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. Author manuscript; available in PMC: 2019 Jul 14.
Published in final edited form as: Nat Neurosci. 2019 Jan 14;22(2):180–190. doi: 10.1038/s41593-018-0293-z

Figure 3. TDP-43 regulates stathmin-2 mRNA levels by repressing premature polyadenylation.

Figure 3.

(a) RNA-seq reads mapped to the genomic region of stathmin-2 reveal incorporation of a new exon, originated from intron 1, into the mature stathmin-2 mRNA. Red arrows indicate the intronic region of aberrant splicing (exon 2a) in SH-SY5Y cells upon TDP-43 depletion (upper panels) or expression of mutant TDP-43 (lower panels). Experiment was repeated independently three times (TDP-43 depletion) or twice (expression of mutant TDP-43) with similar results. (b) Representative RT-PCR (left, experiment was repeated 3 times independently with similar results) and qPCR (right, n=3 biologically independent experiments) analyses confirmed expression of the new spliced mRNA isoform containing exon 2a upon TDP-43 depletion (mean=8.7, P=0.00078) or mutation (mean=3.8, P=0.0001). The location of primers in exons 1 and 2a is shown. RT-PCR of the CENP-A transcript was used as loading control for RT-PCR, TFRC and GAPDH were used as qPCR normalizers (***p<0.001, two-tailed t-test, SEM). For uncropped gel images, see Supplementary Fig. 10. (c) Schematic representation of stathmin-2 pre-mRNA (upper panel) and alternative RNA isoforms (lower panels) in normal cells (i) or cells with TDP-43 deficiency (ii). Constitutive exons are represented by black boxes, exon 2a is in red and the thin red box represents the newly acquired 3’UTR. Light grey boxes represent 3’ or 5’ UTRs of normal stathmin-2 mRNA. (d) Sequence of exon 2a is shown in red, including the embedded in-frame UAG codon generating a premature stop codon. Highlighted are potential TDP-43 binding sites located 127 upstream of the alternative polyadenylation signal. (e) 3’ end sequencing by reverse transcription using oligo(dT)-VN primers confirmed exon 2a as the terminal exon of short stathmin-2 mRNA. (f) Genome browser track obtained from cross-linking and immuno-precipitation (iCLIP) for TDP-43 in human SH-SY5Y cells 9, revealed TDP-43 physical binding to exon 2a, located in intron 1 of stathmin-2 pre-mRNA.