Proximity-dependent and proximity-independent trans-splicing in mammalian cells.
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.
Viles
Kristi D
KD
Department of Surgery, Duke University Medical Center, Duke University, Durham, North Carolina 27517, USA.
Sullenger
Bruce A
BA
D000818:Animals; D002460:Cell Line; D005091:Exons; D006801:Humans; D012322:RNA Precursors / Q000378:metabolism*; D051381:Rats; D017381:Spliceosomes; D020040:Trans-Splicing; D014158:Transcription, Genetic
D012322:RNA Precursors