will be sought by a dual selection and screen for derepressed mutants. rRNAs, transcribed in the nucleolus, and ribosomal proteins, synthesized in the cytoplasm, assemble into ribosomes in the nucleolus, a long studied but poorly understood structure prominently visible in the nucleus. We will study the structure and function of two yeast nucleolar proteins necessary for ribosome assembly: Drs1p, a putative RNA dependent ATPase or RNA helicase, and Nop4p, a protein that contains four RNA recognition motifs(RRMs). An ATPase or helicase like Drs1p or a protein with multiple RNA binding sites, like Nop4p, might facilitate rearrangements of RNA structure or nucleate assembly sites during processing and assembly of rRNAs into nascent ribosomes. (1) We will define more precisely steps in the production of 25S rRNA in which Drs1p and Nop4p function. (2) RNA ligands of these two proteins and proteins that interact with Drs1p and Nop4p will be identified by genetic and biochemical approaches. (3) We will determine whether Drs1p is an RNA dependent ATPase or RNA helicase, and analyze functions of each of its auxiliary motifs. (4) We will assay the function of each RRM in Nop4p, as well as that of each auxiliary motif or Nop4p.
Showing the most recent 10 out of 63 publications
