The human cell nucleus appears to be organized at multiple levels in a manner that is intimately linked to important nuclear functions such as DNA replication and gene expression. A global description of the nuclear organization of the expressed human genome at the single-gene level is necessary to investigate the functional significance of this organization, but is lacking in part due to the fact that the current methods used to investigate this problem lack either gene-level resolution or are limited to the analysis of a handful of genes or RNAs at one time. The studies described herein propose to evaluate ribozyme-mediated RNA tagging as a fundamentally different approach to probing the global organization of the expressed genome at the single gene level. In this approach, genes expressed near the chromosomal locus of a trans-splicing ribozyme are identified by transfer of an RNA tag from the ribozyme to the cellular RNA. RNA tagging is an ideal genome-scale technology to apply to the question of expressed genome organization because it should be capable of simultaneously probing the entire expressed genome within a defined nuclear region, and because it will directly take advantage of the human genomic maps and DNA sequences for data analysis and interpretation. Additionally, this RNA tagging approach should generate both expressed genome organization maps and chromosome region-specific tagged RNA libraries that can be easily disseminated to the research community. The objectives of this proposal are: 1.) To construct human cell lines expressing trans-splicing ribozyme probes. 2.) To employ trans-splicing ribozymes to assess the global organization of the expressed human genome at the single-gene level. 3.) To induce ribozyme-expressing cells to undergo terminal differentiation, and assess the affect of this differentiation on the organization of the expressed genome. The completion of such analysis will provide the needed proof of principle that ribozyme-mediated RNA tagging is a genome-scale technology that can be used to elucidate the global organization of the expressed human genome the single-gene level.
