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Most human pre-mRNAs are  cis -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. This  trans -splicing reaction can be exploited by pre- trans -splicing molecules (PTMs), which are incapable of  cis -splicing. PTM-mediated  trans -splicing has been utilized to repair mutant RNAs as a novel approach to gene therapy. Herein we explore how the site of PTM expression influences  trans -splicing activity. We stably inserted a PTM expression cassette into the genome of HEK293 cells, generating clonal lines with single, unique insertion sites. We analyzed  trans -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 for  trans -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 to  trans -splice with them is consistent with the observation that pre-mRNA  cis -splicing occurs cotranscriptionally. Interestingly, we identified several cellular pre-mRNAs in one localized area that serve as  trans -splicing substrates irrespective of the PTM expression site. Thus, we find multiple cellular pre-mRNAs require PTM expression in close proximity to  trans -splice while others do not.

Proximity-dependent and proximity-independent trans-splicing in mammalian cells