ALTERED RNA COMPARTMENTATION IN CARCINOGENESIS

This proposal continues investigations into altered RNA compartmentation associated with early stages of carcinogenesis. In the preceding grant period, two basic lines of investigation were pursued. First, the putative nuclear scaffold (NS) NTPase involved in RNA transport, here termed p46, was identified as the N-terminus of lamins A/C. Second, RNA sequences which appear in cytoplasmic RNA following carcinogen treatment were found to consist of a subfamily of B2 sequences. This B2 subfamily corresponds to a subfamily of human Alu sequences, which is actively transcribed and involved in retrotranspositional human mutations. B2 transcripts showing altered compartmentation are 170-360 nt pol III "sense" transcripts, and the altered compartmentation occurs in welldelineated foci in rat liver. Our basic hypothesis is that altered compartmentation of B2 (Alu-like) transcripts is an initiation event, which immortalizes cells and predisposes them to subsequent promotional/progressional alterations. It is proposed thataltered B2 compartmentation results from structural/functional abnormalities produced by altered expression of p46. Two independent objectives, which are likely to be interrelated, are to determine: 1. THE FUNCTIONAL IMPORTANCE OF ALTERED COMPARTMENTATION OF B2 TRANSCRIPTS. Bxperiments will define: A) Posttranscriptional alterations in B2 transcripts which relate to their altered compartmentation; B) Modulation of B2 transcript levels will be accomplished using novel strategies, and effects on cell growth, differentiation and immortalization/transformation will be investigated; C) Whether altered B2 compartmentation is associated with genomic instability and retrotransposition, using 3 distinct approaches. 2) THE ROLE OF ELEVATED NS NTPase IN EARLY STAGES OF CARCINOGENESIS. The importance of NS NTPase activity in RNA transport has been documented, and we have identified the putative NTPase as the N-terminus of lamins A/C (p46). Production of p46 appears to be accomplished by a nuclear multicatalytic proteinase,, which removes the nuclear localization signal (NLS). We have created a modified p46 construct by inserting an epitope tag and NLS into the linker region between coils l and 2. This construct will be tested for ATPase/kinase activities, and in cell culture for effects on: A) nuclear structure; B) NS NTPase & RNA capping; C) cell growth, differentiation, and immortalization/transformation; and D) B2 compartmentation.