Frame-disrupting mutations elicit pre-mRNA accumulation independently of frame disruption

Abstract

The T-cell receptor (TCR) and immunoglobulin (Ig) genes are unique among vertebrate genes in that they undergo programmed rearrangement, a process that allows them to generate an enormous array of receptors with different antigen specificities. While crucial for immune function, this rearrangement mechanism is highly error prone, often generating frameshift or nonsense mutations that render the rearranged TCR and Ig genes defective. Such frame-disrupting mutations have been reported to increase the level of TCRbeta and Igmicro pre-mRNA, suggesting the hypothesis that RNA processing is blocked when frame disruption is sensed. Using a chimeric gene that contains TCRbeta sequences conferring this upregulatory response, we provide evidence that pre-mRNA upregulation is neither frame- nor translation-dependent; instead, several lines of evidence suggested that it is the result of disrupted cis elements necessary for efficient RNA splicing. In particular, we identify the rearranging VDJ(beta) exon as being uniquely densely packed with exonic-splicing enhancers (ESEs), rendering this exon hypersensitive to mutational disruption. As the chimeric gene that we developed for these studies generates unusually stable nuclear pre-mRNAs that accumulate when challenged with ESE mutations, we suggest it can be used as a sensitive in vivo system to identify and characterize ESEs.

Figure 1.

Nonsense and frameshift mutations upregulate pre-mRNA. (A) Schematic diagram showing how the TCR/TPI chimeric gene construct was generated from the parental TCRβ and TPI gene constructs. (B) Schematic diagram of the TCR/TPI chimeric constructs used for the transfection experiments in (C). The position of the nonsense and frameshift mutations in A^N1, A^I1+ and A^N2 are indicated (at codons 91 and 100 and 194, respectively). Stop signs indicate the position of in-frame termination codons. (C) RNase protection analysis of total cellular RNA (10 µg) from HeLa cells transiently transfected with the constructs described in (B). Probe 1 (B) was used to detect both pre- and mature mRNA. Neomycin (Neo) mRNA, an independent transcription unit expressed from the TCR/TPI plasmids, serves as an internal control for transfection efficiency. (D, E) Quantification of mature mRNA (D) and pre-mRNA (E) levels from cells transfected as in (C). The values were determined from three or more experiments and normalized to Neo mRNA level. Pre-mRNA and mature mRNA levels from construct A were arbitrarily set to 100. Error bars indicate standard error.

Figure 2.

Pre-mRNA upregulation requires the VDJ exon. (A) Schematic diagrams of the constructs transfected in (B) (see text for further details of these constructs). Note that A^N2, B^N, C^N and D^N all have the same nonsense mutation at the same position in exon 6 (indicated with a stop sign). (B) RNase protection analysis of total cellular RNA (10 µg) harvested from HeLa cells transiently transfected with the constructs shown. Probe 2 (A) was used to detect the pre-mRNA; quantification was done as in Figure 1 (average of two experiments). The values below the gel were quantified as in Figure 1D and E from two experiments. Pre-mRNA levels for each wild-type construct were arbitrarily set to 100.

Figure 3.

The upregulated pre-mRNAs are partially spliced. (A) Schematic diagram of construct A, indicating the position of the intron probes used for the northern blot analysis in (B) and (C). (B) Northern blot analysis of total cellular RNA (10 µg) isolated from HeLa cells stably transfected with construct A. The schematics indicate the introns present in the pre-mRNAs in each band, based on band migration and their hybridization with the different probes. The bands corresponding to spliced IVS-A, -B, -C and -D migrated at a position consistent with their expected sizes (∼0.5, ∼0.8, ∼0.25 and ∼0.1 nt, respectively). The Δ denotes 28S rRNA that cross-hybridized with all probes and was present in nontransfected HeLa cells (data not shown); it is a broad band that co-migrated with the high-molecular-weight TCR/TPI pre-mRNAs. The § denotes ∼1.8–2.0-kb transcripts that hybridized with all the intron probes; their size is consistent with them being 3′ cleavage intermediates that have either IVS-A and -B at their 5′ terminus (as a result of 5′ splice-site cleavage but not 3′ splice-site cleavage). IVS-C and -D are present in a fraction of these § transcripts presumably because these small introns are sometimes retained. The asterisk indicates a transcript whose size and hybridization characteristics suggest it is a partially spliced TCR/TPI pre-mRNA lacking the β-actin intron, which is upstream of IVS-A (data not shown). (C) Northern blot analysis of nuclear and cytoplasmic fraction RNA from HeLa cells stably transfected with the constructs shown and hybridized with probe E. U, unspliced pre-mRNA; P, partially spliced pre-mRNA; M, mature mRNA. The blots shown in (B) and (C) are representative of two or more independent blots.

Figure 4.

Nonsense and frameshift mutations in the VDJ exon trigger pre-mRNA upregulation. (A) Schematic diagram indicating the location of the nonsense and frameshift mutations introduced into construct A. Constructs A^N1, A^N3 and A^N4 have nonsense mutations at codons 91, 112 and 146, respectively. Construct A^I2+ has a 1-nt insertion at codon 114 (indicated by an inverted triangle) that generates the downstream PTC shown. (B–D) Quantification of RNase protection analysis performed on total cellular RNA (10 µg) harvested from HeLa cells transiently transfected with the constructs shown. Probes 1 and 3 (A) were used to detect mature/IVS-A+ pre-mRNA and IVS-B+ pre-mRNA, respectively. Values were quantified from three or more independent experiments by the approach described in Figure 1. Error bars indicate standard error.

Figure 5.

Missense mutations in the VDJ exon trigger pre-mRNA upregulation. (A) Schematic diagram indicating the location of missense mutations (A^M1, A^M3 and A^M4) introduced at the same nucleotide positions as the nonsense mutations in A^N1, A^N3 and A^N4, respectively (Figure 4A). (B–D) Quantification of RNase protection analysis performed as described in Figure 4. (E) Splicing rate as measured by pre-mRNA-to-mRNA ratio. The ratios are calculated from the values in (B) and (D); the ratio for construct A is arbitrarily set to 1.

Figure 6.

Both frame-disrupting and frame-neutral insertions and deletions elicit pre-mRNA upregulation. (A) Schematic diagrams of construct A variants harboring either a 1-nt insertion at codon 114 (+1, construct A^I2+), a 1-nt deletion at codon 146 (−1, construct A^D1+), both the 1-nt insertion and deletions (+1/−1, construct A^I2D1) or a 3-nt insertion (+3, construct A^I3). The location of the downstream PTC generated by the +1 and −1 mutations are shown. (B) RNase protection analysis performed using probe 1 (A) and a Neo probe (Figure 1) on total cellular RNA (10 µg) harvested from HeLa cells transiently transfected with constructs shown. (C, D) Quantification of mature mRNA (C) and pre-mRNA (D) levels from cells analyzed as in (B). Values are the average of three experiments, determined as described in Figure 1. Error bars indicate standard error. (E) Splicing rate as measured by pre-mRNA-to-mRNA ratio, determined as in Figure 5E, using the values in (C) and (D).

Figure 7.

Evidence that the pre-mRNA upregulatory response is independent of protein synthesis and UPF1. (A) Schematic diagram denoting the locations of the nonsense mutations in constructs A^N1 and A^N2 (also in Figure 1). (B, C) Quantification of RNase protection analysis performed on total cellular RNA (10 µg) harvested from HeLa cells incubated for 6 h with cycloheximide (CHX). Prior to CHX treatment, the cells were transiently transfected with the constructs shown, as well as a β-globin expression vector as an internal control, and cultured for 2 days. Probe 1 (A) was used to detect both the pre-mRNA and mature mRNA. The values shown are the average of two independent experiments that were normalized with β-globin mRNA, the internal control. Error bars indicate standard error. (D–F) Quantification of RNase protection analysis performed on total cellular RNA (10 µg) harvested from HeLa cells transiently transfected with a UFP1 siRNA to deplete UPF1 levels or a Luciferase (Luc) siRNA as a negative control. Probes 1 and 3 (A) were used to detect mature/IVS-A+ pre-mRNA and IVS-B+ pre-mRNA, respectively. Values were quantified from three independent experiments by the approach described in Figure 1.

Figure 8.

Nonsense mutations in exons 5 and 6 trigger strong NMD but modest or no pre-mRNA upregulation. (A) Schematic diagram indicating the location of the nonsense and missense mutations introduced into construct A. Constructs A^N5/^M5, A^N6/^M6/^M6′, A^N7/^M7/^M7′, A^N8/^M8/^M8′ and A^D2+ have nonsense (N) or missense (M or M^′) mutations at codons 164, 191, 192, 195 and 190, respectively. M and M′ are distinct missense mutations. (B–E) Quantification of RNase protection analysis performed on total cellular RNA (10 µg) harvested from HeLa cells transiently transfected with the constructs shown. Probes 1, 3 and 4 (A) were used to detect mature/IVS-A+ pre-mRNA, IVS-B+ pre-mRNA and IVS-D+ pre-mRNA, respectively. Values were quantified from three or more independent experiments by the approach described in Figure 1. Error bars indicate standard error. (F) Splicing rate as measured by pre-mRNA-to-mRNA ratio, determined as in Figure 5E, using the values in (B–E).

Figure 9.

Location of all mutations introduced into the TCR/TPI chimeric gene. (A) Nonsense and frameshift mutations. Figures 1, 4, 6 and 8 indicate their codon location. Note that the schematic diagram is of the mature mRNA and thus introns are not shown. (B) Missense and frame-neutral mutations. Figures 5, 6 and 8 indicate their codon location.