The broad goal of this proposal is to elucidate the structural details of the numerous protein-RNA interactions that are essential in the recognition and modification of eukaryotic messenger RNA. The nascent eukaryotic transcript is subjected to numerous modifications before the journey out of the nucleus. As transcripts are necessarily diverse in sequence, general recognition of the single stranded RNA messages must be sequence non-specific. Modifications at the 5' end of the message aid in transcript recognition by providing unique molecular handles for cellular proteins. In this proposal, we will focus on the recognition of one such molecular handle (the 5' m7G cap), the further modification of this handle (the O2-methylation of the first transcribed nucleotide), and the general sequence non- specific recognition of the transcript itself. These events will be studied in three model systems by combining the structural information of X-ray crystallographic analysis with the functional information obtained from biochemical assays. The concerted application of structural and functional analysis on structurally diverse but functionally similar proteins is a powerful method for elucidating structural features essential to protein function.
Specific aim 1 is to elucidate the structural determinants of m7G cap binding and discrimination using the cap-specific RNA methyltransferase VP39 and the guanosine specific ribonuclease T1 as model systems.
Specific aim 2 is to test the mechanism for single-stranded sequence non-specific RNA binding observed in a recent VP39/RNA co-crystal structure and then to determine the generality of this mechanism through the structural and biochemical characterization of a monoclonal anti-RNA antibody D44.
Specific aim 3 examines the structural details and mechanism of O2 ribose RNA methylation by inducing catalysis in a VP39/RNA co- crystal. The results of these studies will prove invaluable in the analysis of current functional information and future structural information obtained from cellular RNA processing machinery.
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