LOCATING THE REGIONS OF AN ADML 3' INTRON RNA ANALOGUE BOUND TO PUF60 RRMS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Alternative splicing is a major mechanism for RNA processing which significantly contributes to the expanded complexity of the expression of human genome. In metazoan, the early-stage recognition of the 3' pre-mRNA intron sequence by RNA binding proteins (RNABPs) is considered to be essential for specifying intron/exon- definitions and priming the pre-mRNA for the later stages of spliceosome assembly. PUF60 is such an RNABP which may promote alternative splicing patterns. We determined the dissociation constants of PUF60 toward the AdML 3' intron sequence to be at the uM-range, with the binding affinity predominantly provided by its two central RRMs. We have also obtained a 1.91 A crystal structure of the two RRMs of PUF60 in complex with AdML3' intron sequence, which reveals a homo-dimeric organization, where each participating PUF60 monomer captures a nucleic acid tract, and the PUF60 dimer places two nucleic acid tracts in parallel with opposing directionalities. However, where PUF60 RRMs bind on the RNA analogue has not been located because two of the bound bases have poor electron densities. Therefore, we have grown new crystals of PUF60 RRMs in complex with Bromine-modified RNA analogues, with the aim to identify the bound bases. CHESS beamlines hold the potential to help us visualize the Bromine modification on our RNA analogues, and therefore allow us to locate the bound regions and propose a more precise model for the PUF60 RRMs mediated RNA interactions.