Proximity-dependent and proximity-independenttrans-splicing in mammalian cells

Most human pre-mRNAs arecis-spliced, removing introns and joining flanking exons of the same RNA molecule. However, splicing of exons present on separate pre-mRNA molecules can also occur. Thistrans-splicing reaction can be exploited by pre-trans-splicing molecules (PTMs), which are incapable ofcis-splicing. PTM-mediatedtrans-splicing has been utilized to repair mutant RNAs as a novel approach to gene therapy. Herein we explore how the site of PTM expression influencestrans-splicing activity. We stably inserted a PTM expression cassette into the genome of HEK293 cells, generating clonal lines with single, unique insertion sites. We analyzedtrans-splicing to the gene where the PTM was integrated, as well as genes neighboring these loci. We observed some pre-mRNAs only serve as substrates fortrans-splicing when they are expressed in immediate proximity to the PTM expression site. The need for PTMs to be in close proximity with pre-mRNAs totrans-splice with them is consistent with the observation that pre-mRNAcis-splicing occurs cotranscriptionally. Interestingly, we identified several cellular pre-mRNAs in one localized area that serve astrans-splicing substrates irrespective of the PTM expression site. Thus, we find multiple cellular pre-mRNAs require PTM expression in close proximity totrans-splice while others do not.