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1.
Nucleic Acids Res ; 44(2): 838-51, 2016 Jan 29.
Article in English | MEDLINE | ID: mdl-26531823

ABSTRACT

Differentiating erythroblasts execute a dynamic alternative splicing program shown here to include extensive and diverse intron retention (IR) events. Cluster analysis revealed hundreds of developmentally-dynamic introns that exhibit increased IR in mature erythroblasts, and are enriched in functions related to RNA processing such as SF3B1 spliceosomal factor. Distinct, developmentally-stable IR clusters are enriched in metal-ion binding functions and include mitoferrin genes SLC25A37 and SLC25A28 that are critical for iron homeostasis. Some IR transcripts are abundant, e.g. comprising ∼50% of highly-expressed SLC25A37 and SF3B1 transcripts in late erythroblasts, and thereby limiting functional mRNA levels. IR transcripts tested were predominantly nuclear-localized. Splice site strength correlated with IR among stable but not dynamic intron clusters, indicating distinct regulation of dynamically-increased IR in late erythroblasts. Retained introns were preferentially associated with alternative exons with premature termination codons (PTCs). High IR was observed in disease-causing genes including SF3B1 and the RNA binding protein FUS. Comparative studies demonstrated that the intron retention program in erythroblasts shares features with other tissues but ultimately is unique to erythropoiesis. We conclude that IR is a multi-dimensional set of processes that post-transcriptionally regulate diverse gene groups during normal erythropoiesis, misregulation of which could be responsible for human disease.


Subject(s)
Erythroblasts/physiology , Erythropoiesis/genetics , Gene Expression Regulation , Introns , Cation Transport Proteins/genetics , Cell Differentiation/genetics , Cell Nucleus/genetics , Cells, Cultured , Cluster Analysis , Codon, Nonsense , Erythroblasts/cytology , Exons , Humans , Introns/genetics , Microfilament Proteins/genetics , Mitochondrial Proteins/genetics , Nonsense Mediated mRNA Decay , Phosphoproteins/genetics , RNA Splice Sites , RNA Splicing Factors , Ribonucleoprotein, U2 Small Nuclear/genetics , Spectrin/genetics
2.
Nucleic Acids Res ; 42(6): 4031-42, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24442673

ABSTRACT

Alternative pre-messenger RNA splicing remodels the human transcriptome in a spatiotemporal manner during normal development and differentiation. Here we explored the landscape of transcript diversity in the erythroid lineage by RNA-seq analysis of five highly purified populations of morphologically distinct human erythroblasts, representing the last four cell divisions before enucleation. In this unique differentiation system, we found evidence of an extensive and dynamic alternative splicing program encompassing genes with many diverse functions. Alternative splicing was particularly enriched in genes controlling cell cycle, organelle organization, chromatin function and RNA processing. Many alternative exons exhibited differentiation-associated switches in splicing efficiency, mostly in late-stage polychromatophilic and orthochromatophilic erythroblasts, in concert with extensive cellular remodeling that precedes enucleation. A subset of alternative splicing switches introduces premature translation termination codons into selected transcripts in a differentiation stage-specific manner, supporting the hypothesis that alternative splicing-coupled nonsense-mediated decay contributes to regulation of erythroid-expressed genes as a novel part of the overall differentiation program. We conclude that a highly dynamic alternative splicing program in terminally differentiating erythroblasts plays a major role in regulating gene expression to ensure synthesis of appropriate proteome at each stage as the cells remodel in preparation for production of mature red cells.


Subject(s)
Alternative Splicing , Erythropoiesis/genetics , Cells, Cultured , Erythroblasts/metabolism , Erythroid Cells/cytology , Erythroid Cells/metabolism , Humans , Nonsense Mediated mRNA Decay , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Transcriptome
3.
Nat Struct Mol Biol ; 20(12): 1434-42, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24213538

ABSTRACT

Alternative splicing (AS) enables programmed diversity of gene expression across tissues and development. We show here that binding in distal intronic regions (>500 nucleotides (nt) from any exon) by Rbfox splicing factors important in development is extensive and is an active mode of splicing regulation. Similarly to exon-proximal sites, distal sites contain evolutionarily conserved GCATG sequences and are associated with AS activation and repression upon modulation of Rbfox abundance in human and mouse experimental systems. As a proof of principle, we validated the activity of two specific Rbfox enhancers in KIF21A and ENAH distal introns and showed that a conserved long-range RNA-RNA base-pairing interaction (an RNA bridge) is necessary for Rbfox-mediated exon inclusion in the ENAH gene. Thus we demonstrate a previously unknown RNA-mediated mechanism for AS control by distally bound RNA-binding proteins.


Subject(s)
Alternative Splicing/physiology , RNA, Messenger/metabolism , RNA-Binding Proteins/physiology , Animals , Base Pairing , Base Sequence , Binding Sites , Cell Line , Conserved Sequence , Humans , Kinesins/genetics , Kinesins/metabolism , Mice , Microfilament Proteins/genetics , Microfilament Proteins/metabolism , Models, Genetic , Nucleic Acid Conformation , RNA Splicing Factors , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , Regulatory Sequences, Ribonucleic Acid
4.
J Biol Chem ; 286(8): 6033-9, 2011 Feb 25.
Article in English | MEDLINE | ID: mdl-21156798

ABSTRACT

Mammalian pre-mRNA alternative splicing mechanisms are typically studied using artificial minigenes in cultured cells, conditions that may not accurately reflect the physiological context of either the pre-mRNA or the splicing machinery. Here, we describe a strategy to investigate splicing of normal endogenous full-length pre-mRNAs under physiological conditions in live mice. This approach employs antisense vivo-morpholinos (vMOs) to mask cis-regulatory sequences or to disrupt splicing factor expression, allowing functional evaluation of splicing regulation in vivo. We applied this strategy to gain mechanistic insight into alternative splicing events involving exons 2 and 16 (E2 and E16) that control the structure and function of cytoskeletal protein 4.1R. In several mouse tissues, inclusion of E16 was substantially inhibited by interfering with a splicing enhancer mechanism using a target protector morpholino that blocked Fox2-dependent splicing enhancers in intron 16 or a splice-blocking morpholino that disrupted Fox2 expression directly. For E2, alternative 3'-splice site choice is coordinated with upstream promoter use across a long 5'-intron such that E1A splices almost exclusively to the distal acceptor (E2dis). vMOs were used to test the in vivo relevance of a deep intron element previously proposed to determine use of E2dis via a two-step intrasplicing model. Two independent vMOs designed against this intronic regulatory element inhibited intrasplicing, robustly switching E1A splicing to the proximal acceptor (E2prox). This finding strongly supports the in vivo physiological relevance of intrasplicing. vMOs represent a powerful tool for alternative splicing studies in vivo and may facilitate exploration of alternative splicing networks in vivo.


Subject(s)
Alternative Splicing/drug effects , Oligoribonucleotides, Antisense/pharmacology , RNA, Messenger/biosynthesis , Alternative Splicing/genetics , Animals , Blood Proteins/biosynthesis , Blood Proteins/genetics , E1A-Associated p300 Protein/biosynthesis , E1A-Associated p300 Protein/genetics , Exons/genetics , Mice , Microfilament Proteins , RNA, Messenger/genetics
5.
Blood ; 113(14): 3363-70, 2009 Apr 02.
Article in English | MEDLINE | ID: mdl-19196664

ABSTRACT

Differentiating erythroid cells execute a unique gene expression program that insures synthesis of the appropriate proteome at each stage of maturation. Standard expression microarrays provide important insight into erythroid gene expression but cannot detect qualitative changes in transcript structure, mediated by RNA processing, that alter structure and function of encoded proteins. We analyzed stage-specific changes in the late erythroid transcriptome via use of high-resolution microarrays that detect altered expression of individual exons. Ten differentiation-associated changes in erythroblast splicing patterns were identified, including the previously known activation of protein 4.1R exon 16 splicing. Six new alternative splicing switches involving enhanced inclusion of internal cassette exons were discovered, as well as 3 changes in use of alternative first exons. All of these erythroid stage-specific splicing events represent activated inclusion of authentic annotated exons, suggesting they represent an active regulatory process rather than a general loss of splicing fidelity. The observation that 3 of the regulated transcripts encode RNA binding proteins (SNRP70, HNRPLL, MBNL2) may indicate significant changes in the RNA processing machinery of late erythroblasts. Together, these results support the existence of a regulated alternative pre-mRNA splicing program that is critical for late erythroid differentiation.


Subject(s)
Alternative Splicing/genetics , Erythropoiesis/genetics , Gene Expression Regulation , RNA Precursors/genetics , Cell Differentiation/genetics , Cells, Cultured , Erythroblasts/metabolism , Erythroblasts/physiology , Exons , Gene Expression Profiling , Humans , Models, Biological , Oligonucleotide Array Sequence Analysis , Protein Conformation , Proteins/chemistry , Proteins/metabolism , RNA Precursors/metabolism
6.
J Biol Chem ; 281(18): 12468-74, 2006 May 05.
Article in English | MEDLINE | ID: mdl-16537540

ABSTRACT

Activation of protein 4.1R exon 16 (E16) inclusion during erythropoiesis represents a physiologically important splicing switch that increases 4.1R affinity for spectrin and actin. Previous studies showed that negative regulation of E16 splicing is mediated by the binding of heterogeneous nuclear ribonucleoprotein (hnRNP) A/B proteins to silencer elements in the exon and that down-regulation of hnRNP A/B proteins in erythroblasts leads to activation of E16 inclusion. This article demonstrates that positive regulation of E16 splicing can be mediated by Fox-2 or Fox-1, two closely related splicing factors that possess identical RNA recognition motifs. SELEX experiments with human Fox-1 revealed highly selective binding to the hexamer UGCAUG. Both Fox-1 and Fox-2 were able to bind the conserved UGCAUG elements in the proximal intron downstream of E16, and both could activate E16 splicing in HeLa cell co-transfection assays in a UGCAUG-dependent manner. Conversely, knockdown of Fox-2 expression, achieved with two different siRNA sequences resulted in decreased E16 splicing. Moreover, immunoblot experiments demonstrate mouse erythroblasts express Fox-2. These findings suggest that Fox-2 is a physiological activator of E16 splicing in differentiating erythroid cells in vivo. Recent experiments show that UGCAUG is present in the proximal intron sequence of many tissue-specific alternative exons, and we propose that the Fox family of splicing enhancers plays an important role in alternative splicing switches during differentiation in metazoan organisms.


Subject(s)
Blood Proteins/physiology , DNA-Binding Proteins/metabolism , Microtubule-Associated Proteins/physiology , RNA-Binding Proteins/metabolism , Repressor Proteins/metabolism , Transcription Factors/metabolism , Amino Acid Motifs , Base Sequence , Blood Proteins/metabolism , Cell Differentiation , Cytoskeletal Proteins , Down-Regulation , Erythroblasts/metabolism , Exons , Gene Expression Regulation , HeLa Cells , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , Humans , Introns , Membrane Proteins , Microtubule-Associated Proteins/metabolism , Molecular Sequence Data , RNA Splicing Factors , RNA, Small Interfering/metabolism
7.
Nucleic Acids Res ; 33(2): 714-24, 2005.
Article in English | MEDLINE | ID: mdl-15691898

ABSTRACT

Previous studies have identified UGCAUG as an intron splicing enhancer that is frequently located adjacent to tissue-specific alternative exons in the human genome. Here, we show that UGCAUG is phylogenetically and spatially conserved in introns that flank brain-enriched alternative exons from fish to man. Analysis of sequence from the mouse, rat, dog, chicken and pufferfish genomes revealed a strongly statistically significant association of UGCAUG with the proximal intron region downstream of brain-enriched alternative exons. The number, position and sequence context of intronic UGCAUG elements were highly conserved among mammals and in chicken, but more divergent in fish. Control datasets, including constitutive exons and non-tissue-specific alternative exons, exhibited a much lower incidence of closely linked UGCAUG elements. We propose that the high sequence specificity of the UGCAUG element, and its unique association with tissue-specific alternative exons, mark it as a critical component of splicing switch mechanism(s) designed to activate a limited repertoire of splicing events in cell type-specific patterns. We further speculate that highly conserved UGCAUG-binding protein(s) related to the recently described Fox-1 splicing factor play a critical role in mediating this specificity.


Subject(s)
Alternative Splicing , Introns , Phylogeny , Regulatory Sequences, Ribonucleic Acid , Animals , Base Sequence , Brain/metabolism , Chickens/genetics , Conserved Sequence , Dogs , Exons , Humans , Mice , Rats , Tetraodontiformes/genetics , Tissue Distribution
8.
Genomics ; 84(4): 637-46, 2004 Oct.
Article in English | MEDLINE | ID: mdl-15475241

ABSTRACT

The EPB41 (protein 4.1) genes epitomize the resourcefulness of the mammalian genome to encode a complex proteome from a small number of genes. By utilizing alternative transcriptional promoters and tissue-specific alternative pre-mRNA splicing, EPB41, EPB41L2, EPB41L3, and EPB41L1 encode a diverse array of structural adapter proteins. Comparative genomic and transcript analysis of these 140- to 240-kb genes indicates several unusual features: differential evolution of highly conserved exons encoding known functional domains interspersed with unique exons whose size and sequence variations contribute substantially to intergenic diversity; alternative first exons, most of which map far upstream of the coding regions; and complex tissue-specific alternative pre-mRNA splicing that facilitates synthesis of functionally different complements of 4.1 proteins in various cells. Understanding the splicing regulatory networks that control protein 4.1 expression will be critical to a full appreciation of the many roles of 4.1 proteins in normal cell biology and their proposed roles in human cancer.


Subject(s)
Alternative Splicing , Biological Evolution , Blood Proteins/genetics , Microtubule-Associated Proteins/genetics , RNA Precursors/genetics , RNA Processing, Post-Transcriptional , Animals , Cytoskeletal Proteins , Exons , Humans , Introns , Membrane Proteins/genetics , Mice , Microfilament Proteins , Protein Isoforms , Protein Structure, Tertiary , RNA, Messenger/genetics , RNA, Messenger/metabolism
9.
Blood ; 101(10): 4164-71, 2003 May 15.
Article in English | MEDLINE | ID: mdl-12522012

ABSTRACT

Among the alternative pre-mRNA splicing events that characterize protein 4.1R gene expression, one involving exon 2' plays a critical role in regulating translation initiation and N-terminal protein structure. Exon 2' encompasses translation initiation site AUG1 and is located between alternative splice acceptor sites at the 5' end of exon 2; its inclusion or exclusion from mature 4.1R mRNA regulates expression of longer or shorter isoforms of 4.1R protein, respectively. The current study reports unexpected complexity in the 5' region of the 4.1R gene that directly affects alternative splicing of exon 2'. Identified far upstream of exon 2 in both mouse and human genomes were 3 mutually exclusive alternative 5' exons, designated 1A, 1B, and 1C; all 3 are associated with strong transcriptional promoters in the flanking genomic sequence. Importantly, exons 1A and 1B splice differentially with respect to exon 2', generating transcripts with different 5' ends and distinct N-terminal protein coding capacity. Exon 1A-type transcripts splice so as to exclude exon 2' and therefore utilize the downstream AUG2 for translation of 80-kDa 4.1R protein, whereas exon 1B transcripts include exon 2' and initiate at AUG1 to synthesize 135-kDa isoforms. RNA blot analyses revealed that 1A transcripts increase in abundance in late erythroblasts, consistent with the previously demonstrated up-regulation of 80-kDa 4.1R during terminal erythroid differentiation. Together, these results suggest that synthesis of structurally distinct 4.1R protein isoforms in various cell types is regulated by a novel mechanism requiring coordination between upstream transcription initiation events and downstream alternative splicing events.


Subject(s)
Alternative Splicing , Cytoskeletal Proteins , Exons , Membrane Proteins , Neuropeptides , Proteins/genetics , Animals , Base Sequence , Blotting, Northern , Consensus Sequence , DNA Primers , Erythroblasts/metabolism , Gene Amplification , Humans , Mice , Molecular Sequence Data , Polymerase Chain Reaction , Promoter Regions, Genetic , Protein Isoforms/genetics , Sequence Alignment , Transcription, Genetic
10.
EMBO J ; 21(22): 6195-204, 2002 Nov 15.
Article in English | MEDLINE | ID: mdl-12426391

ABSTRACT

A physiologically important alternative pre-mRNA splicing switch, involving activation of protein 4.1R exon 16 (E16) splicing, is required for the establishment of proper mechanical integrity of the erythrocyte membrane during erythropoiesis. Here we identify a conserved exonic splicing silencer element (CE(16)) in E16 that interacts with hnRNP A/B proteins and plays a role in repression of E16 splicing during early erythropoiesis. Experiments with model pre-mRNAs showed that CE(16) can repress splicing of upstream introns, and that mutagenesis or replacement of CE(16) can relieve this inhibition. An affinity selection assay with biotinylated CE(16) RNA demonstrated specific binding of hnRNP A/B proteins. Depletion of hnRNP A/B proteins from nuclear extract significantly increased E16 inclusion, while repletion with recombinant hnRNP A/B restored E16 silencing. Most importantly, differentiating mouse erythroblasts exhibited a stage-specific activation of the E16 splicing switch in concert with a dramatic and specific down-regulation of hnRNP A/B protein expression. These findings demonstrate that natural developmental changes in hnRNP A/B proteins can effect physiologically important switches in pre-mRNA splicing.


Subject(s)
Alternative Splicing , Cytoskeletal Proteins , Erythroid Precursor Cells/metabolism , Erythropoiesis/genetics , Gene Expression Regulation/genetics , Gene Silencing , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/biosynthesis , Membrane Proteins , Neuropeptides , Proteins/genetics , RNA Precursors/metabolism , Regulatory Sequences, Nucleic Acid , Animals , Base Sequence , Consensus Sequence , Erythroid Precursor Cells/cytology , Exons/genetics , HeLa Cells , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/genetics , Humans , Introns/genetics , Mice , Molecular Sequence Data , Mutagenesis , Protein Binding , RNA Precursors/genetics , RNA-Binding Proteins/metabolism , Sequence Alignment , Sequence Homology, Nucleic Acid , Species Specificity , Transfection , Vertebrates/genetics , Xenopus laevis
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