Introduction

The vast majority of human genes contain introns that are removed from the precursor messenger RNA (pre-mRNA) during pre-mRNA splicing to generate mature mRNAs. Splicing requires conserved splice site consensus sequences at the intron/exon borders as well as diverse cis-acting elements within exons and introns (1,2). Disease-causing mutations within the splice sites or exonic elements frequently disrupt splicing, resulting in loss of function of the affected allele (3,4). In addition, single nucleotide polymorphisms (SNPs) have the potential to affect splicing efficiency and modify disease severity (5) or responsiveness and side effects to pharmaceuticals (6).

The most direct approach to determine whether disease-causing mutations or SNPs are associated with splicing is to perform a reverse transcription PCR (RT-PCR) analysis on RNA from the relevant tissue(s) of affected individuals. However, tissue samples are often not available. An alternative approach is to test the effects of the mutation on splicing using minigenes, in which the relevant genomic segment is cloned within a plasmid between an upstream ubiquitous transcriptional promoter and a downstream gene segment necessary for mRNA 3′ end formation.

In addition to their utility for analyzing the effects of mutations and allelic variants on splicing efficiency, minigenes have been a mainstay of investigations to identify the cis elements and trans-acting factors that regulate tissue-specific alternative splicing (7,8). Detailed strategies for the use of minigenes to investigate a variety of different questions involving constitutive and alternative splicing have been described (8,9).

Here we describe a versatile plasmid vector for the identification and characterization of cis-acting elements that affect splicing efficiency and splicing regulation. Genomic segments cloned into this vector can be used to (i) test whether mutations genetically linked with disease affect the splicing efficiency of an adjacent exon or cause cryptic splicing; (ii) test whether SNPs located within or adjacent to an exon affects its splicing efficiency; (iii) perform functional analyses of computationally identified putative splicing elements; (iv) identify cis-acting elements that enhance or repress basal splicing efficiency of specific exons; (v) identify cis-acting elements required for cell-specific splicing regulation; and (vi) determine the responsiveness of alternative exons and flanking introns to overexpression and depletion of specific regulators and identify regions required for responsiveness.

Materials and Methods

Plasmids

RHCglo (Figure 1A) was constructed by PCR using priming oligonucleotides containing restriction sites that are unique to the plasmid. The exon (Figure 1B) was derived from oligonucleotides that when annealed generated 5′ overhangs compatible with BamHI and XhoI restriction sites. RtauWT, RtauN279K, and RtauΔ280K were made by replacing the RHCglo exon with three derivatives of MAPT exon 10 via the BamHI/XhoI sites. Exons were PCR-amplified using forward primers containing either unmodified or modified sequence [tauE10wtf (5′-ATATATGGATCCGTGCAGATAATTAATAAGAA-3′), tauE10N279Kf (5′ -ATATATGGATCCGTGCAGATAATTAAGAAGAAGCT-3′), tau10del280Kf (5′-TATATGGATCCGTGCAGATAATTAATAAGCTGGATCTT-3′)], and a reverse primer with unmodified sequence tauE10wtr (5′-ATATATCTCGAGACTGCCGCCTCCCGGGACGT3′) amplified on commercially available human genomic DNA. The 5′ splice site was modified using a “doped” oligonucleotide [GLO5 (5′-GGCAGTCTCGAGGTTGKYATCAAGGTTA-3′) as the forward primer in a PCR with a reverse primer located downstream of the XbaI site (Figure 1A). The PCR product was used to replace the XhoI/XbaI fragment. The oligonucleotide generated four possible 5′ splice site sequences, and all four clones were identified by sequencing.

Figure 1. RHCglo splicing reporter plasmid. (A) RHCglo showing all restriction sites that are unique to the plasmid. The middle exon is immediately flanked upstream and downstream by the last and first 91 and 73 nucleotides (nt) of human β-globin intron 1, respectively. The distal upstream segment of intron 1 contains introns 1 and 3 of chicken skeletal troponin I (Click to enlarge)

RTB300 contains a genomic segment from human cardiac troponin T (cTNT) and was described previously (10). The flanking intronic segments from this minigene were PCR-amplified using oligonucleotides containing appropriate restriction sites and inserted between SalI/BamHI and XhoI/XbaI restriction sites to construct RF300 (see Figure 1A and 3). The four amino acid insertion (LYLQ) and 41-kDa protein isoforms of human CUG binding protein 1 (CUG-BP1) and muscleblind like 1 (MBNL1), respectively, were expressed with N-terminal FLAG® epitopes. Myotonic dystrophy protein kinase (DMPK) mRNAs containing 960 interrupted CUG repeats were expressed from a previously described minigene (11).