Detection of brain- and spleen-specific β-adducin exons in rats and mice

<p><b>Copyright information:</b></p><p>Taken from "Brain-specific promoter and polyadenylation sites of the β-adducin pre-mRNA generate an unusually long 3′-UTR"</p><p>Nucleic Acids Research 2006;34(1):243-253.</p><p>Published online 9 Jan 2006</p><p>PMCID:PMC1326019.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> () Schematic representation of the riboprobes used in the RNase protection experiment shown in (B and C). The input probe and the size of the protected fragments for each probe and each tissue are indicated. ( and ) The spleen and brain probes shown in (A) were used in (B and C), respectively. Thirty micrograms of total spleen and brain RNA were annealed to the probe, digested with RNase, run in a polyacrylamide denaturing gel and autoradiographed. Lanes 1 and 7 correspond to the undigested probe (input probe), lanes 6 and 7 are radioactive molecular weight markers, lanes 2–3 and 9–10 spleen RNA, and lanes 4–5 and 11–12 brain RNAs. The arrows indicate the protected fragments. () Primer extension experiment with a primer annealing in the boundary between the constitutive 99 bp and the 217 bp exons (exons 2 and 3), that are present in both the brain and spleen forms of the β-adducin mRNA. The arrows indicate the primer extension products observed in brain and spleen RNAs. Lanes 15–16 and 17–18 correspond to spleen and brain RNAs, respectively. Lane 14 is a radioactive molecular weight marker, and lane 18 is a one-lane Sanger sequence using the same primer used in the experiment. () Northern blot experiment of cerebellum, brain and spleen mouse RNA (lanes 19–21, respectively) with a probe corresponding to the mouse brain-specific exon. The position of the 28S and 18S rRNA is indicated

Exon First Nucleotide Mutations in Splicing: Evaluation of <i>In Silico</i> Prediction Tools

<div><p>Mutations in the first nucleotide of exons (E⁺¹) mostly affect pre-mRNA splicing when found in AG-dependent 3′ splice sites, whereas AG-independent splice sites are more resistant. The AG-dependency, however, may be difficult to assess just from primary sequence data as it depends on the quality of the polypyrimidine tract. For this reason, <i>in silico</i> prediction tools are commonly used to score 3′ splice sites. In this study, we have assessed the ability of sequence features and <i>in silico</i> prediction tools to discriminate between the splicing-affecting and non-affecting E⁺¹ variants. For this purpose, we newly tested 16 substitutions <i>in vitro</i> and derived other variants from literature. Surprisingly, we found that in the presence of the substituting nucleotide, the quality of the polypyrimidine tract alone was not conclusive about its splicing fate. Rather, it was the identity of the substituting nucleotide that markedly influenced it. Among the computational tools tested, the best performance was achieved using the Maximum Entropy Model and Position-Specific Scoring Matrix. As a result of this study, we have now established preliminary discriminative cut-off values showing sensitivity up to 95% and specificity up to 90%. This is expected to improve our ability to detect splicing-affecting variants in a clinical genetic setting.</p></div

Analyzed sequences.

<p>The sequences are ordered according to the length of their longest PPS. Exons whose splicing was shown to depend on intact E⁺¹ position are underlined. The PPS are singly underlined and other polypyrimidine stretches are dashed underlined. Sites of mutations are showed in bold. Seq. = sequence. (A) Sequences of the test set. (B) Sequences of the borderline set.</p

Results of the splicing minigene analyses.

<p>RT-PCR analysis of the literature-derived E⁺¹ variations. The splicing affecting sequences are underlined. (A) The test set sequences. cDNA bands originating from <i>BTK</i> exon 10 mutated minigene are numbered as follows: 1) cryptic 3′ss utilization 31 nt upstream of the authentic splice site (the aberrant exon starts at c.840-31G), 2) normally spliced RNA. (B) The borderline set sequences.</p

Predicted value ranges in the test set of G⁺¹ mutations obtained from the instruments evaluating the overall strength of the 3′splice site.

<p>Computational predictions for experimentally confirmed test set of splicing-affecting and non-affecting G⁺¹ mutations were subjected to statistical analysis using the Mann-Whitney test (see Table S1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089570#pone.0089570.s006" target="_blank">File S1</a> for details). Significant differences (p<0.05) are marked in bold. Diff. = difference, M-W test = Mann-Whitney test, perc. = percentile.</p

Results of combined predictions in discrimination of G⁺¹-dependent 3′ss from G⁺¹-independent 3′ss.

a<p>Each combined prediction was considered positive if two or more of the three predicted values exceeded the herein proposed cut-off values of the individual tools. ^bThe sensitivity and specificity was calculated using Fu-mut set of G⁺¹ mutations, containing artificially mutated PPTs. ^cThe sensitivity and specificity calculated using combined sets: test set and borderline set of G⁺¹ mutations. Py25 = number of pyrimidines in the 25 nucleotides upstream from splice site; ME s.d. = difference between wild type and mutant sequence scores predicted by MaxEnt program; ME p.d. = difference between wild type and mutant sequence percentiles predicted by MaxEnt program; PSSM s.d.: accordingly.</p

Selected results of <i>in silico</i> predictions and other parameters of AG-dependent and AG-independent splice sites.

<p>The boxplots show the values, median and interquartile range of the values predicted for the test set sequences. The values of scores are shown in the original units of the tools, differences are counted as ratios of the absolute score (or percentile) difference to the wild type score (or percentile). Other values are simply numbers of nucleotides. Asterisks indicate statistically significant differences between the two sets of values (*at p<0.05; **at p<0.01; ***at p<0.001). Abbreviations: Ab = group of sequence variants that lead to aberrant splicing; diff. = difference; Non = group of sequence variants that do not disrupt the process of splicing; nt = nucleotide(s); perc. = percentile; Py in 25 nt = number of pyrimidines in 25 nucleotides upstream of acceptor splice site.</p

Analysis of the two E⁺¹ mutations of the <i>BTK</i> gene, O13 (c.1482G>T) and HK08 (c.1883G>A).

<p>(A) Schematic sequences of mutated acceptor splice sites. Introns are shown in lower-case, and exons are shown in capital letters. Mutated nucleotides are bold and underlined. The PPS are singly underlined and other polypyrimidine stretches are dashed underlined. (B) RT-PCR of minigenes transfected into HeLa cells. cDNA bands originating from O13 mutated minigene are numbered as follows: 1) cryptic 3′ss utilization 81 nt upstream of the authentic splice site (the aberrant exon starts at c.1350-81G), 2) normally spliced RNA, 3) skipping of mutated exon. (C) RT-PCR from RNA extracted from patients’ blood. P = patient’s sample, HC = healthy control sample. The O13 cDNA bands are numbered as in (B).</p

Comparison of value ranges describing particular intronic parameters in the test set of G⁺¹ mutations.

<p>The BS and PPT parameters were predicted using prediction programs provided by Kol et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089570#pone.0089570-Kol1" target="_blank">[10]</a> or Schwartz et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089570#pone.0089570-Schwartz1" target="_blank">[11]</a> using the Sroogle engine. Computational predictions and other sequence parameters for an experimentally confirmed test set of splicing-affecting and non-affecting G⁺¹ mutations were subjected to statistical analysis using the Mann-Whitney test (see Table S1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089570#pone.0089570.s006" target="_blank">File S1</a> for details). Significant differences (p<0.05) are marked in bold. dist. = distance, BS = branch point site, M-W test = Mann-Whitney test, perc. = percentile, PPS = the longest uninterrupted polypyrimidine stretch Py = number of pyrimidines (in 25 or 50 nt upstream from 3′ss).</p

Proposed cut-off values for the <i>in silico</i> tools that discriminate AG-dependent 3′ss from AG-independent 3′ss.

a<p>The cut-off values were proposed according to the predicted values obtained using the test set of naturally occurring G⁺¹ mutations (as used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089570#pone-0089570-t001" target="_blank">Tables 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089570#pone-0089570-t002" target="_blank">2</a>). ^bThe sensitivity and specificity calculated using Fu-mut set of G⁺¹ mutations, containing artificially mutated PPTs. ^cThe sensitivity and specificity calculated using the original test set of G⁺¹ mutations. The predicted scores and percentiles below the cut-off values and the differences between the predicted values for wild type and mutant sequences above the cut-off values are supposed to pertain to variants prone to affect splicing.</p