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Published online before print April 25, 2008
RNA, DOI: 10.1261/rna.384808
 Copyright © 2008 RNA Society
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Proximity-dependent and proximity-independent trans-splicing in mammalian cells

Kristi D. Viles1,2,3 and Bruce A. Sullenger1,2,3

1 Department of Surgery, Duke University Medical Center, Duke University, Durham, North Carolina 27517, USA
2 University Program in Genetics and Genomics, Duke University Medical Center, Duke University, Durham, North Carolina 27710, USA
3 Duke Center for Translational Research, Duke University Medical Center, Duke University, Durham, North Carolina 27710, USA

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.

Keywords: RNA; trans-splicing; splicing

Received November 17, 2007 ; accepted February 1, 2008.

Reprint requests to: Bruce A. Sullenger, Duke Center for Translational Research, Duke University Medical Center, Medical Sciences Research Building #2, P.O. Box 103035, Duke University, Durham, NC 27710, USA; e-mail: bruce.sullenger{at}duke.edu; fax: (919) 684-6492.

Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.384808.


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