5' exon replacement and repair by spliceosome-mediated RNA trans-splicing

Abstract

Spliceosome-mediated RNA trans-splicing (SMaRT) has been used previously to reprogram mutant endogenous CFTR and factor VIII mRNAs in human epithelial cell and tissue models and knockout mice, respectively. Those studies used 3' exon replacement (3'ER); a process in which the distal portion of RNA is reprogrammed. Here, we also show that the 5' end of mRNA can be completely rewritten by 5'ER. For proof-of-concept, and to test whether 5'ER could generate functional CFTR, we generated a mutant minigene target containing CFTR exons 10-24 (deltaF508) and a mini-intron 10, and a pretrans-splicing molecule (targeted to intron 10) containing CFTR exons 1-10 (+F508), and tested these two constructs in 293T cells for anion efflux transport. Cells cotransfected with target and PTM showed a consistent increase in anion efflux, but there was no response in control cells that received PTM or target alone. Using a LacZ reporter system to accurately quantify trans-splicing efficiency, we tested several unique PTM designs. These studies provided two important findings as follows: (1) efficient trans-splicing can be achieved by binding the PTM to different locations in the target, and (2) relatively few changes in PTM design can have a profound impact on trans-splicing activity. Tethering the PTM close to the target 3' splice site (as opposed to the donor site) and inserting an intron in the PTM coding resulted in a 65-fold enhancement of LacZ activity. These studies demonstrate that (1) SMaRT can be used to reprogram the 5' end of mRNA, and (2) efficiency can be improved substantially.

FIGURE 1.

CFTR model constructs and illustration of trans-splicing by 5′ exon replacement. (A) Detailed structure of a 5′ER PTM (CFTR-PTM11), and a mini-gene target (CFTR-T11). The PTM lacks a poly(A) signal and the target lacks a methionine initiator codon. Modified codons are engineered in exons 9 and 10 of the PTM, and exons 11 and 12 of the target to aid in distinguishing trans-spliced products from cis-spliced or endogenous CFTR products. The position of the three oligonucleotide primers (CF1, CF93, and CF111) used in RT–PCR experiments are indicated by arrows. The diagram shows the PTM binding to the 5′ splice site of intron 10 of the minigene target at the pre-mRNA level. (B) Demonstration of trans-splicing by RT–PCR. Cells were transfected with either PTM plus target, or PTM, target, or vector (pc3.1DNA) alone, or PTM plus target, but without the reverse transcription step (four different negative controls). Cis-spliced (ΔF508) products were detected with primers CF1 and CF111 (right arrow), and trans-spliced products with primers CF93 and CF111 (left arrow). (BD) Binding domain.

FIGURE 2.

RNA trans-splicing by 5′ exon replacement generates functional ion channels in human cells. HEK 293T cells were transfected with either target alone (CFTR-T11), PTM alone (CFTR-PTM30), or with target and PTM. Cell populations were incubated in medium containing ¹²⁵Iodine, and then tested for the presence of ion channel efflux activity (in min⁻¹) by stimulation with forskolin and IBMX at intervals of 3 min (time points 7, 10, 13, and 16 min). Forskolin is predicted to elevate cAMP levels and stimulate cAMP-dependant ion channel activity. Only those monolayers that received a PTM and target showed substantial increases in anion efflux at three time-points (arrows). Values represent mean ± standard error of two independent transfections, with the exception of the 4-min time point for PTM plus target, which represents a single data point. (BD) Binding domain.

FIGURE 3.

Model system to optimize trans-splicing efficiency. (A) Schematic diagram showing a LacZ PTM binding to a chimeric LacZ-ΔF508-CFTR target at the pre-mRNA level. (B) Trans-splicing efficiency by 5′ exon replacement is increased by binding to more than one splice site. The panels above the histogram shows the location of four different PTM-binding domains (CF-PTM42, CF-PTM43, CF-PTM44, CF-PTM45) targeted to different regions of a defective LacZ-ΔF508 target (LacZCF10m). CF-PTM45 and CF-PTM45(+) are identical, except that the latter has a poly(A) signal and the former does not. The histogram below the five panels compares the repair efficiency of the five PTMs using an in-solution β-galactosidase assay. Transfections, preparation of cell lysates, and assessment of activity was as described previously (Puttaraju et al. 2001). Values are the mean of two independent experiments (± standard error) and are expressed as units of β-galactosidase activity per milligram of total protein. PTMs and the target are cloned in pc3.1DNA(−). The vector-alone sample refers to plasmid pc3.1DNA(−) that does not contain either a PTM or a target. Each transfection was performed with 2 μg of target plasmid and 2 μg of PTM plasmid. All samples contained the same amount of plasmid DNA; PTM-alone and target-alone samples were balanced to 4 μg with vector pc3.1DNA(−). (BD) Binding domain; (SS) splice site; (+), with bGH poly(A) signal; (−), without bGH polyA signal.

FIGURE 4.

Targeting the PTM closer to the intended 3′ splice site results in a substantial increase in trans-splicing efficiency. The panel above the histogram shows a schematic of two different PTMs and the location of their binding domain with respect to the target acceptor site. The binding domain of CF-PTM53 is located ~400 bases downstream of CF-PTM50. Both PTMs have a spacer sequence that is shorter than all previous constructs by 50 bases. Values are mean ± standard error of three separate experiments. (BD) Binding domain.

FIGURE 5.

Inserting an intron in the PTM coding unit results in a significant improvement in trans-splicing efficiency. The two PTMs shown in the panel above the histogram are identical, except that CF-PTM59 has a 543-bp CFTR mini-intron 9 in the LacZ coding unit. The 3′ LacZ exon in CF-PTM59 is 124 bp long. Values are mean ± standard error of two separate experiments. (BD) Binding domain.