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The distribution of phosphorylated SR proteins and alternative splicing are regulated by RANBP2.

ABSTRACT: The mammalian cell nucleus is functionally compartmentalized into various substructures. Nuclear speckles, also known as interchromatin granule clusters, are enriched with SR splicing factors and are implicated in gene expression. Here we report that nuclear speckle formation is developmentally regulated; in certain cases phosphorylated SR proteins are absent from the nucleus and are instead localized at granular structures in the cytoplasm. To investigate how the nuclear architecture is formed, we performed a phenotypic screen of HeLa cells treated with a series of small interfering RNAs. Depletion of Ran-binding protein 2 induced cytoplasmic intermediates of nuclear speckles in G1 phase. Detailed analyses of these structures suggested that a late step in the sequential nuclear entry of mitotic interchromatin granule components was disrupted and that phosphorylated SR proteins were sequestered in an SR protein kinase-dependent manner. As a result, the cells had an imbalanced subcellular distribution of phosphorylated and hypophosphorylated SR proteins, which affected alternative splicing patterns. This study demonstrates that the speckled distribution of phosphorylated pre-mRNA processing factors is regulated by the nucleocytoplasmic transport system in mammalian cells and that it is important for alternative splicing.

SUBMITTER: Saitoh N

PROVIDER: S-EPMC3302738 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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The distribution of phosphorylated SR proteins and alternative splicing are regulated by RANBP2.

Saitoh Noriko N Sakamoto Chiyomi C Hagiwara Masatoshi M Agredano-Moreno Lourdes T LT Jiménez-García Luis Felipe LF Nakao Mitsuyoshi M

Molecular biology of the cell 20120119 6

The mammalian cell nucleus is functionally compartmentalized into various substructures. Nuclear speckles, also known as interchromatin granule clusters, are enriched with SR splicing factors and are implicated in gene expression. Here we report that nuclear speckle formation is developmentally regulated; in certain cases phosphorylated SR proteins are absent from the nucleus and are instead localized at granular structures in the cytoplasm. To investigate how the nuclear architecture is formed, ...[more]

PMID: 22262462

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Project description:BackgroundSeveral recent studies indicate that alternative splicing in Arabidopsis and other plants is a common mechanism for post-transcriptional modulation of gene expression. However, few analyses have been done so far to elucidate the functional relevance of alternative splicing in higher plants. Representing a frequent and universal subtle alternative splicing event among eukaryotes, alternative splicing at NAGNAG acceptors contributes to transcriptome diversity and therefore, proteome plasticity. Alternatively spliced NAGNAG acceptors are overrepresented in genes coding for proteins with RNA-recognition motifs (RRMs). As SR proteins, a family of RRM-containing important splicing factors, are known to be extensively alternatively spliced in Arabidopsis, we analyzed alternative splicing at NAGNAG acceptors in SR and SR-related genes.ResultsIn a comprehensive analysis of the Arabidopsis thaliana genome, we identified 6,772 introns that exhibit a NAGNAG acceptor motif. Alternative splicing at these acceptors was assessed using available EST data, complemented by a sequence-based prediction method. Of the 36 identified introns within 30 SR and SR-related protein-coding genes that have a NAGNAG acceptor, we selected 15 candidates for an experimental analysis of alternative splicing under several conditions. We provide experimental evidence for 8 of these candidates being alternatively spliced. Quantifying the ratio of NAGNAG-derived splice variants under several conditions, we found organ-specific splicing ratios in adult plants and changes in seedlings of different ages. Splicing ratio changes were observed in response to heat shock and most strikingly, cold shock. Interestingly, the patterns of differential splicing ratios are similar for all analyzed genes.ConclusionNAGNAG acceptors frequently occur in the Arabidopsis genome and are particularly prevalent in SR and SR-related protein-coding genes. A lack of extensive EST coverage can be compensated by using the proposed sequence-based method to predict alternative splicing at these acceptors. Our findings indicate that the differential effects on NAGNAG alternative splicing in SR and SR-related genes are organ- and condition-specific rather than gene-specific.

Dataset Information

The distribution of phosphorylated SR proteins and alternative splicing are regulated by RANBP2.

Publications

The distribution of phosphorylated SR proteins and alternative splicing are regulated by RANBP2.

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