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

Abstract

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.

FIGURE 1.

(A) Trans-splicing expression cassette (pCIZ). Diagram of the untargeted trans-splicing expression cassette pCIZ. The positions of the CMV promoter, branchpoint sequence (BPS), polypyrimidine tract (PyT), 3′splice site (3′ss), 3′ exon (partial LacZ) tag, and the rat pre-proinsulin (PPI) polyadenylation signal are all noted. The blue “A” within the BPS begins the nucleophilic attack on the 5′ss to begin the splicing reaction. The intronic sequence is derived from adenovirus type II. (B) Trans-splicing competes with cis-splicing reactions. Trans-splicing of the LacZ tag onto cellular RNA transcripts takes place within the endogenous spliceosome and competes with cis-splicing. For both, splicing of exons and removal of introns proceeds via two trans-esterification reactions (steps 1 and 2 for cis-splicing and steps 1′ and 2′ for trans-splicing. A trans-spliced pre-mRNA transcript will be “tagged” with the LacZ as its 3′ exon. (C) Diagram of the retroviral construct with the 3′ PTM cloned into the U3 region of the LTR. The trans-splicing molecule (pCIZ) was PCR amplified with primers that included two restriction enzyme sites—BglII and MluI. These sites were cut and used to ligate pCIZ into integrating the LTR of the retroviral vector (N2A) (Hantzopoulos et al. 1989). The regions covered by the neomycin and Northern, and Southern probes are shown. Half arrows indicate the location of inverse primers used to find the integration site within the genome of HEK293 cells. U3, R, and U5 make up the LTR of the construct. Expression of the pCIZ trans-splicing molecule is driven by a CMV promoter and includes an intronic region, a BPS, PyT, a consensus 3′ss, and a 3′ exon. The exon here is a partial sequence from LacZ and PPI designates the rat PPI signal.

FIGURE 2.

(A) Integration of the retroviral vector carrying the trans-splicing molecule. Southern blot showing unique integration bands from several clonal populations. Genomic DNA was digested with BglII and hybridized with a probe covering the intron/LacZ region (Fig. 1c). The 4.5-kb band is a constant band throughout the populations. Populations used for further investigation are in bold and marked with an asterisk. (B) Scheme showing integrating retrovirus with the trans-splicing construct and how the constant and variable bands are derived. The variable band (>1.8 kb) will be 1.8 kb plus the size of the genomic piece cut with BglII. The constant band (4.5 kb) and a probe to the neomycin (Neo) gene (data not shown) were used as Southern controls. The pCIZ integrated construct is represented with a “CMV” rectangle, a black bar (intron region), and a “Z” rectangle (LacZ and the PPI, pre-proinsulin polyadenylation signal region).

FIGURE 3.

Diagram of integration sites of N2A-pCIZ. The population integrated into each gene is noted. The gene name, chromosome position, and total number of exons within the gene are noted for each schematic given. The sizes of exons (squares) and introns (bars) are shown. The integration site and direction are indicated by the small dark gray box (LacZ/PPI) and by the small checkered box with an arrow (CMV). Small half-arrows show the position of primers used in PCR reactions. The checkered/open bars show exons (checkered bar) trans-spliced and tagged with the LacZ exon (open bar tail). The endogenous 5′ss sequence (EXON | intron) involved in trans-slicing are also shown. Bases matching the cis-splicing consensus 5′ donor sequence (^C _AAG | gt^a _gagt) are underlined bold. The largest intron within the gene is indicated with italic numbers.

FIGURE 4.

(A) Representative agarose gels of trans-spliced RT-PCR products from genes involved in integration. All bands were cut out of gel, cleaned, and sequenced. (Left panel) Small arrowheads indicate trans-spliced products. Other bands are nonspecific RT-PCR products. (Right panel) Diagram shows the location of primers (half arrows) used to find trans-spliced products and a schematic representation of the RT-PCR amplified product. Forward primers to the first exon within each gene and primers to the exon preceding the largest intron were used for the RT-PCR reactions. (B) Example of results obtained from sequencing RT-PCR bands. (Left panel) Sequence chromatogram showing the end of ROBO2 exon2 and the beginning of the LacZ sequence (indicated by an arrow). (Right panel) Example of sequence chromatogram of an unreacted PTM with the intron/LacZ junction indicated by an arrow.

FIGURE 5.

Genes along chromosome 12. Distance (bp) from Z11 is noted. The asterisks denote trans-spliced transcripts from tables above.