Permeable trypanosome cells as a model system for transceiption and trans-splicing

摘要

We have established conditions for Trypanosoma brucei permeable cells to study transcription and transsplicing. We found that the concentration of monovalent and, to a lesser extent, divalent ions plays a critical role for the expression of a number of different genes. Most remarkably, the synthesis of the spliced leader (SL) RNA was optimal at 20 mM KCI, whereas higher potassium concentrations were inhibitory. In addition, MgCL₂ concentrations above 3 mM led to the accumulation of a 3′ end shortened SL RNA species, which has been previously reported not to participate in trans-splicing (20). Using conditions optimal for the synthesis of the SL RNA, we observed accurate transsplicing of newly-synthesized α-tubulin RNA. Moreover, we detected the SL intron both joined to high molecular weight RNAs in the form of branched Y-structures and as a free linear molecule, which rapidly turned over. Furthermore, ionic concentrations that inhibit the synthesis of the SL RNA produced exclusively unspliced α-tubulin RNA, thus demonstrating that transcription and trans-splicing can be uncoupled.