Dysregulation of Alternative Poly-adenylation as a Potential Player in Autism Spectrum Disorder

Abstract

We present here the hypothesis that alternative poly-adenylation (APA) is dysregulated in the brains of individuals affected by Autism Spectrum Disorder (ASD), due to disruptions in the calcium signaling networks. APA, the process of selecting different poly-adenylation sites on the same gene, yielding transcripts with different-length 3' untranslated regions (UTRs), has been documented in different tissues, stages of development and pathologic conditions. Differential use of poly-adenylation sites has been shown to regulate the function, stability, localization and translation efficiency of target RNAs. However, the role of APA remains rather unexplored in neurodevelopmental conditions. In the human brain, where transcripts have the longest 3' UTRs and are thus likely to be under more complex post-transcriptional regulation, erratic APA could be particularly detrimental. In the context of ASD, a condition that affects individuals in markedly different ways and whose symptoms exhibit a spectrum of severity, APA dysregulation could be amplified or dampened depending on the individual and the extent of the effect on specific genes would likely vary with genetic and environmental factors. If this hypothesis is correct, dysregulated APA events might be responsible for certain aspects of the phenotypes associated with ASD. Evidence supporting our hypothesis is derived from standard RNA-seq transcriptomic data but we suggest that future experiments should focus on techniques that probe the actual poly-adenylation site (3' sequencing). To address issues arising from the use of post-mortem tissue and low numbers of heterogeneous samples affected by confounding factors (such as the age, gender and health of the individuals), carefully controlled in vitro systems will be required to model the effect of calcium signaling dysregulation in the ASD brain.

Keywords: RNA–seq; alternative poly-adenylation; autism spectrum disorder; calcium signaling; transcription.

FIGURE 1

A model for the hypothesis of APA dysregulation in the autistic brain. Our hypothesis suggests that a disruption of regulation of calcium levels in the cell affects the kinetics and pausing of Pol II, resulting in aberrant selection of poly-adenylation sites during the transcription process. In this simplified schematic, an excess of calcium in the autistic cell is signaled to the nucleus increasing the speed of Pol II and leading to preferential selection of the distal over the proximal poly-adenylation sites. This, in turn, results in the inclusion of regulatory elements in the 3′ UTR, such as binding sites for miRNA or RNA-binding proteins (RBP), at times when their presence is undesirable. These elements affect the translation efficiency or degradation rate of the mRNA in question potentially leading to less protein product, or interfere with localization signals leading to misplaced protein. As we have no evidence for specific genes/proteins playing a major role in the hypothesis, we have deliberately avoided labeling the molecules in this cartoon of our model. It is also important to highlight that this is only one possible scenario that is compatible with our hypothesis but other possibilities exist, e.g., calcium levels may go down instead of up, the transcription process may lead to shorter rather than longer mRNAs, the amount of protein produced may or may not be affected etc. In fact, it is likely that several possibilities co-exist and are applicable to different genes, depending on the presence and levels of other factors, as well as the sequence signals integral to the mRNAs in question.

FIGURE 2

Publicly available transcriptomic data supports a model of APA dysregulation in the ASD brain. (A–D) RNA-seq data for ASD and control post-mortem brain samples from four publicly available datasets show statistically significant differential APA events, as revealed by the software DaPars-v.0.9 (Masamha et al., 2014). Box-and-whisker plots depict the distribution of Percentage Distal poly(A) site Usage Index (PDUI) values within a set of samples, grouped by condition (gray:control; red:ASD). The PDUI value is calculated for each gene passing the coverage thresholds of DaPars (number of genes quoted in brackets) and is a measure of the preference of using the distal over the proximal poly(A) site (PDUI is 1, if all reads are assigned to the distal site and 0, if all reads are assigned to the proximal site). Only statistically significant events (adjusted p-value < 0.05, Fisher exact test) are included in the box-and-whisker plots. There is a statistically significant preference for longer 3′ UTRs (larger PDUI values) in the ASD group as compared with the control group in both the Ellis and Voineagu datasets (p-value < 2.2e-16 and p-value = 6.2e-12 respectively for one-sided KS test between control and ASD group mean PDUIs). The Irimia and Li datasets show the opposite trend (p-values are 0.0018 and 0.075 correspondingly) but much smaller numbers of differential events have been identified in these datasets (see also F of this figure). (E) “Fast” mutants of Pol II affect the selection of the poly-adenylation site in a way reminiscent of the effects observed in ASD brain samples. Box-and-whisker plots show the distribution of PDUI values for genes showing differential APA events between “fast” (orange) and “slow” (green) mutants of Pol II (data from (Fong et al., 2014)). There is a clear preference for longer 3′ UTRs (larger PDUI values) in the case of the fast mutant (p-value = 2.9e-06, KS test), reminiscent of the ASD group in the Ellis and Voineagu datasets (A,C). (F) Samples originating from brain gray matter display larger variations in PDUI values compared with samples from white matter (p-value < 2.2e-16; unpaired one-sided Wilcoxon rank-sum test), offering an explanation for why we find so few differential APA events in the Li dataset (which originates from corpus callosum, an area of the brain dominated by white matter). The box-and-whisker plots display the range of PDUI variances calculated by DaPars for 1038 genes from the Mills dataset. The dataset originates from (Mills et al., 2015) and comprises three samples derived from white matter and three samples derived from gray matter. The plot suggests that samples from white matter can be expected to show relatively little variation in their PDUI values. (G,H) Genes associated with greater variance in APA site selection among neurotypical subjects also show larger PDUI group mean differences between ASD and control subjects. Box-and-whiskers plots of the standard deviation of PDUI value within the control group are shown for genes with the largest difference in APA site selection between ASD and control groups (top 25% of the distribution of absolute values of PDUI group mean differences) and genes with the smallest difference (bottom 25% of the same distribution). Plots are shown for the Ellis (G) and Irimia (H) datasets but plots for the Voineagu and Li datasets are similar (not shown). Genes with large between-group PDUI differences show much larger variability in the PDUI value within the control group in both the Ellis and Irimia datasets (p-value = < 2.2e-16 for both datasets; unpaired, one-sided Wilcoxon rank-sum test). All plots were generated using the R statistical software suite. The boxplot whiskers extend in all cases to the most extreme data point, which lies at no more than 1.5 times the interquartile range from the box.

FIGURE 3

GOseq enrichment of Reactome pathways among genes with differential APA events. Barplot of adjusted p-values for the enrichment of 19 Reactome pathways over-represented among genes with differential APA events in both Ellis and Voineagu datasets. The pathways have been grouped according to their main classification within Reactome, with signal transduction being the most commonly occurring classification. The vertical line indicates the chosen significance cut-off (adjusted p-value < 0.05).