Intron retention resulting from a silent mutation in the VWF gene that structurally influences the 5' splice site

Abstract

Disease-associated silent mutations are considered to affect the accurate pre-messenger RNA (mRNA) splicing either by influencing regulatory elements, leading to exon skipping, or by creating a new cryptic splice site. This study describes a new molecular pathological mechanism by which a silent mutation inhibits splicing and leads to intron retention. We identified a heterozygous silent mutation, c.7464C>T, in exon 44 of the von Willebrand factor (VWF) gene in a family with type 1 von Willebrand disease. In vivo and ex vivo transcript analysis revealed an aberrantly spliced transcript, with intron 44 retained in the mRNA, implying disruption of the first catalytic step of splicing at the 5' splice site (5'ss). The abnormal transcript with the retained intronic region coded a truncated protein that lacked the carboxy-terminal end of the VWF protein. Confocal immunofluorescence characterizations of blood outgrowth endothelial cells derived from the patient confirmed the presence of the truncated protein by demonstrating accumulation of VWF in the endoplasmic reticulum. In silico pre-mRNA secondary and tertiary structure analysis revealed that this substitution, despite its distal position from the 5'ss (85 bp downstream), induces cis alterations in pre-mRNA structure that result in the formation of a stable hairpin at the 5'ss. This hairpin sequesters the 5'ss residues involved in U1 small nuclear RNA interactions, thereby inhibiting excision of the pre-mRNA intronic region. This study is the first to show the allosteric-like/far-reaching effect of an exonic variation on pre-mRNA splicing that is mediated by structural changes in the pre-mRNA.

Figure 1

RT-PCR products on agarose gel. (A) Schematic scale of the coding region of VWF (exons 2-52) with the primer positions designed for amplification of the full-length VWF mRNA and corresponding amplicon segments. Agarose gel electrophoresis image shows the 14 overlapping RT-PCR products of VWF using total RNA from the IP’s blood as template, under thermocycling conditions with 2 minutes of extension time. The sequence chromatogram of segments 12 and 13 (both covering exon 44 carrying the silent mutation c.7464C>T) demonstrate single-peak manifestation of wt nucleotide C, c.7464C, indicating a fail in amplification of the mutant transcript. Lanes 13 and 14 were run in a separate gel but with similar running conditions. (B) RT-PCR products of segment 12 amplified with primers residing in exon 39 and exons 45/46 boundary, and with increased extension time (6 minutes) of thermocycling. RT-PCR products of RNA obtained from blood of the IP and her mother demonstrate a smaller product (920 bp) relevant to the normal transcript and an aberrant larger fragment (3130 bp) corresponding to the retained intron 44 in mRNA, whereas RT-PCRs using RNA from 5 healthy control subjects as template show only the smaller normal fragment (lanes 1-5). (C) RT-PCR amplification using allele-specific primers to confirm intron 44 retention. The primer combinations and expected amplicon sizes, if intron 44 is retained, are as follows: segment 1 (1437 bp), forward primer in exons 40/41 boundary and reverse primer targeted in intron 44, 861 nucleotides downstream of the exon 44 (lane 1); and segments 2, 3 and 4 (length 1873, 1683, and 896 bp, respectively), forward primers directed in intron 44 in 3 different positions (+787, +977, and +1764) and reverse primer in exons 48/49 boundary (lanes 2, 3, and 4). Sequence analysis of the cDNA segment 1 exhibited monoallelic presentation of the mutant variant T (c.7464T) in the sequence chromatogram, indicating that the aberrant transcript is derived solely from the mutant allele. Lane M represents the molecular weight marker (1-kb ladder).

Figure 2

Subcellular distribution and expression of VWF in the BOECs isolated from the IP and healthy donors. (A) Characteristics of the BOECs via staining of the cell-specific markers VWF (light green) and PECAM (red) with secondary antibodies conjugated with Alexa Fluor-488 and Alexa Fluor-594, respectively. However, only 64 of 100 inspected IP BOECs emitted light green fluorescent signals, representing production of VWF protein. In the VWF-expressing IP BOECs, VWF staining is mostly diffuse, accumulating around the nucleus of the cells, whereas in normal BOECs, VWF can be seen as distinct elongated structures, indicating storage in WPBs. The BOECs isolated from the IP’s mother illustrate a combination of VWF stored in WPBs and diffuse staining. The white box points out secreting VWF strings in normal BOECs that are not visible in the IP-derived BOECs. Bars represent 20 µm. (B) Diffuse staining observed in BOECs obtained from the IP and her mother (carrying the mutation c. 7464C>T) was colocalized with protein disulfide isomerase (PDI). Staining was performed using primary antibodies anti-VWF (left channel, light green) and anti-PDI (ER marker; middle, red) and secondary antibodies conjugated with Alexa Fluor-488 and Alexa Fluor-555, respectively. Colocalization of VWF and PDI staining is illustrated in the right channel (Merge). Bars represent 10 µm. (C) Bar graph of the mean of VWF:Ag levels in the medium and lysates of BOECs obtained from the IP, her mother (3 independent experiments, N = 3), and 3 healthy donors (each 3 independent experiments, N = 9). The mean VWF:Ag was determined after 10× concentration of the collected medium and cell lysates of confluent BOECs in 75 cm² flasks. Error bars indicate the standard deviation. (D) Western blot analysis of BOEC intracellular VWF after electrophoresis on 4% to 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Left lane shows both pre-pro-VWF and mature VWF in the lysate of the normal BOECs after 10× concentration. Middle and right lanes are representative of the IP’s BOEC lysates after 10× and 30× concentrations, respectively. WPBs, Weibel-Palade bodies.

Figure 3

Ab initio models of pre-mRNA and docking analysis. (A) Ab initio structures corresponding to wt (i) and mutated (ii) pre-mRNA sequences generated on iFold. The structure is depicted in stick format and shown in cyan. The mutated residue location is depicted in blue and marked with a lavender-shaded area. Hydrogen bonds are depicted as magenta dots throughout panels A-C. The backbone of the region corresponding to residues that bind to U1 snRNA is red, whereas the bases are yellow. This region is also marked by the lavender-shaded area. (B) Region corresponding to U1 snRNA binding for the wt (i) and mutated (ii) pre-mRNA models at a closer view. Color coding is as observed in panel A. The residues that bind to U1 snRNA are numbered in the mutated sequence structure model. In the wt sequence model, the unbound residues corresponding to U1 snRNA binding are marked with lavender-shaded regions. (C) Coarse-grained depiction of the dock of U1 snRNA over the wt (i) and mutated (ii) pre-mRNA sequence structure. Because the depiction is coarse-grained, the entire model is depicted only as a beaded trace. The trace is blue for the pre-mRNA sequence, with only the region corresponding to U1 snRNA binding shown in red. The different putative U1 snRNA docked structures are colored differently. Because only 1 putative dock was observed for the wt sequence, U1 snRNA is shown as light green in the dock between the U1 snRNA and wt pre-mRNA structures. The inset images provide a closer view of the U1 snRNA binding region on the pre-mRNA structures. The proximal regions in the U1 snRNA and wt pre-mRNA structures are marked by the lavender-shaded area.

Figure 4

Secondary structure prediction of pre-mRNA sequence. Secondary structure of the wt pre-mRNA sequence (A) and the mutated pre-mRNA sequence (B) predicted by mfold. Many of the 5′ donor splice site residues (5 residues: −1U, +1G, +2U, +3A, and +7C) that bind to U1 snRNA were observed to be free in the wt sequence but prebound or physically constrained in a double-stranded form in the mutated sequence. The regions corresponding to residues that bind to U1 snRNA are marked by the lavender-shaded area. The mutated residue locations are also marked by the magenta-shaded area and identified by a thick arrow. The inset images in each panel correspond to a close-up view of the region of pre-mRNA that binds to U1 snRNA in the 3-dimensional model of the wt and mutated structures generated from mfold secondary structure prediction on the RNAComposer Web server. The model structure is depicted in stick format (light green). The residues that bind to U1 snRNA are shown in blue. Hydrogen bonds are depicted as magenta dots.

Figure 5

Hypothetical U1 snRNA–pre-mRNA complex structure. The main image represents the secondary structure depiction of the hypothetical U1 snRNA–pre-mRNA complex derived from the RNAcofold server. The inset images show the secondary structure and the tertiary structure generated from the same secondary structure prediction in closer detail. In the secondary structure (lower inset), the residues of U1 snRNA are shown in dark green, whereas the residues of the pre-mRNA participating in the interaction with U1 snRNA are red. The canonical base pairing is shown by lines, whereas noncanonical ones are represented by a blue dot. The tertiary structure (upper inset) is depicted in stick format. U1 snRNA is shown in light green, whereas the pre-mRNA interacting residues are red. The rest of the pre-mRNA is gray.