A mutagenesis screen to develop mouse models for neurological diseases led to our recent finding that neurodegeneration could arise from an epistatic mutation affecting transfer RNA (tRNA) biogenesis and function. Loss of function of GTPBPP2, a protein that we showed resolves stalled ribosomes caused by insufficiency of a tRNA, results in a severe phenotype only when present together with a single C50U mutation in a central nervous system (CNS)-specific tRNAArgUCU, n-Tr20. Mutation of this tRNA, which constituted almost 60% of the brain tRNAArgUCU pool, caused a dramatic decrease in processing and thus aminoacylation, and led to ribosomal pausing at AGA codons on cerebellar transcripts. Without GTPBP2 to recycle stalled ribosomes, disease developed with pronounced locomotor defects and neuron loss in the cerebellum beginning at four weeks of age. Neurons also degenerated in other brain regions beginning at 6 weeks of age. While the brain- specificity of phenotypes is consistent with the expression pattern of tRNAArgUCU, there were notable differences in the processing of the C50U tRNAArgUCU depending on the region (cerebellum < cortex, hippocampus; postnatal day 30 < day 0 or 10). Therefore, we postulate that tissue- or development-specific distinctions in the inventory of enzymes that acts on tRNAs are key determinants for disease etiology. It is in this regard that we focus on RNase P, which catalyzes removal of the 5?? leader of pre-tRNAs. Although mammalian liver RNase P functions as a ribonucleoprotein (RNP) consisting of a ribozyme and 10 protein cofactors, there is growing evidence that RNase P may be subject to remodeling especially in the brain. Thus, altered substrate recognition could stem from structural alterations either in the pre-tRNA substrate or in RNase P. These findings provide the framework for our hypotheses: (i) the C50U tRNAArgUCU mutant has a destabilized structure that disables 5?? processing by RNase P, and (ii) spatiotemporal variations in brain RNase P make-up engender defects in processing pre-tRNAs with mutations. We will test these ideas by pursuing two aims. First, we will test the activity of partially purified mouse cerebellar RNase P towards wild-type (WT) and C50U pre- tRNAArgUCU. We will also use chemical/enzymatic probing and high-resolution structural studies to map structural differences between the WT and mutant pre-tRNAArgUCU. Second, we will perform expression analysis and RNP affinity purification to investigate the make-up of brain RNase P from different regions at specific developmental stages. Collectively, our studies will lead to the first biochemical characterization of mammalian brain RNase P and test the novel idea that spatiotemporal variations in the catalytic repertoire that acts on brain tRNAs as well as other non-coding RNAs partly hold the key to delineating how defects in RNA metabolism might result in neurological disorders.
Understanding and treating neurological diseases is a high priority in the United States and globally. Defective biogenesis of transfer RNAs, which are needed for decoding the genetic blueprint, is an etiologic basis for these diseases. Our goal is to test the idea that spatiotemporal remodeling of an enzyme critical for transfer RNA maturation is a contributing factor in neurological disorders.
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| Jarrous, Nayef; Gopalan, Venkat (2010) Archaeal/eukaryal RNase P: subunits, functions and RNA diversification. Nucleic Acids Res 38:7885-94 |
