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Nova regulates brain-specific splicing to shape the synapse

Abstract

Alternative RNA splicing greatly increases proteome diversity and may thereby contribute to tissue-specific functions. We carried out genome-wide quantitative analysis of alternative splicing using a custom Affymetrix microarray to assess the role of the neuronal splicing factor Nova in the brain. We used a stringent algorithm to identify 591 exons that were differentially spliced in the brain relative to immune tissues, and 6.6% of these showed major splicing defects in the neocortex of Nova2−/− mice. We tested 49 exons with the largest predicted Nova-dependent splicing changes and validated all 49 by RT-PCR. We analyzed the encoded proteins and found that all those with defined brain functions acted in the synapse (34 of 40, including neurotransmitter receptors, cation channels, adhesion and scaffold proteins) or in axon guidance (8 of 40). Moreover, of the 35 proteins with known interaction partners, 74% (26) interact with each other. Validating a large set of Nova RNA targets has led us to identify a multi-tiered network in which Nova regulates the exon content of RNAs encoding proteins that interact in the synapse.

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Figure 1: Microarray probe design and ASPIRE method for data analysis.
Figure 2: Comparative analysis of microarray data.
Figure 3: Validation of microarray data by RT-PCR.
Figure 4: Relative representation of selected Gene Ontology functions for the total set of control exons from genes expressed in brain that were tested on the microarray (total brain-expressed); the 54 Nova2 regulated exons (wild-type (WT) versus Nova2 knockout (KO) neocortex) validated to date (including five previously reported8,9); and the exons predicted to be differentially spliced in brain versus immune tissues (shared |ΔI| > 0.2), in neocortex versus spinal cord (|ΔI| > 0.24) and in spleen versus thymus (|ΔI| > 0.24).
Figure 5: The synaptic module of 41 proteins encoded by validated Nova-regulated RNAs (see also Table 1).

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Acknowledgements

We thank L.Y. Jan, B. Taneri and Y. Xia for comments on the manuscript; X. Wang for designing the website with the microarray data; S.A. Fung-Ho for help with figure design; A. Titov and T. Vojtko for help with the Pathway Assist program; and all members of the lab for their help and insight throughout this work. This work was supported by grants from the US National Institutes of Health (R.B.D.) and the Howard Hughes Medical Institute, the tumor immunology program of Cancer Research Institute (J.U.), an MSTP grant (J.S.) and a Human Frontiers Science Program Fellowship (M.R.). R.B.D. is an Investigator of the Howard Hughes Medical Institute.

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Correspondence to Robert B Darnell.

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Supplementary information

Supplementary Fig. 1

Representation of microarray design and data for APLP2 gene. (PDF 789 kb)

Supplementary Fig. 2

Distribution of comparative data from Nova-2 and Nova-1 wt/ko, Nova-2 wt brain/thymus and Nova-2 brain subregion sample comparisons. (PDF 992 kb)

Supplementary Fig. 3

Comparative microarray data analysis. (PDF 667 kb)

Supplementary Fig. 4

RT-PCR validation of Nova-regulated exons in Nova-2 wt/ko neocortex. (PDF 4406 kb)

Supplementary Fig. 5

The role of Nova in brain-specific splicing regulation. (PDF 504 kb)

Supplementary Fig. 6

New types of Nova-regulated alternative splicing choices. (PDF 1595 kb)

Supplementary Table 1

Primers for RT-PCR and real-time RT-PCR. (PDF 48 kb)

Supplementary Table 2

GoMiner analysis of genes regulated by splicing or steady state. (PDF 437 kb)

Supplementary Methods (PDF 300 kb)

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Ule, J., Ule, A., Spencer, J. et al. Nova regulates brain-specific splicing to shape the synapse. Nat Genet 37, 844–852 (2005). https://doi.org/10.1038/ng1610

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