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. 2024 Feb:100:104953.
doi: 10.1016/j.ebiom.2023.104953. Epub 2024 Jan 5.

Intron retention as a productive mechanism in human MAPT: RNA species generated by retention of intron 3

Daniel Ruiz-Gabarre  1 , Laura Vallés-Saiz  2 , Almudena Carnero-Espejo  3 , Isidro Ferrer  4 , Félix Hernández  2 , Ramon Garcia-Escudero  5 , Jesús Ávila  6 , Vega García-Escudero  7
Affiliations

Intron retention as a productive mechanism in human MAPT: RNA species generated by retention of intron 3

Daniel Ruiz-Gabarre et al. EBioMedicine. 2024 Feb.

Abstract

Background: Tau is a microtubule-binding protein encoded by the MAPT gene. Tau is essential for several physiological functions and associated with pathological processes, including Alzheimer's disease (AD). Six tau isoforms are typically described in the central nervous system, but current research paints a more diverse landscape and a more nuanced balance between isoforms. Recent work has described tau isoforms generated by intron 11 and intron 12 retention. This work adds to that evidence, proving the existence of MAPT transcripts retaining intron 3. Our aim is to demonstrate the existence of mature MAPT RNA species that retain intron 3 in human brain samples and to study its correlation with Alzheimer's disease across different regions.

Methods: Initial evidence of intron-3-retaining MAPT species come from in silico analysis of RNA-seq databases. We further demonstrate the existence of these mature RNA species in a human neuroepithelioma cell line and human brain samples by quantitative PCR. We also use digital droplet PCR to demonstrate the existence of RNA species that retain either intron 3, intron 12 or both introns.

Findings: Intron-3-retaining species are even more prominently present that intron-12-retaining ones. We show the presence of MAPT transcripts that retain both introns 3 and 12. These intron-retaining species are diminished in brain samples of patients with Alzheimer's disease with respect to individuals without dementia. Conversely, relative abundance of intron-3- or intron-12-retaining MAPT species with respect to double-retaining species as well as their percentage of expression with respect to total MAPT are increased in patients with Alzheimer's disease, especially in hippocampal samples. Among these TIR-MAPT species, TIR3+12 double truncation allows better classification potential of Alzheimer's disease samples. Moreover, we find a significant increase in intron-3- or intron-12-retaining species and its relative abundance with respect to double-retaining MAPT species in cerebellum in contrast to frontal lateral cortex and hippocampus in individuals with no signs of dementia.

Interpretation: Intron retention constitutes a potential mechanism to generate Tau isoforms whose mature RNA expression levels correlate with Alzheimer's pathology showing its potential as a biomarker associated to the disease.

Funding: This research was funded by the Spanish Ministry of Science, Innovation and Universities: PGC2018-096177-B-I00 (J.A.); Spanish Ministry of Science and Innovation (MCIN): PID2020-113204GB-I00 (F.H.) and PID2021-123859OB-100 from MCIN/AEI/10.13039/501100011033/FEDER, UE (J.A.). It was also supported by CSIC through an intramural grant (201920E104) (J.A.) and the Centre for Networked Biomedical Research on Neurodegenerative Diseases (J.A.). The Centro de Biología Molecular Severo Ochoa (CBMSO) is a Severo Ochoa Center of Excellence (MICIN, award CEX2021-001154-S).

Keywords: Alternative splicing; Alzheimer's disease; Intron retention; Tau; Tau isoforms; Tauopathies.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
RNA-seq study of Intron-3-retaining MAPT in human brain donors without dementia. (a) RNA-seq data from GTEx was analysed. A total of 363 samples of frontal cortex, dorsolateral prefrontal cortex, and hippocampus from 180 human brain donors without dementia were used. mRNA level values [log2 (TPM)] of the canonical MAPT gene (total-MAPT), MAPT gene with intron 12 retention (All TIR12-MAPT), and additional specific MAPT-derived transcripts, including intron retention for introns 12 or 3 are shown. Red lines represent mean mRNA levels and 95% confidence intervals (CI); and black dots mark single values. Black dots accumulating near log2 (tpm) = −10 as a black line represent the number of samples that do not express those species. All TIR12-MAPT are all transcripts that retain intron 12. TIR12-MAPT2-3-10- are RNA species including intron 12 but excluding exons 2, 3 and 10, as reported in. TIR12-MAPT2+3+4A+6+10+ includes exons 2, 3, 4a, 6 and 10 in addition to the constitutive ones and intron 12 retention, while TIR12-MAPT2+3-10+ includes exons 2 and 10 but excludes exon 3 while retaining intron 12. TIR3-MAPT refers to RNA species that retain only intron 3, while TIR3+12-MAPT represents all species that retain both intron 3 and intron 12. Kruskal–Wallis test with Dunn's correction for multiple comparison analysis was used to compare the differences between groups and P-values are represented. (b) Percentage of samples expressing each of the transcripts detected in (a). (c) DNA nucleotide sequence of intron-3-retaining MAPT species, excluding exons 2, 3 and 10, with (TIR3+12-MAPT) and without (TIR3-MAPT) simultaneous retention of intron 12. The sequence highlighted in violet represents the part of intron 3 that is retained, while the one highlighted in orange represents the part of intron 12 that is retained in TIR3+12-MAPT. The green-coloured sequence is the one that can be found in canonical MAPT transcripts not retaining intron 12. (d) Amino acid sequence of the hypothetical tau isoform resulting from intron 3 or both introns 3 and 12 retention with only three microtubule-binding repetitions (3 R), with respect to its full-length counterpart (Tau 3R0N or Tau 352). Again, green-coloured sequences represent canonical tau, while violet and orange represent the translation of the retained portion of introns 3 and 12, respectively. (e) Schematic representation of the protein isoforms that would arise from intron-retaining transcripts. The violet dashed area represents the portion of the protein arising from the translation of the portion of intron 3 that is retained, while the dashed orange one represents the equivalent portion from intron 12. All the protein representations include a label for the fragment of protein that would be generated by the translation of exon 1 (E1), exon 4 (E4), exon 13 (E13), and the part of introns 3 and 12 that are retained (I3 and I12) to highlight the differences between isoforms. NW-Tau: N-terminal W-Tau. DW-Tau: Double W-Tau.
Fig. 2
Fig. 2
Intron-3-retaining MAPT RNA species are confirmed to exist and are more prominent in the human brain than in cultured cells. (a) Schematic representation of the MAPT gene sequence from exon 3 (E3) to exon 7 (E7) and the hybridisation sites for oligonucleotides detecting intron-3-retaining MAPT species: TIRI3-Fw, TIRI3-Rv1 and TIRI3-Rv2. Dashed arrows represent intron-spanning sequences on oligonucleotides. (b) qPCR results showing detection of TIR3-MAPT cDNA in SK-N-MC human neuroepithelioma cells (n = 6) with respect to frontal cortex samples from control, participants without dementia (n = 4), using the reverse oligonucleotide TIRI3-Rv1. (c) Confirmation of silencing efficiency for shRNA against MAPT in comparison to scramble shRNA (n = 3), proving MAPT can be effectively silenced. (d) Confirmation of TIR3-MAPT silencing using the same shRNA against MAPT (n = 3), confirming TIR3-MAPT's identity as MAPT species. These results are shown using both TIRI3-Rv1 and TIRI3-Rv2. Unpaired t-tests were performed to compare among groups using Welch's correction for unequal variances or the Mann–Whitney test when the data distribution was not Gaussian and equal variances. Scatter plot graphs show error-bars representing 95% confidence intervals (CI) and the corresponding P-values when P < 0.05.
Fig. 3
Fig. 3
Intron-3-retaining RNA species in healthy controls and patients with AD. (a) Comparison of intron-3-retaining cDNA levels between control participants without dementia, and patients with AD in hippocampus, frontal lateral cortex, and cerebellum. Oligonucleotide TIRI3-Rv1 was used. (b) Analogous analysis to that carried out on (a) for total MAPT, comparing control and AD subjects across three different regions. HPC: hippocampus (n = 10 control subjects and 23 patients with Alzheimer's disease). FLC: frontal lateral cortex (n = 10 control subjects and 26 patients with Alzheimer's disease). CBL: cerebellum (n = 5 samples used as control and 5 patients with advanced Alzheimer's disease). Two-way ANOVA test was done to rule out interaction between variables and one-way ANOVA with Tukey's correction for multiple comparison was performed to examine the differences between the means of three brain regions when there was a Gaussian distribution of the data or Kruskal-Wally's test with Dunn's correction for multiple comparison when the distribution of data was non-Gaussian. Unpaired t-test was performed to compare between control and AD using the Mann–Whitney test when the data distribution was not Gaussian. Scatter plot graphs show error bars representing 95% confidence intervals (CI) and the corresponding P-values between control and AD and between different brain areas when P < 0.05.
Fig. 4
Fig. 4
Changes in different intron-retaining MAPT RNA species between individuals without dementia and patients with AD. (a-c) Relative RNA copy number of different intron-retaining MAPT species, TIR3-MAPT (a), TIR12-MAPT (b), and TIR3+12-MAPT (c), measured by ddPCR normalised with β-tubulin (TubB). (d-h) Absolute RNA levels measured in copies per nanogram of RNA of TIR3-MAPT (d), TIR12-MAPT (e), TIR3+12-MAPT (f), total-MAPT (g), and β-tubulin (h). (i-j) Relative RNA copy number ratios of different intron-retaining MAPT species, TIR3-MAPT/TIR12-MAPT (i), TIR3-MAPT/TIR3+12-MAPT (j), and TIR12-MAPT/TIR3+12-MAPT (k), measured by ddPCR. (l-n) Percentage of different intron-retaining MAPT species, TIR3-MAPT (l), TIR12-MAPT (m) and TIR3+12-MAPT (n), with respect to total-MAPT RNA copy number. Graphs show results of samples from frontal lateral cortex in orange (FLC: n = 10 control subjects and 26 patients with Alzheimer's disease, AD) and hippocampus in green (HPC: n = 10 control subjects and 23 patients with Alzheimer's disease, AD). Two-way ANOVA test was done to rule out interaction between variables and t-test analysis was performed to examine the differences between the means of two groups using Welch's correction when unequal variances or Mann–Whitney test when data distribution was non-Gaussian, and equal variance or Kolmogorov–Smirnov test when unequal variances. Scatter plot graphs show error-bars representing 95% confidence intervals (CI) and the corresponding P-values when P < 0.05.
Fig. 5
Fig. 5
Diagnostic relevance of TIR-MAPT species for Alzheimer's disease. ROC curves of the classification potential of patients with AD and individuals without dementia using ddPCR values (copies/ng) of each MAPT isoform, total-MAPT and β-tubulin measured in brain samples of frontal lateral cortex (FLC) in orange (a-e) and hippocampus (HPC) in green (f-j). Graphs also show values of area under the ROC curve (AUC), 95% confidence intervals (CI), and P-values of each ROC curve using Wilson/Brown method.
Fig. 6
Fig. 6
Relative abundance of TIR3-MAPT species in different brain regions of asymptomatic patients. (a-c) Relative RNA copy number of different intron-retaining MAPT species, TIR3-MAPT (a), TIR12-MAPT (b), and TIR3+12-MAPT (c), measured by ddPCR normalized with respect to β-tubulin. (d-h) Absolute RNA levels measured in copies per nanogram of RNA of TIR3-MAPT (d), TIR12-MAPT €, TIR3+12-MAPT (f), total-MAPT (g), and β-tubulin (h). (i-j) Relative RNA copy number ratios of different intron-retaining MAPT species, TIR3-MAPT/TIR12-MAPT (i), TIR3-MAPT/TIR3+12-MAPT (j), and TIR12-MAPT/TIR3+12-MAPT (k), measured by ddPCR. (l-n) Percentage of different intron-retaining MAPT species, TIR3-MAPT (l), TIR12-MAPT (m), and TIR3+12-MAPT (n), with respect to total MAPT RNA copy number. Graphs show results of samples from frontal lateral cortex in orange (FLC: n = 12), hippocampus in green (HPC: n = 12) and cerebellum in pink (CBL: n = 5) of patients with no clinical signs of dementia, including controls and Braak stages I and II. One-way ANOVA test with Tukey's correction for multiple comparisons was performed to examine the differences between the means of the groups. When non-Gaussian distribution of data, Kruskal–Wallis test for multiple comparison with Dunn's correction was done. When unequal variances, Brown–Forsythe and Welch ANOVA test was performed. Scatter plot graphs show error-bars representing 95% confidence intervals (CI) and the corresponding P-values when P< 0.05.
Fig. 7
Fig. 7
Schematic representation of the W-Tau family. (a) Representation of the protein sequence of isoforms retaining introns 3, 12 or both, in comparison with full-length tau. Tau 352 represents full-length tau, excluding exons 2, 3, and 10 (3R0N). 3 R (and 0 N) variants are represented for the members of the W-Tau family, but we cannot rule out that 4 R equivalents would exist, potentially. Dashed orange fragments represent the region of intron 12 translated upon intron 12's retention, whose amino acid sequence is detailed above the drawing of CW-Tau. Dashed violet fragments represent the region that would be translated if RNA species retaining intron 3 synthesise proteins, with its 33-residue specific sequence detailed below NW-Tau's illustration. (b) Representation of different exons 1, 2, or 3-exon 4 junctions depending on the splicing variants or intron 3-exon 4 junction and their translations into protein. None of the combinations alters the reading frame of exon 4.
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