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

Journal Article

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.

Full Text

Duke Authors

Cited Authors

  • Viles, KD; Sullenger, BA

Published Date

  • June 2008

Published In

Volume / Issue

  • 14 / 6

Start / End Page

  • 1081 - 1094

PubMed ID

  • 18441053

Electronic International Standard Serial Number (EISSN)

  • 1469-9001

Digital Object Identifier (DOI)

  • 10.1261/rna.384808

Language

  • eng

Conference Location

  • United States