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NOVA2-mediated RNA regulation is required for axonal pathfinding during development.

Saito Y, Miranda-Rottmann S, Ruggiu M, Park CY, Fak JJ, Zhong R, Duncan JS, Fabella BA, Junge HJ, Chen Z, Araya R, Fritzsch B, Hudspeth AJ, Darnell RB - Elife (2016)

Bottom Line: The neuron specific RNA-binding proteins NOVA1 and NOVA2 are highly homologous alternative splicing regulators.NOVA proteins regulate at least 700 alternative splicing events in vivo, yet relatively little is known about the biologic consequences of NOVA action and in particular about functional differences between NOVA1 and NOVA2.Thus we have discovered that NOVA2 uniquely regulates alternative splicing of a coordinate set of transcripts encoding key components in cortical, brainstem and spinal axon guidance/outgrowth pathways during neural differentiation, with severe functional consequences in vivo.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Neuro-Oncology, Howard Hughes Medical Institute, The Rockefeller University, New York, United States.

ABSTRACT
The neuron specific RNA-binding proteins NOVA1 and NOVA2 are highly homologous alternative splicing regulators. NOVA proteins regulate at least 700 alternative splicing events in vivo, yet relatively little is known about the biologic consequences of NOVA action and in particular about functional differences between NOVA1 and NOVA2. Transcriptome-wide searches for isoform-specific functions, using NOVA1 and NOVA2 specific HITS-CLIP and RNA-seq data from mouse cortex lacking either NOVA isoform, reveals that NOVA2 uniquely regulates alternative splicing events of a series of axon guidance related genes during cortical development. Corresponding axonal pathfinding defects were specific to NOVA2 deficiency: Nova2-/- but not Nova1-/- mice had agenesis of the corpus callosum, and axonal outgrowth defects specific to ventral motoneuron axons and efferent innervation of the cochlea. Thus we have discovered that NOVA2 uniquely regulates alternative splicing of a coordinate set of transcripts encoding key components in cortical, brainstem and spinal axon guidance/outgrowth pathways during neural differentiation, with severe functional consequences in vivo.

No MeSH data available.


Related in: MedlinePlus

NOVA2 unique alternative splicing events of axon guidance related genes in E18.5 mice cortex.Left Diagrams showing RefSeq annotation genes, changed alternative splicing events, RNA-seq results of wild-type (grey) and Nova2-/- (blue), NOVA2 CLIP clusters (light blue), RNA-seq results of wild-type (grey) and Nova1-/- (red), and NOVA1 CLIP clusters (pink). Right panels and graphs showing RT-PCR results and quantification data of Ahrgef12, Ppp3cb, Neo1, and Rock1 in E18.5 wild-type, Nova2-/-, and Nova1-/- mice cortex, respectively. **p<0.01 (n=3, Tukey’s multiple comparison test). Data are presented as mean ± SD.DOI:http://dx.doi.org/10.7554/eLife.14371.018
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fig4s1: NOVA2 unique alternative splicing events of axon guidance related genes in E18.5 mice cortex.Left Diagrams showing RefSeq annotation genes, changed alternative splicing events, RNA-seq results of wild-type (grey) and Nova2-/- (blue), NOVA2 CLIP clusters (light blue), RNA-seq results of wild-type (grey) and Nova1-/- (red), and NOVA1 CLIP clusters (pink). Right panels and graphs showing RT-PCR results and quantification data of Ahrgef12, Ppp3cb, Neo1, and Rock1 in E18.5 wild-type, Nova2-/-, and Nova1-/- mice cortex, respectively. **p<0.01 (n=3, Tukey’s multiple comparison test). Data are presented as mean ± SD.DOI:http://dx.doi.org/10.7554/eLife.14371.018

Mentions: We explored the biologic pathways regulated by NOVA1 and NOVA2 with gene ontology (GO) analysis on the transcripts harboring NOVA-regulated alternative exons. Among the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway terms associated with NOVA2-regulated genes (FDR<0.05) were axon guidance signaling (FDR=0.0039) and adherens junctions signaling (FDR = 0.017) (Figure 3—figure supplement 2 and 3). In the GO terms associated with NOVA2-regulated genes (FDR<0.05), axonal projection related terms (cell morphogenesis, neuron projection morphogenesis, axonogenesis, cell morphogenesis involved in neuron differentiation, and cell projection morphogenesis) were also enriched (Figure 3—source data 1). NOVA2-dependent alternative splicing events of axon guidance related genes were validated by semi-quantitative RT-PCR with RNA prepared from E18.5 wild-type Nova2-/-, and Nova1-/- cortex (Figure 4 and Figure 4—figure supplement 1). The alternative splicing events (Dcc exon 17, Slit2 exon 28b, Robo2 exons 6b and 21, Epha5 exon 7, Arhgef12 exon 4, Ppp3cb exon 10b, Neo1 exon 26, and Rock1 exon 27b) were significantly changed in Nova2-/- but not in Nova1-/- mice cortex, coinciding with RNA-seq results. No association was detected in the NOVA1-regulated alternative exons GO or KEGG pathways terms, likely due to the smaller number of NOVA1-regulated transcripts in cortex.10.7554/eLife.14371.017Figure 4.NOVA2 unique alternative splicing events of axon guidance related genes in E18.5 mice cortex.


NOVA2-mediated RNA regulation is required for axonal pathfinding during development.

Saito Y, Miranda-Rottmann S, Ruggiu M, Park CY, Fak JJ, Zhong R, Duncan JS, Fabella BA, Junge HJ, Chen Z, Araya R, Fritzsch B, Hudspeth AJ, Darnell RB - Elife (2016)

NOVA2 unique alternative splicing events of axon guidance related genes in E18.5 mice cortex.Left Diagrams showing RefSeq annotation genes, changed alternative splicing events, RNA-seq results of wild-type (grey) and Nova2-/- (blue), NOVA2 CLIP clusters (light blue), RNA-seq results of wild-type (grey) and Nova1-/- (red), and NOVA1 CLIP clusters (pink). Right panels and graphs showing RT-PCR results and quantification data of Ahrgef12, Ppp3cb, Neo1, and Rock1 in E18.5 wild-type, Nova2-/-, and Nova1-/- mice cortex, respectively. **p<0.01 (n=3, Tukey’s multiple comparison test). Data are presented as mean ± SD.DOI:http://dx.doi.org/10.7554/eLife.14371.018
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Related In: Results  -  Collection

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fig4s1: NOVA2 unique alternative splicing events of axon guidance related genes in E18.5 mice cortex.Left Diagrams showing RefSeq annotation genes, changed alternative splicing events, RNA-seq results of wild-type (grey) and Nova2-/- (blue), NOVA2 CLIP clusters (light blue), RNA-seq results of wild-type (grey) and Nova1-/- (red), and NOVA1 CLIP clusters (pink). Right panels and graphs showing RT-PCR results and quantification data of Ahrgef12, Ppp3cb, Neo1, and Rock1 in E18.5 wild-type, Nova2-/-, and Nova1-/- mice cortex, respectively. **p<0.01 (n=3, Tukey’s multiple comparison test). Data are presented as mean ± SD.DOI:http://dx.doi.org/10.7554/eLife.14371.018
Mentions: We explored the biologic pathways regulated by NOVA1 and NOVA2 with gene ontology (GO) analysis on the transcripts harboring NOVA-regulated alternative exons. Among the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway terms associated with NOVA2-regulated genes (FDR<0.05) were axon guidance signaling (FDR=0.0039) and adherens junctions signaling (FDR = 0.017) (Figure 3—figure supplement 2 and 3). In the GO terms associated with NOVA2-regulated genes (FDR<0.05), axonal projection related terms (cell morphogenesis, neuron projection morphogenesis, axonogenesis, cell morphogenesis involved in neuron differentiation, and cell projection morphogenesis) were also enriched (Figure 3—source data 1). NOVA2-dependent alternative splicing events of axon guidance related genes were validated by semi-quantitative RT-PCR with RNA prepared from E18.5 wild-type Nova2-/-, and Nova1-/- cortex (Figure 4 and Figure 4—figure supplement 1). The alternative splicing events (Dcc exon 17, Slit2 exon 28b, Robo2 exons 6b and 21, Epha5 exon 7, Arhgef12 exon 4, Ppp3cb exon 10b, Neo1 exon 26, and Rock1 exon 27b) were significantly changed in Nova2-/- but not in Nova1-/- mice cortex, coinciding with RNA-seq results. No association was detected in the NOVA1-regulated alternative exons GO or KEGG pathways terms, likely due to the smaller number of NOVA1-regulated transcripts in cortex.10.7554/eLife.14371.017Figure 4.NOVA2 unique alternative splicing events of axon guidance related genes in E18.5 mice cortex.

Bottom Line: The neuron specific RNA-binding proteins NOVA1 and NOVA2 are highly homologous alternative splicing regulators.NOVA proteins regulate at least 700 alternative splicing events in vivo, yet relatively little is known about the biologic consequences of NOVA action and in particular about functional differences between NOVA1 and NOVA2.Thus we have discovered that NOVA2 uniquely regulates alternative splicing of a coordinate set of transcripts encoding key components in cortical, brainstem and spinal axon guidance/outgrowth pathways during neural differentiation, with severe functional consequences in vivo.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Neuro-Oncology, Howard Hughes Medical Institute, The Rockefeller University, New York, United States.

ABSTRACT
The neuron specific RNA-binding proteins NOVA1 and NOVA2 are highly homologous alternative splicing regulators. NOVA proteins regulate at least 700 alternative splicing events in vivo, yet relatively little is known about the biologic consequences of NOVA action and in particular about functional differences between NOVA1 and NOVA2. Transcriptome-wide searches for isoform-specific functions, using NOVA1 and NOVA2 specific HITS-CLIP and RNA-seq data from mouse cortex lacking either NOVA isoform, reveals that NOVA2 uniquely regulates alternative splicing events of a series of axon guidance related genes during cortical development. Corresponding axonal pathfinding defects were specific to NOVA2 deficiency: Nova2-/- but not Nova1-/- mice had agenesis of the corpus callosum, and axonal outgrowth defects specific to ventral motoneuron axons and efferent innervation of the cochlea. Thus we have discovered that NOVA2 uniquely regulates alternative splicing of a coordinate set of transcripts encoding key components in cortical, brainstem and spinal axon guidance/outgrowth pathways during neural differentiation, with severe functional consequences in vivo.

No MeSH data available.


Related in: MedlinePlus