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Quantitative analysis of cryptic splicing associated with TDP-43 depletion
© The Author(s). 2017
Received: 18 November 2016
Accepted: 17 May 2017
Published: 26 May 2017
Reliable 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.
We developed an analysis pipeline to discover and quantify cryptic exon inclusion and applied it to publicly available human and murine RNA-sequencing data.
We 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.
Our 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.
KeywordsRNA-seq Cryptic exons Splicing TDP-43
Splicing depends on the reliable recognition and subsequent removal of non-coding intronic sequence by the multi-protein spliceosome complex. Boundaries that demarcate exons from introns are first recognised by the binding of the U1 small nuclear RNA (snRNA) to the 5′ splice site at the beginning of the intron and the U2 snRNA binding to the polypyrimidine tract and 3′ splice site at the end of the intron. The two snRNAs then interact with each other and the intronic sequence is removed via a series of transesterification reactions . During splicing, regulatory sequences in the nascent RNAs recruit RNA-binding proteins (RBPs) to shape mRNAs, selecting or omitting particular exons by acting to enhance or repress splicing.
Due to the long length and reduced evolutionary conservation of intronic sequences, pairs of 3′ and 5′ splice sites can emerge randomly to create potentially new exons. These cryptic exons (also known as pseudoexons) arise due to mutations that create new splice sites or remove the existing binding sites for splicing repressors. These type of mutations have also been implicated in a number of genetic diseases [2–5]. Inclusion of a cryptic exon, untested by evolution, can destabilise the transcript or radically alter the eventual protein structure. The former can occur by the nonsense-mediated decay (NMD) pathway, which occurs when a transcript has a premature termination codon introduced either directly by the cryptic exon or by a subsequent shift of reading frame [6, 7].
Cryptic exons can also emerge from transposable elements. One such example are Alu elements, the predominant transposable element in primates which are often found within introns in the antisense direction . The consensus Alu sequence consists of two arms joined by an adenine-rich linker ending with a poly-adenine tail. When transcribed in the antisense direction these uridine-rich sequences can act as cryptic polypyrimidine tracts and only a few mutations are required to convert them into viable exons in a process termed exonisation . De novo mutations that lead to Alu exonisation have been found in a range of diseases [3, 4, 10, 11] suggesting a need for regulation of potentially damaging Alu exons. Alu exonisation is repressed by the RNA binding protein hnRNP C, which competes with the spliceosome component protein U2AF65, the partner of the U2 snRNA, for binding cryptic 3′ splice sites . Due to the potentially negative effects of incorporation of new exons, aberrant recognition of cryptic exons needs to be repressed.
TDP-43 is an RNA-binding protein encoded by the TARDBP gene. Loss of TDP-43 from the nucleus accompanied by TDP-43 positive inclusions in the cytoplasm of cortical and spinal cord neurons is the hallmark pathology of amyotrophic lateral sclerosis (ALS) as well as the majority of cases of frontotemporal dementia (FTD) . In addition, missense mutations in TARDBP can cause familial ALS . These findings point to a central role of TDP-43 in the aetiology of ALS and FTD.
Rare ALS-causing mutations have been found in another RNA-binding protein, FUS  further raising the possibility that the impairment of RNA processing is a central cause of ALS. TDP-43 was first shown to repress the inclusion of exon 9 in the CFTR gene by binding to long UG-rich sequences  and subsequently shown to act as both a splicing enhancer and repressor [17–19]. TDP-43 and FUS have both been shown to bind a set of overlapping RNA targets . Transcriptome-wide studies of the effects of TDP-43 depletion, overexpression or mutation have demonstrated widespread changes in gene expression and splicing [21–24]. Of particular interest are long intron containing genes, which are dramatically downregulated during both TDP-43 and FUS depletion [20, 23].
Recently, Ling and colleagues observed the inclusion of cryptic exons when TDP-43 was depleted in HeLa or mouse embryonic stem cells . These cryptic exons were shown to originate from poorly evolutionarily conserved sequence and shared no positions between the two species. These findings raise the possibility that impaired exon recognition contributes to TDP-43’s role in ALS aetiology. We therefore aimed to replicate and expand the findings of Ling and colleagues. Firstly, we undertook a quantitative genome-wide analysis of cryptic exon patterns, defining objective criteria that take advantage of biological replicates when available. Secondly, we applied this computational strategy to seven datasets (four human and three murine models) to systematically quantify cryptic RNA alterations associated with depletion of TDP-43. In addition we also investigated FUS in order to determine whether modulation of cryptic splicing was a common feature of RBPs implicated in ALS. We also analysed hnRNP C, as it had been previously shown to repress cryptic exons. Lastly, we used independent protein-RNA interaction datasets, conservation data, repeat element annotation and splice site scoring to investigate the potential mechanisms linking TDP-43 depletion with the cryptic exon phenomenon.
List of accessions. For the ENCODE RNA-seq libraries, the control and target depletion samples are listed under separate accessions
Mouse adult brain
K562 total RNA
K562 total RNA
Mouse adult brain
Mouse embryonic brain 1
Mouse embryonic brain 2
Human neural stem cells
Human SH-SY5Y 1
Human SH-SY5Y 2
Processed eCLIP data (previously described by ) was downloaded from the ENCODE project. The narrowPeaks bed format was used with the first nucleotide of the cluster defined as the peak. Peak coordinates from iCLIP and eCLIP were converted to the hg38 and mm10 builds using the LiftOver tool from UCSC.
Cryptic splicing definition
Splicing aware alignment software such as STAR cut short reads that originate from a spliced transcript and align the pieces separately, marking the distance between them as a splice junction. Splice junctions can be used to reaffirm known splicing patterns or infer novel splicing. We define cryptic splicing as the emergence or relative increase in splice junctions that splice from known splice sites to unannotated positions within introns. This increase correlates with the depletion of a particular RNA binding protein. Different repositories have different levels of proof for annotating exons but we define an annotated exon as one listed in the Ensembl list of transcripts (release 82).
Cryptic splicing discovery with the CryptEx pipeline
Due to the diversity in the quality of published RNA-seq data, the cryptic splicing discovery pipeline was designed to be used on any RNA-seq library, whether single or paired end, stranded or unstranded, total RNA or polyA-selected RNA. However, when high quality RNA-seq data is available (paired end reads >100bp sequenced with >30M reads per sample), we instead suggest using one of the several recently released tools that can identify and classify unannotated splice junctions such as JunctionSeq , Leafcutter , MAJIQ  or SGSeq .
The inherent flexibility of our pipeline results in a large number of false positive hits which have to be aggressively filtered downstream. The initial cryptic exon discovery pipeline was written in Bash, using SAMTools (version 1.2)  and BEDTools (version 2.25.0) . The statistical testing for differential cryptic exon usage was carried out using the well-known DEXSeq framework . All code for downstream processing and filtering of cryptic hits was written in the R language (version 3.1.1) using the Biostrings, data.table, DEXSeq, dplyr, GenomicRanges, ggplot2, gridExtra, optparse, plyr, stringr, and tidyr packages. All the code for reproducing this paper is available in a GitHub repository .
In order to discover all possible splice junctions that travel into the intron we first extracted all spliced reads from each aligned bam file using SAMTools, filtering out any secondary alignments. To extract only the spliced reads that overlap an annotated exon we then performed an intersection in BEDTools with a flattened list of exons, created using the dexseq prepare annotation.py Python script included with the DEXSeq package . An inverse intersection was then performed with the same exon list to retain only the spliced reads which do not bridge two annotated exons. The intronic mapping sections of each read were split off from the rest and retained. In each dataset all intronic mapping spliced reads from each sample were grouped together irrespective of condition. Split reads that were within 500bp of each other were merged into larger intervals, hereby referred to as tags. This ideally captures both the upstream and downstream splice junction to a central cryptic cassette exon. To keep only the tags that are splicing within the gene body another intersection was performed with a list of introns. This was generated from the same flattened exon file by an R script written by Devon Ryan . The tags were then incorporated into the flattened list of exons. The reads that overlap annotated exons and tags were counted using HTSeq  on the default settings, ignoring PCR duplicate reads. The read counts were used to calculate differential usage of each exon with DEXSeq.
All the cryptic tags with an adjusted P-value (false discovery rate) < 5% and a | log2(fold change)| > 0.6 were extracted from the DEXSeq results table (see Additional file 1: Figure S1). The splice junctions from the alignment of each sample were used to work out the coordinates of the canonical junction that spans the intron within which the cryptic tag is or isn’t spliced in control samples. Using splice junctions from the depletion condition samples, the upstream and downstream junctions that connect the adjacent annotated exons to the cryptic tag were re-discovered and quantified. Any cryptic example that did not have at least one upstream or downstream junction per sample or had fewer than ten canonical splice junctions was removed. These junctions were used to calculate per-condition mean Percent Spliced In (PSI) values which are a ratio of cryptic splicing over the sum of cryptic and canonical splicing . As a number of cryptic splicing events are present at a low level in control samples, ∆PSI values were created for both upstream and downstream splicing for each tag. This is the difference in PSI between the depletion samples and the control samples. Any cryptic tag that had either an upstream or downstream ∆PSI < 5% was removed.
The coordinates of each cryptic tag were flanked by 100 base pairs on either side to capture binding around the putative splice sites. In order to compare the overlap between cryptic exons and RNA-protein binding peaks, two sets of null exons were created for comparison, which maintain the same length as their corresponding cryptic exon but sample either the intronic sequence outside of the flanked exon or that of the adjacent introns within the same gene if available. Overlaps between exons and iCLIP and eCLIP peaks were calculated using BedTools.
Motif enrichment analysis
FASTA sequence was generated for the cryptic exons flanked by 100 nucleotides either side and submitted to the MEME web tool  under the default settings. The analysis was repeated using the HOMER algorithm  on RNA mode. Motifs were created using WebLogo . Frequencies of the 16 possible dinucleotides were compared between flanked cryptic exon sequences with adjacent intron sequences from the same gene.
Transposable element enrichment
Lists of transposable elements in human and mouse (hg38 and mm10 respectively) were previously generated by the RepeatMasker tool  and were downloaded from UCSC. Overlap between different transposable elements and the cryptic exons was calculated in each orientation using BedTools.
PhyloP compares the sequence alignments of multiple species to produce per base conservation scores . Average conservation score per cryptic tag was calculated using bigWigSummary (UCSC) for both human and mouse data. The lists of splice junctions created by STAR when aligning each sample were used to identify the coordinates of the exons adjacent to the cryptic exon. The randomly sampled intronic sequence from the cryptic-containing intron was used as a negative control.
For each sample the number of reads overlapping each gene were counted using HTSeq using the same Ensembl gene models as before. Differentially expressed genes were identified using DESeq2  at a false discovery rate of 10%.
Protein prediction analysis
Any cryptic exon which did not fall within the coding sequence of a transcript was omitted. Splice junctions generated by STAR were used to determine the upstream and downstream exons adjacent to each cryptic exon. Only cryptic exons that had both upstream and downstream splice junctions (cassette exons) were kept. The upstream and downstream exons were matched to their corresponding annotated exon in the Ensembl transcript file for each species to assign the correct reading frame for translation. Nucleotide sequences for transcripts either including or excluding the central cryptic exon were created and translated in silico using the R Biostrings package. Premature termination codons (PTCs) were identified from the translated sequence and defined as escaping nonsense mediated decay (NMD) if they occurred within 50 nucleotides of the downstream exon-exon junction. Frameshifts were identified by comparing the sequence of the downstream exon with and without the central cryptic exon. A null distribution of PTC-containing or frame shifted transcripts was created by generating central exons from random nucleotide sequence, keeping the length distribution the same. For each species this was repeated 100 times.
Splice junction scoring
The strength of 5′ and 3′ splice sites was calculated for the human cryptic exons using maxEnt . Higher scores indicate the increased log odds of a given splice site being a true splice site. The 5′ splice site is defined as the last 3 nucleotides of the upstream exon flanked by 6 intronic nucleotides, of which the first two are invariably GU. The 3′ splice site is defined as the last 20 intronic nucleotides of which the final two are invariably AG, flanked by the first 3 nucleotides of the downstream exon. The splice sites of annotated exons were used as a positive control. Randomly generated sequence with invariant AG or GT was used as a negative control. Paired t-tests were carried out to test the direction of change between the cryptic and annotated splice sites for each class of cryptic exon.
Depletion of TDP-43 but not FUS results in cryptic exons
All RNA-sequencing data used in this study
Ling, 2015 
Ling, 2015 
Polymenidou, 2011 
Chiang, 2010 
Lagier-Tourenne, 2012 
Zarnack, 2013 
Figure 1b lists the counts of both pre-classification cryptic regions (“unfiltered output”) and the post-classification cryptic exons. Comparing the two human ENCODE K562 cell line TDP-43 depletion datasets (3-4), the poly-A selected mRNA-Seq dataset yielded far more splicing events than the total RNA dataset, presumably due to polyA selection leading to a higher coverage of mature spliced mRNA species. In total 95 human cryptic exons were discovered and classified, with the majority only detected in the mRNA-seq dataset. 11 cryptic splicing events were shared between datasets 3 and 4 (Fig. 1c). Of the 26 human cryptic exons reported by Ling, 12 were seen in at least one of the two datasets 3 and 4.
Both mouse datasets differ in both cell type (adult striatum in dataset 5 vs embryonic stem (ES) cell in dataset 6) and read depth (35-60M in dataset 5 vs 2-10M in dataset 6). 52 cryptic exons were identified in total, with 46 detected in the adult striatum and 15 in ES cells, with 6 exons observed in both. Of the 46 cryptic splicing events identified in murine samples by Ling et al, 13 were detected in at least one of datasets 5 and 6. Side by side visual inspection (Additional file 2: Figure S2 and Additional file 3: Figure S3) suggests that differences in library preparation and read depth are behind the low concordance rates in both human and mouse, as cryptic exons detected in the higher depth dataset (K562 mRNA and mouse adult brain) can be observed by eye in the lower depth dataset (K562 total RNA and mouse ES cell). These exons currently fail to be detected by the CryptEx algorithm.
No cryptic splicing events were shared between human and mouse as previously reported . Note that to report overlap with Ling and colleagues (datasets 1 and 2), the raw data was unsuitable for our cryptic exon discovery pipeline due to a lack of biological replicate samples. Instead the sequence data was aligned and the splice junctions generated by the aligner were used to classify previously reported cryptic exons.
In contrast, while a large number of novel splicing events were observed in the FUS depletion datasets, our algorithm only classified 3 in mouse and 1 in human as cryptic exons. FUS depletion was not observed to produce any cassette-like cryptic exons in either species. Additional file 1: Figure S1 visualises the full unclassified output of the pipeline and illustrates the diversity of splicing alterations that occur upon RBP depletion. Additional file 2: Figure S2 and Additional file 3: Figure S3 comprise of screenshots of every reported cryptic exon from the IGV browser in mouse and human respectively. Additional file 4: Tables S1 and S2 (ST1/ST2) list the coordinates of each cryptic exon along with the results of each experiment.
To verify that the cryptic exons were not included under normal conditions, we looked for evidence of the 52 total mouse cryptic exons in a set of 9 mouse tissues previously published in . For the 95 total human cryptic exons we looked for evidence of inclusion in 49 normal human tissues collected by the GTEx project . For each cryptic exon we looked for junctions that splice to and from the cryptic exon and divided by the counts of a junction that spans the length of it, to produce a ratio of cryptic exon inclusion. Only 4 mouse and 3 human cryptic exons showed any evidence of being included at all in normal tissues, albeit at low levels and with very high variability (Additional file 5: Tables S5 and S6). This suggests that the cryptic exons we discovered are generally not included in normal tissues and that they can only be seen during the depletion of TDP-43.
Cryptic exons are enriched in TDP-43 binding motifs and iCLIP peaks
For the remainder of our study, cryptic exons were grouped into unions of all cassette-like exons and extension events discovered in human and mouse, totalling 95 human and 52 murine cryptic exons. We then explored whether TDP-43 binding could explain the observed splicing changes in RNA-Seq data, as observed by Ling and colleagues. We took two complementary and genome-wide approaches: (i) searching for enriched motifs in the RNA sequence including and surrounding the cryptic exons and (ii) correlating the positions of cryptic exons with TDP-43 protein-RNA interaction data.
Individual nucleotide resolution UV crosslinking followed by immunoprecipitation (iCLIP) allows for precise nucleotide-resolution observation of which RNA species interact with a particular RNA-binding protein [12, 54]. We downloaded all publicly available TDP-43 iCLIP data from the iCount repository (http://icount.biolab.si/). We then intersected each set of iCLIP peaks with our cryptic exons, surrounded by 100 base pairs of flanking sequence either side. The proportion of overlapping cryptic exons was compared to the proportion of overlap between iCLIP peaks and two classes of null exon; the first created from the surrounding intronic sequence outside of each flanked cryptic and the second from an adjacent intron (Fig. 2c). If TDP-43 binding was uniform throughout an intron or gene then we would expect to see similar proportions of overlap in each. However, both species show an enrichment in TDP-43 binding peaks specific to the cryptic exons in every iCLIP dataset used, with as much as 25% of human cryptic exons and 50% of mouse cryptic exons overlapping at least one iCLIP peak each (Fig. 2d; both species P < 10−16; proportion test).
Cryptic exons are marginally enriched in transposable elements
Motivated by previous findings of cryptic exons associated with hnRNP C depletion and caused by exonisation of antisense Alu elements [12, 55], we investigated whether TDP-43 induced cryptic exons preferentially overlap specific families of transposable elements and/or class of repetitive sequences. Transposable/repeat element annotations were obtained using the RepeatMasker software and these features were split by family and orientation. Although Alu elements are a subfamily within the primate SINE element family, we included them separately given the prior hnRNP C result. As transposable elements are abundant in the genome, the number of overlapping exons was compared as before to the overlap in the whole intron and adjacent introns.
Cryptic exons are poorly conserved and their inclusion acts to destabilise their host transcript
We also investigated the consequences of inclusion of cryptic exons on translation of the transcript. The inclusion of a premature termination codon (PTC) into a transcript could lead to either mRNA degradation by the nonsense-mediated decay (NMD) pathway. Alternatively, if the PTC falls within 50 nucleotides upstream of an exon-exon junction then NMD can be evaded, leading to the production of a truncated protein . These two outcomes are compounded by the possibility of a cryptic exon shifting the reading frame of the downstream exon, leading either to more PTCs or the creation of a benign transcript of a different function. This gives cryptic exon inclusion six functional outcomes that can destabilise either the RNA or the downstream protein. We tested for all these outcomes by translating the predicted transcripts in silico. In this analysis we only considered the”cassette-like” cryptic exons that had both upstream and downstream splice junctions. Of the 31 human cryptic exons tested, 21 (67%) are predicted to destabilise their host transcript through the NMD pathway (Fig. 4b). Five cryptic exons are predicted to impart new functionality whereas five others are predicted to contain premature stop codons but evade NMD, creating truncated proteins upon translation. However, comparing these results to randomly generated sequence of the same length demonstrates that this distribution of outcomes can occur purely by chance. The 23 mouse cryptic exons tested behave similarly, with four cryptic exons creating functional proteins, one creating a new downstream protein, six leading to protein truncation and 12 being degraded by NMD (Fig. 4b). The random sequences were similarly distributed. Together this suggests that the cryptic exons are not enriched in particular sequence features and upon inclusion behave like randomly chosen sequences. This disruption of multiple mRNA species may have an effect at the protein level and explain the toxicity seen in response to TDP-43 depletion.
Cryptic exon containing genes are downregulated
Furthermore, we performed the same analysis for each dataset with the cryptic exon containing genes that were only found in the other dataset of the same species (Fig. 5b). Surprisingly, in the mouse ES cell dataset 6 there was an enrichment of downregulated genes that contain cryptic exons only detectable in the mouse adult brain dataset 5 (P < 0.001; hypergeometric test). Visual inspection of these 10 introns in the mouse ES cell data (Additional file 2: Figure S2) suggests that seven of them may harbour cryptic exons in the ES cell data that are currently undetectable by the CryptEx algorithm.
Human cryptic exons are driven by the recognition of strong splice sites that are normally repressed
Cryptic exons are bound by other RNA - binding proteins
We have designed an analytical strategy to identify cryptic splicing that takes advantage of biological replicates in RNA sequencing data. We have applied this tool to a set of human and murine TDP-43 depletion datasets, as well as datasets that deplete hnRNP C or FUS. Our results are consistent with the previous findings that depletion of TDP-43 or hnRNP C leads to the inclusion of novel cryptic exons in both human and mouse, albeit with hnRNP C depletion leading to a far greater number of events than TDP-43. Although FUS undoubtedly plays an important role in splicing and mRNA stability and shares a number of targets with TDP-43 , the low number of cryptic exons observed due to FUS depletion suggests that it does not play a major role in cryptic splicing and is a key point of differentiation with TDP-43. This is despite the FUS and TDP-43 data analysed being produced under the same conditions for both mouse and human.
Further examination of TDP-43 linked exons suggests they tend to possess the necessary UG-rich sequence elements to be bound by TDP-43 and using iCLIP data we observed that a subset of the cryptic exons are bound by TDP-43 in vivo. We went on to investigate the origins of these TDP-43 bound cryptic exons, as has been done for the targets of hnRNP C. We observed that unlike hnRNP C linked cryptic exons, which invariably originate from antisense Alu elements, TDP-43 linked cryptic exons do not originate from any single family of transposable element. Furthermore their sequences show very low species conservation, akin to random intronic sequence, but remarkably they contain splice sites very close in strength to those of their adjacent annotated exons. Our differential expression analysis suggests that the bulk of cryptic exon containing genes are significantly downregulated upon TDP-43 depletion. We hypothesize that while some cassette-like cryptic exons will create functional or truncated proteins, most will lead to premature stop codons and nonsense-mediated decay of the inclusion transcript. The large number of exon extensions discovered by CryptEx may similarly lead to frameshifting and nonsense-mediated decay upon inclusion but may alternatively be sequestered in the nucleus and degraded by the mRNA surveillance pathway, as has been observed for retained introns . A number of cassette-like cryptic exons could be not be spliced in silico to any annotated coding exons, suggesting a role for cryptic exon inclusion in to upstream open reading frames and 3′ untranslated regions, both of which have been observed to provide a site for modulation of gene expression by introducing premature termination codons [61, 62].
Consistent with this possibility, a recent proteomic study of TDP-43 depletion in human SH-SY5Y cells  that protein levels were changed for 3 of the 95 human cryptic exon containing genes. Two, HUWE1 and GOLGB1 had protein levels that were 8 and 31% of the control cells respectively whereas the third, HNRNPH3 was found to be 7-fold increased under TDP-43 depletion. Interestingly, the cryptic exon discovered in HNRNPH3 falls upstream of the start codon whereas those found in HUWE1 and GOLGB1 are predicted to trigger NMD by inclusion into the coding sequence (see Additional file 4: Table S1). Another gene with cryptic splicing seen in both K562 datasets and in the initial Ling HeLa data, AGRN, was recently shown to be decreased at the protein level in the cerebrospinal fluid of ALS patients compared to healthy controls and other neurological diseases . Correct splicing of AGRN has shown to be crucial for the formation of the neuromuscular junction .
Our current understanding of TDP-43’s role in cryptic splicing is that of a safeguard against the inclusion of potentially damaging intronic sequence into transcripts. However, the relationship between the UG-rich sequences and the strong 5′ and 3′ splice sites and their changes over evolutionary time are unknown as we observed no conserved cryptic exons between human and mouse.
Using publicly available ENCODE eCLIP data, we identified a number of RNA binding proteins that also bind subsets of human cryptic exons under normal conditions, that is, in the presence of TDP-43. It is unsurprising that the splicing factors U2AF35 and U2AF65 are enriched as they preferably bind pyrimidine-rich 3′ splice site sequences, which all cryptic exons appear to possess. That only 10–15% of cryptic exons show U2AF35/65 binding may be due to competition from TDP-43 in a manner similar to that seen between hnRNP C and U2AF65. TIA-1 is an exciting finding due to its role in the formation of stress granules, key regulators of RNA stability . In addition, IGF2BP1 has been reported as binding to TDP-43 in HEK293T and HeLa cell extracts [57, 58], whereas SRSF7 was reported as binding to TDP-43 in mouse N2A cells . None of the observed proteins have been reported to change their protein level in response to TDP-43 depletion .
As the majority of cryptic exons are predicted to lead to nonsense-mediated decay of the inclusion transcript it seems peculiar that we can observe these transcripts at all. We hypothesise that cryptic splicing may be much more widespread than can be observed by RNA sequencing due to the highly efficient nature of nonsense- mediated decay. Over half of all cryptic exon genes are significantly downregulated in each dataset (Fig. 2b), suggesting that cryptic exon inclusion may be a key mechanism in the widespread changes in RNA expression that occur upon TDP-43 depletion. An alternative hypothesis is there is an interaction between TDP-43 and hnRNPC with the NMD machinery, which reduces the efficiency of degradation that is not shared by FUS. Experiments depleting both hnRNP C and the NMD component UPF1 have increased the number of hnRNP C-associated cryptic exons even further , suggesting that these experiment should be repeated for TDP-43 and FUS to better understand the relationship between cryptic splicing and mRNA degradation. Differences between the number of cryptic exons between TDP-43 and hnRNP C depletion may also relate to this, as well as differences in strength of depletion. Both TDP-43 and FUS proteins are known to bind their own mRNAs [68, 69] and so may compensate protein levels in response to shRNA knockdown.
Two genes, ATG4B and GPSM2, have previously been demonstrated to have cryptic exon inclusion RNA transcripts in ALS patient brain samples, suggesting a role for cryptic splicing in disease . Our analysis also identified a cryptic exon in ATG4B in human cells, but not GPSM2; however we did not analyse human brain data. By expanding the list of cryptic exons, it will be interesting to explore whether these are also dysregulated in ALS patient brains. However, such analysis may prove challenging owing to the likely small concentrations of RNA originating from diseased cells in brain homogenate and the likelihood of degradation by NMD. Alternate strategies may involve mass spectrometry screens for the subset of cryptic exon containing genes that escape the NMD process and are translated into functional or truncated proteins. Such proteins may represent useful biomarkers for TDP-43 mislocalization and therefore ALS pathology.
A recent paper has used a complementary bioinformatic method to increase the number of cryptic exons seen in the Ling data . This study extends the cryptic splicing phenomenon to RBM17, another RNA-binding protein. This finding makes the very low number of cryptic exons observed in the FUS depletion datasets even more surprising,
We confirm the presence of cryptic exons after TDP-43 depletion and show they have a negative impact on the genes they reside in, leading to decreased expression levels. Further work is warranted to determine the relevance of cryptic exons to ALS and FTD pathogenesis.
The authors would like to thank all members of the Plagnol, Pritchard, Fratta and Isaacs labs for their assistance and discussion. We are extremely grateful for the RNA-seq, iCLIP and eCLIP data made freely available by the Cleveland, Gravely, Wong, Ule and Yeo labs. We are also grateful to Yang Li and Jonathan Pritchard for generously providing the processed GTEx and mouse tissue junctions.
J.H. is supported by a Medical Research Council and Brain Research Trust PhD studentship. The work of W.E. is supported by the Wellcome Trust (103760/Z/14/Z) and the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001002), the UK Medical Research Council (FC001002), and the Wellcome Trust (FC001002). P.F. is funded by a Medical Research Council/Motor Neuron Disease Association LEW Fellowship and by the NIHR UCLH BRC. A.M.I. is funded by Alzheimers Research UK, the Motor Neuron Disease Association and the European Research Council.
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JH and VP wrote all the code used to analyse the data with input from WE. JH, VP, PF, and AMI designed the experiments. JH wrote the manuscript with discussion and approval from all authors.
The authors declare that they have no competing interests.
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