A Deep Exon Cryptic Splice Site Promotes Aberrant Intron Retention in a Von Willebrand Disease Patient.

A translationally silent single nucleotide mutation in exon 44 (E44) of the von Willebrand factor (VWF) gene is associated with inefficient removal of intron 44 in a von Willebrand disease (VWD) patient. This intron retention (IR) event was previously attributed to reordered E44 secondary structure that sequesters the normal splice donor site. We propose an alternative mechanism: the mutation introduces a cryptic splice donor site that interferes with the function of the annotated site to favor IR.

Unannotated splicing regulatory elements in deep intron space.

Deep intron space harbors a diverse array of splicing regulatory elements that cooperate with better-known exon-proximal elements to enforce proper tissue-specific and development-specific pre-mRNA processing. Many deep intron elements have been highly conserved through vertebrate evolution, yet remain poorly annotated in the human genome. Recursive splicing exons (RS-exons) and intraexons promote noncanonical, multistep resplicing pathways in long introns, involving transient intermediate structures that are greatly underrepresented in RNA-seq datasets.

Selective effects of protein 4.1N deficiency on neuroendocrine and reproductive systems.

Protein 4.1N, a member of the protein 4.1 family, is highly expressed in the brain.

Antisense targeting of decoy exons can reduce intron retention and increase protein expression in human erythroblasts.

The decoy exon model has been proposed to regulate a subset of intron retention (IR) events involving predominantly larger introns (>1 kb). Splicing reporter studies have shown that decoy splice sites are essential for activity, suggesting that decoys act by engaging intron-terminal splice sites and competing with cross-intron interactions required for intron excision. The decoy model predicts that antisense oligonucleotides may be able to block decoy splice sites in endogenous pre-mRNA, thereby reducing IR and increasing productive gene expression.

pre-mRNA splicing, revisited.

In this issue of , Ji et al have identified a novel regulatory mechanism that ensures proper splicing of human pre-messenger RNA (pre-mRNA).

An important class of intron retention events in human erythroblasts is regulated by cryptic exons proposed to function as splicing decoys.

During terminal erythropoiesis, the splicing machinery in differentiating erythroblasts executes a robust intron retention (IR) program that impacts expression of hundreds of genes. We studied IR mechanisms in the splicing factor gene, which expresses ∼50% of its transcripts in late erythroblasts as a nuclear isoform that retains intron 4. RNA-seq analysis of nonsense-mediated decay (NMD)-inhibited cells revealed previously undescribed splice junctions, rare or not detected in normal cells, that connect constitutive exons 4 and 5 to highly conserved cryptic cassette exons within the intron.

Circulating primitive erythroblasts establish a functional, protein 4.1R-dependent cytoskeletal network prior to enucleating.

Hematopoietic ontogeny is characterized by distinct primitive and definitive erythroid lineages. Definitive erythroblasts mature and enucleate extravascularly and form a unique membrane skeleton, composed of spectrin, 4.1R-complex, and ankyrinR-complex components, to survive the vicissitudes of the adult circulation.

RNA splicing during terminal erythropoiesis.

Purpose Of Review: Erythroid progenitors must accurately and efficiently splice thousands of pre-mRNAs as the cells undergo extensive changes in gene expression and cellular remodeling during terminal erythropoiesis. Alternative splicing choices are governed by interactions between RNA binding proteins and cis-regulatory binding motifs in the RNA. This review will focus on recent studies that define the genome-wide scope of splicing in erythroblasts and discuss what is known about its regulation.

Developmental regulation of RNA processing by Rbfox proteins.

The Rbfox genes encode an ancient family of sequence-specific RNA binding proteins (RBPs) that are critical developmental regulators in multiple tissues including skeletal muscle, cardiac muscle, and brain. The hallmark of Rbfox proteins is a single high-affinity RRM domain, highly conserved from insects to humans, that binds preferentially to UGCAUG motifs at diverse regulatory sites in pre-mRNA introns, mRNA 3'UTRs, and pre-miRNAs hairpin structures. Versatile regulatory circuits operate on Rbfox pre-mRNA and mRNA to ensure proper expression of Rbfox1 protein isoforms, which then act on the broader transcriptome to regulate alternative splicing networks, mRNA stability and translation, and microRNA processing.

A dynamic intron retention program enriched in RNA processing genes regulates gene expression during terminal erythropoiesis.

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.

A dynamic alternative splicing program regulates gene expression during terminal erythropoiesis.

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.

Rbfox proteins regulate alternative mRNA splicing through evolutionarily conserved RNA bridges.

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.

Deep intron elements mediate nested splicing events at consecutive AG dinucleotides to regulate alternative 3' splice site choice in vertebrate 4.1 genes.

Distal intraexon (iE) regulatory elements in 4.1R pre-mRNA govern 3' splice site choice at exon 2 (E2) via nested splicing events, ultimately modulating expression of N-terminal isoforms of cytoskeletal 4.1R protein.

Rbfox-regulated alternative splicing is critical for zebrafish cardiac and skeletal muscle functions.

Rbfox RNA binding proteins are implicated as regulators of phylogenetically-conserved alternative splicing events important for muscle function. To investigate the function of rbfox genes, we used morpholino-mediated knockdown of muscle-expressed rbfox1l and rbfox2 in zebrafish embryos. Single and double morphant embryos exhibited changes in splicing of overlapping sets of bioinformatically-predicted rbfox target exons, many of which exhibit a muscle-enriched splicing pattern that is conserved in vertebrates.

Efficient in vivo manipulation of alternative pre-mRNA splicing events using antisense morpholinos in mice.

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.

Comprehensive characterization of expression patterns of protein 4.1 family members in mouse adrenal gland: implications for functions.

The members of the protein 4.1 family, 4.1R, 4.

Alternative pre-mRNA splicing switches modulate gene expression in late erythropoiesis.

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.

Intrasplicing coordinates alternative first exons with alternative splicing in the protein 4.1R gene.

In the protein 4.1R gene, alternative first exons splice differentially to alternative 3' splice sites far downstream in exon 2'/2 (E2'/2). We describe a novel intrasplicing mechanism by which exon 1A (E1A) splices exclusively to the distal E2'/2 acceptor via two nested splicing reactions regulated by novel properties of exon 1B (E1B).

A correlation with exon expression approach to identify cis-regulatory elements for tissue-specific alternative splicing.

Correlation of motif occurrences with gene expression intensity is an effective strategy for elucidating transcriptional cis-regulatory logic. Here we demonstrate that this approach can also identify cis-regulatory elements for alternative pre-mRNA splicing. Using data from a human exon microarray, we identified 56 cassette exons that exhibited higher transcript-normalized expression in muscle than in other normal adult tissues.

Fox-2 splicing factor binds to a conserved intron motif to promote inclusion of protein 4.1R alternative exon 16.

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.

High frequency of alternative first exons in erythroid genes suggests a critical role in regulating gene function.

The human genome uses alternative pre-mRNA splicing as an important mechanism to encode a complex proteome from a relatively small number of genes. An unknown number of these genes also possess multiple transcriptional promoters and alternative first exons that contribute another layer of complexity to gene expression mechanisms. Using a collection of more than 100 erythroid-expressed genes as a test group, we used genome browser tools and genetic databases to assess the frequency of alternative first exons in the genome.

Evolutionarily conserved coupling of transcription and alternative splicing in the EPB41 (protein 4.1R) and EPB41L3 (protein 4.1B) genes.

Recent studies have shown that transcription and alternative splicing can be mechanistically coupled. In the EPB41 (protein 4.1R) and EPB41L3 (protein 4.

The splicing regulatory element, UGCAUG, is phylogenetically and spatially conserved in introns that flank tissue-specific alternative exons.

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.

Putative tumor suppressor protein 4.1B is differentially expressed in kidney and brain via alternative promoters and 5' alternative splicing.

Protein 4.1B has been reported as a tumor suppressor in brain, but not in kidney, despite high expression in both tissues. Here we demonstrate that N-terminal variability in kidney and brain 4.

Differential domain evolution and complex RNA processing in a family of paralogous EPB41 (protein 4.1) genes facilitate expression of diverse tissue-specific isoforms.

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.