Sub-cellular localisation and trans-splicing of fusion transcripts in mouse embryonic stem cells containing gene trap insertions

• Main Author: Sleeman, Judith E.
• Published: University of Edinburgh 1995
• Subjects: 572.8
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.662030

A gene trap vector designed to trap the 3' UTRs of endogenous genes was used in mouse embryonic stem (ES) cells to assess the range of RNA localization patterns than can be directed by these regions. Conventional gene trap vectors which form fusion transcripts containing the 5' regions of endogenous genes were also used both in ES cells and in fibroblasts to investigate the possibility that RNA localization signals also exist in these regions. Using vectors that contain a splice acceptor site to trap the 5' regions of genes, a number of cell lines showed nuclear localization of the fusion transcript. These nuclear transcripts contain intron sequences from the gene trap vector, indicating that they are inefficiently or incorrectly spliced. In two of these cell lines, although most of the <I>βgeo</I> transcript remained unspliced, a proportion was processed by an accurate inter-molecular splicing reaction, joining sequences from a variety of endogenous mouse genes to the vector splice acceptor site. Each of these cell lines was shown to contain a single site of integration of the vector, within the 5' external transcribed spacer (5'ETS) of a ribosomal transcription unit. These integrations are predicted to lead to the synthesis of poII transcripts containing the ribosomal 5'ETS followed by the splice acceptor, <I>βgeo</I> and polyadenylation signal from the gene trap vector. These poII transcripts, containing protein coding sequences linked to a splice acceptor site with no upstream splice donor are analogous to the VSG and PARP transcription units that are <I>trans</I>-spliced in trypanosomes. This analogy suggests that a mechanism similar to the <I>trans</I>-splicing of trypanosome VSG and PARP genes is being used in these ES cell lines to produce translated products from gene trap insertions into RNA poII transcription units.