Alternative splicing of autism-related microexons in genes postulated to be regulated by nSR100




Taryn Brahmsteadt

Introduction

Autism spectrum disorder (ASD) refers to a range of conditions characterized by challenges with social interaction, communication, behavior, and sensory sensitivities. The symptoms associated with ASD can result in mild social impairment to severe disability. Approximately 1 in 68 children has been identified to have some form of ASD (Christensen. 2012.). Neuronal characteristics of autism have also been identified, including altered synaptic transmission and neuronal excitability. De novo missense mutations, de novo likely gene-disrupting mutations, and copy number variants have been found to be contributing factors to some ASD diagnoses (Iossifov et al. 2014.; Krumm et al. 2015.).

Microexons, exons between 3 and 27 nucleotides long, have been found to be frequently misregulated in individuals with ASD. 30% of microexons have been found to be misregulated in the brains of individuals with ASD, compared to 5% of longer exons; 90% of these misregulated microexons have also been found to display neural-differential regulation (Irimia et al. 2014). Exclusion of microexons is more likely to occur in the brains of individuals with ASD than in the brains of individuals unaffected by ASD. The neural splicing factor nSR100 facilitates the inclusion of most neural microexons (Irimia et al. 2014). Neuronal activation, characteristic of autistic brains, decreases nSR100 levels and therefore increases microexon exclusion. Altered synaptic transmission and neuronal excitability has been shown to occur in nSR100 deficient mice. nSR100 deficient mice also exhibited altered social behavior and autistic-like behaviors (Quesnel-Valličres et al. 2016).

If autism-related genes containing microexons are regulated by nSR100, the same pattern of alternative splice variants would be expected to occur. In order to determine whether the same patterns were exhibited in the same cell line by multiple genes, four genes were analyzed in three cell lines.


Materials and Methods
RNA was extracted from three unique cell lines:
THP1, Hep G2, A549.
RT-PCR was run in duplicate for each cell line with primer sets for four microexon containing genes:
AGRN, ITSN1, ABI1, RAPGEF6.
Gel electrophoresis was used to visualized RT-PCR results on a 4% agarose gel.
RT-PCR products were purified using QIAquick PCR purification protocol.
Purified RT-PCR product was sent to GeneWiz for sequencing:
AGRN, ITSN1, ABI1.
Sequencing was analyzed and aligned using the programs ApE and Clustal Omega, respectively.


Results

AGRN and ITSN1 each exhibited only one band, which sequencing showed to be the splice variant lacking the microexon, in all three cell lines. ABI1 showed two bands of similar intensities in cell lines THP1 and A549, but in the cell line HepG2 the splice variant lacking the microexon exhibited a more intense band. Analysis of RAPGEF6 indicated the presence of two bands in the cell lines HepG2 and A549, but only showed one apparent band in the cell line THP1. The RAPGEF6 splice alternative containing the microexon exhibited a more intense band in the three cell lines than the splice variant lacking the microexon, which was light in the HepG2 and A549 cell lines, and not visible in the THP1 cell line.

Sequence analysis confirmed the sequences of ABI1, AGRN, and ITSN1. ABI1 sequencing confirmed the presence of two alternative splice variants, one containing the 15 nucleotide microexon and the other lacking the 15 nucleotide microexon. AGRN sequencing confirmed that the observed variant was that in which the 12 nucleotide microexon had been excluded. ITSN1 sequencing also showed that the only present variant did not contain the 15 nucleotide microexon.


Discussion

We observed the same pattern in AGRN and ITSN1 in all cell lines, where there was only one variant and that variant was that in which the microexon had been excluded. ABI1 showed a unique pattern, where all three cell lines exhibited two bands (Figure 1). The two bands were of approximately the same intensity in the THP1 and A549 cell lines, but in the HepG2 cell line the lower band, which does not contain a microexon, was more intense than the microexon-containing band, indicating a different splicing regulator than that of AGRN and ITSN1. Analysis of RAPGEF6 showed a unique pattern because in all three cell lines the band of the splice variant containing the microexon was more intense than the band lacking the microexon. Furthermore, THP1 exhibited a novel pattern where the splice variant containing the microexon was the only visible band (Figure 1). This indicates RAPGEF6 is regulated by a different splicing regulator than the other three genes. Because different splicing patterns were observed among the microexon-containing genes ABI1, ITSN1, AGRN, and RAPGEF6 within specific cells lines, autism-related genes appear to be differentially regulated and may not be dependent on nSR100. If these genes were all regulated by the same splicing factor, they would be expected to exhibit the same splicing patterns for each of the genes within the same cell line.

The sequencing results indicated the expected sequences for the three genes which were sent out for sequencing: ABI1, AGRN, and ITSN1. Analysis of ABI1 showed that the intronic sequences were those that would be expected at the 5’ and 3’ splice sites. Further analysis of these genes and their regulators could allow scientists to elucidate the mechanisms by which microexons tend to be excluded in the brains of individuals with ASD.


References

Chen, Mo, and James L. Manley. “Mechanisms of Alternative Splicing Regulation: Insights from Molecular and Genomics Approaches.” Nature reviews. Molecular cell biology 10.11 (2009): 741–754.
Christensen, Deborah L. "Prevalence and characteristics of autism spectrum disorder among children aged 8 years—autism and developmental disabilities monitoring network, 11 sites, United States, 2012." MMWR. Surveillance Summaries 65 (2016).
Iossifov, Ivan, et al. "The contribution of de novo coding mutations to autism spectrum disorder." Nature 515.7526 (2014): 216-221.
Irimia, et. al. “A Highly Conserved Program of Neuronal Microexons Is Misregulated in Autistic Brains.” Cell 159.7 (2014): 1511-1523.
Krumm, Niklas, et al. "Excess of rare, inherited truncating mutations in autism." Nature genetics 47.6 (2015): 582-588.
O’Roak, Brian J., et al. "Sporadic autism exomes reveal a highly interconnected protein network of de novo mutations." Nature 485.7397 (2012): 246-250.
Quesnel-Valličres, M. Zahra Dargaei, Manuel Irimia, Melanie A. Woodin, Benjamin J. Blencowe, Sabine P. Cordes. “Misregulation of an Activity-Dependent Splicing Network as a Common Mechanism Underlying Autism Spectrum Disorders.” Molecular Cell 64 (2016): 1023–1034
Quesnel-Valličres, M., Irimia, M., Cordes, S.P. and Blencowe, B.J. “Essential roles for the splicing regulator nSR100/SRRM4 during nervous system development.” Genes Dev. 29 (2015): 746-759.
Wang, Eric T. et al. “Alternative Isoform Regulation in Human Tissue Transcriptomes.” Nature 456.7221 (2008): 470–476.
Wang, Yan et al. “Mechanism of Alternative Splicing and Its Regulation.” Biomedical Reports 3.2 (2015): 152–158. PMC.

Figures


Figure 1-RT-PCR results visualized on a 4% agarose gel for electrophoresis. Primers were used for the genes AGRN, ITSN1, ABI1, and RAPGEF6 in the cell lines THP1, Hep G2, and A549.


Figure 2-Alignment of the DNA sequences obtained from GeneWiz via Clustal Omega. The upper sequence is the sequence lacking the 15 nucleotide microexon. The lower strand is the sequence containing the 15 nucleotide microexon. The microexon is highlighted by the red square.


Figure 3-ABI1 showed expected sequences in intronic splice site regions.


Autism spectrum disorder (ASD) is characterized by difficulties with social interaction, communication, behavior, and sensory sensitivities. Neuronal characteristics of autism include altered synaptic transmission and neuronal exitability. Neural microexons are frequently misregulated in individuals with ASD.
Microexons are 3-27 nucleotide exons. The neuronal-specific splicing factor nSR100 facilitates the inclusion of most neural microexons (Irimia et. al. 2014.). Neuronal activation decreases nSR100 levels and therefore increases microexon exclusion. Autistic-like characteristics are observed in nSR100 deficient mice. However, four autism-related genes exhibited unique splicing patterns in three different cell lines, indicating that the inclusion of microexons in these genes is not regulated by the same splicing factor.

Full Paper

Acknowledgments

I would like to thank Dr. Berish Rubin for his support and willingness to share his expertise. I would like to thank Catharina Grubaugh and Anthony Evans for their immense support and enthusiasm, as well as for their endless patience.


This document was last modified 05/17/2017.
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