Quantitative analysis of cryptic splicing associated with TDP-43 depletion

Abstract

BackgroundReliable exon recognition is key to the splicing of pre-mRNAs into mature mRNAs. TDP-43 is an RNA-binding protein whose nuclear loss and cytoplasmic aggregation are a hallmark pathology in amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). TDP-43 depletion causes the aberrant inclusion of cryptic exons into a range of transcripts, but their extent, relevance to disease pathogenesis and whether they are caused by other RNA-binding proteins implicated in ALS/FTD are unknown.MethodsWe developed an analysis pipeline to discover and quantify cryptic exon inclusion and applied it to publicly available human and murine RNA-sequencing data.ResultsWe detected widespread cryptic splicing in TDP-43 depletion datasets but almost none in another ALS/FTD-linked protein FUS. Sequence motif and iCLIP analysis of cryptic exons demonstrated that they are bound by TDP-43. Unlike the cryptic exons seen in hnRNP C depletion, those repressed by TDP-43 cannot be linked to transposable elements. Cryptic exons are poorly conserved and inclusion overwhelmingly leads to nonsense-mediated decay of the host transcript, with reduced transcript levels observed in differential expression analysis. RNA-protein interaction data on 73 different RNA-binding proteins showed that, in addition to TDP-43, 7 specifically bind TDP-43 linked cryptic exons. This suggests that TDP-43 competes with other splicing factors for binding to cryptic exons and can repress cryptic exon inclusion.ConclusionsOur quantitative analysis pipeline confirms the presence of cryptic exons during the depletion of TDP-43 but not FUS providing new insight into to RNA-processing dysfunction as a cause or consequence in ALS/FTD.Electronic supplementary materialThe online version of this article (doi:10.1186/s12920-017-0274-1) contains supplementary material, which is available to authorized users.

HnRNP L represses cryptic exons

“…4A). Likewise, the demonstration that TDP-43 and PTBP1/PTBP2 repress cryptic exons from upstream and downstream intronic positions is consistent with the known activities of these proteins (Ashiya and Grabowski 1997;Pérez et al 1997;Ayala et al 2005;Oberstrass et al 2005;Boutz et al 2007;D'Ambrogio et al 2009;Kuo et al 2009;Xue et al 2009;Chiang et al 2010;Polymenidou et al 2011;Tollervey et al 2011;Kafasla et al 2012;Keppetipola et al 2012;Licatalosi et al 2012;Lukavsky et al 2013;Zhang et al 2013b;Humphrey et al 2017). Therefore, our results suggest that cryptic pseudoexons are repressed by mechanisms similar to standard exons, and that a widespread activity of hnRNP L is repression of strong 5 ′ SSs, presumably through inhibiting replacement of U1 by U6.…”

Mice with endogenous TDP ‐43 mutations exhibit gain of splicing function and characteristics of amyotrophic lateral sclerosis

“…Intriguingly, SEs represent the opposite phenomenon to recently described cryptic exons (CEs), in which TDP‐43 LOF induces the inclusion of otherwise constitutively repressed exons (Ling et al , 2015; Humphrey et al , 2017). We defined cryptic exons (CEs) as exons that are significantly de‐repressed using the reverse criteria from the above skiptic exons: repressed in WT (PSI < 0.05) and present in mutants (∆PSI of > 0.05).…”

“…Remarkably, the skipping of constitutive exons induced by GOF is in contrast to the previously identified TDP‐43 LOF‐induced cryptic exons, which are normally repressed but in LOF are aberrantly included in mRNAs (Ling et al , 2015; Humphrey et al , 2017). …”

Regulating Divergent Transcriptomes through mRNA Splicing and Its Modulation Using Various Small Compounds

“…The functions of TDP-43 include repressing the splicing of nonconserved cryptic exons, maintaining intron integrity and preventing cell death [56,145]. RNA-seq analysis in human postmortem brain with TDP-43 mutations revealed cryptic exon expression [56].…”