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Comparative Study
. 2006 Jan 15;15(2):329-36.
doi: 10.1093/hmg/ddi450. Epub 2005 Dec 15.

Identification of a splicing enhancer in MLH1 using COMPARE, a new assay for determination of relative RNA splicing efficiencies

Dong-Qing Xu  1 , William Mattox
Affiliations
Comparative Study

Identification of a splicing enhancer in MLH1 using COMPARE, a new assay for determination of relative RNA splicing efficiencies

Dong-Qing Xu et al. Hum Mol Genet. .

Abstract

Exonic splicing enhancers (ESEs) are sequences that facilitate recognition of splice sites and prevent exon-skipping. Because ESEs are often embedded within protein-coding sequences, alterations in them can also often be interpreted as nonsense, missense or silent mutations. To correctly interpret exonic mutations and their roles in diseases, it is important to develop strategies that identify ESE mutations. Potential ESEs can be found computationally in many exons but it has proven difficult to predict whether a given mutation will have effects on splicing based on sequence alone. Here, we describe a flexible in vitro method that can be used to functionally compare the effects of multiple sequence variants on ESE activity in a single in vitro splicing reaction. We have applied this method in parallel with conventional splicing assays to test for a splicing enhancer in exon 17 of the human MLH1 gene. Point mutations associated with hereditary non-polyposis colorectal cancer (HNPCC) have previously been found to correlate with exon-skipping in both lymphocytes and tumors from patients. We show that sequences from this exon can replace an ESE from the mouse IgM gene to support RNA splicing in HeLa nuclear extracts. ESE activity was reduced by HNPCC point mutations in codon 659, indicating that their primary effect is on splicing. Surprisingly, the strongest enhancer function mapped to a different region of the exon upstream of this codon. Together, our results indicate that HNPCC point mutations in codon 659 affect an auxillary element that augments the enhancer function to ensure exon inclusion.

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Figures

Figure 1
Figure 1. COMPARE strategy
A schematic diagram of the major steps in the COMPARE strategy is shown. Exon sequences to be tested (white box) are inserted into a plasmid containing sequences of an enhancer-dependent RNA splicing substrate (grey boxes). The original test sequence and variants thereof are transcribed with a bacteriophage RNA polymerase (SP6) in vitro to produce a pool of splicing substrates. Represented in the upper part of the diagram is the production of labeled RNA substrates with various Bst XI linker substitution mutations (black boxes). The entire pool of RNA is subjected to in vitro splicing and RT-PCR amplification with primers on either side of the intron. Products corresponding to unspliced and spliced RNA are isolated and separately labeled with 32P-dNTPs by linear amplification (“hot-stop labeling”) then digested with Bst X1 or another diagnostic restriction enzyme. Fragments of different lengths represent each sequence variant in the original RNA population. The relative representation of these variants in the spliced and unspliced RNA populations can be compared side-by-side after gel electrophoresis. Sequences disrupting ESEs will be underrepresented in the spliced product lane and overrepresented in the unspliced lane.
Figure 2
Figure 2. MLH1 exon 17 contains a splicing enhancer
In the top part of the figure a schematic diagram shows splicing substrates used to test whether MLH1 exon 17 contains a splicing enhancer. Splicing substrates from the mouse IgM gene with its native ESE (μM 1+2), a deletion of the ESE (μMΔ), and a substitution of 88 nt from the exon 17 (μM-MLH1) are represented. In vitro splicing assays carried out in HeLa nuclear extracts are shown below the diagram. In each set of reactions the positions of the larger unspliced and smaller spliced RNAs are indicated by arrows. Three lanes are shown for each substrate. From left to right these are untreated precursor RNA (P), a mock reaction with extract but no ATP (M) and a splicing reaction with ATP (S). Note that the μMΔ substrate which lacks an ESE is not detectably spliced but that μM-MLH1 is spliced at a level similar to μM 1+2.
Figure 3
Figure 3. Linker substitution scanning for the exon 17 splicing enhancer
A series of 23 nt Bst X1 linker substitutions were used to scan exon 17 for splicing enhancer sequences. (A) The position of each substitution is indicated by the dark line under the exon 17 sequences. Capital letters indicate the exon 17 sequences and lower case letters indicate the flanking intron sequences of MLH1. The Bst XI substitution sequences used is in all four substitutions are also shown below the exon. The C>T mutation indicated with the arrow is associated with HNPCC and occurs in codon 659. This mutation disrupts a Taq I site (grey underline) (B) COMPARE analysis of the Bst X1 substitutions and control RNA. Two parallel assays are shown in the first set mutant and control substrates were pooled before transcription (DNA mix) in the second equal levels of transcript were pooled (RNA mix). The reactions compared in each set are Bst X1 restriction digests on hot stop labeled DNA from the original pooled DNA or RNA (I), from unspliced product (U) or spliced product (S). The mean spliced/unspliced ratio (S/U) of signals is shown for each substitution and for the control with standard errors. Percent changes in relation to the C2 control are also shown.
Figure 4
Figure 4. Conventional in vitro splicing assays carried out on the same μM-MLH1 substitutions and control RNAs that were used for COMPARE analysis
Bands indicated are unspliced precursor (U), lariat intermediate (L), spliced product (S), excised intron (I), and 5’ exon intermediate (5’). For each substrate the three lanes correspond to unspliced precursor, and reactions without and with ATP as in Figure 2. In addition to substitutions S1-S4 reactions are shown for control μM-MLH1 substrates both without (C1) and with (C2) an inserted Bst X1 site at the 3’ MLH1/IgM border, the μMΔ. substrate (Δ), the “RNA mix” substrate pool (5R) and the “DNA mix” substrate pool (5D). The percent of spliced product generated is given below each RNA tested.
Figure 5
Figure 5. Deletion mapping of ESE sequences in exon 17
A series of deletions were generated from the μM-MLH1 plasmid and used to produce splicing substrates with exon sequences as shown in the top part of the figure. Solid lines indicate the sequences present. Dashed line indicates deleted sequences. Products from conventional in vitro splicing reactions are shown below the schematic. For each substrate two lanes are shown. In each pair the right lane is a reaction without ATP, the left lane is a reaction carried out with ATP. Percent of spliced product is indicated below the gel. On this gel lariat intermediates run just below unspliced precursor (U+L). The mobilities of spliced product (S), excised intron (I) and 5’ exon intermediate (5’) are also indicated.
Figure 6
Figure 6. Analysis of HNPCC associated exon 17 point mutations
Splicing assays testing the effects of HNPCC mutations on splicing enhancer activity are shown. (A) Conventional splicing assays were carried out on RNA substrates based on μM-MLH1 with various point mutations. The three lanes shown for each mutation are in the same order as in Figure 4. The mobility of unspliced and lariat intermediates are indicated (U/L) as well as spliced RNA (S). Excised intron and 5’ exon migrated faster and are not shown in the figure. The point mutations and the affected codon are indicated above each reaction set. Unaltered μM-MLH1 (wt) and μMΔ (Δ) splicing substrates are included as controls. (B) RNA substrates based on μM-MLH1 (wild type) and a C>T point mutation in codon 659 of this substrate (mutant) were analyzed by COMPARE. The position and sequence of the mutation in the exon is indicated in the schematic as are the locations of the amplification primers (short arrows). Hot stop products were generated as indicated by the arrows with asterisks. Expected Taq I cleavage sites for wild type and mutant products are indicated by dotted vertical lines. Duplicate COMPARE reactions with lanes for the original pool (I), unspliced (U) and spliced (S) products are shown after electrophoresis in parallel.
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