Zebrafish mbnl mutants model physical and molecular phenotypes of myotonic dystrophy.

The muscleblind RNA-binding proteins (MBNL1, MBNL2 and MBNL3) are highly conserved across vertebrates and are important regulators of RNA alternative splicing. Loss of MBNL protein function through sequestration by CUG or CCUG RNA repeats is largely responsible for the phenotypes of the human genetic disorder myotonic dystrophy (DM). We generated the first stable zebrafish (Danio rerio) models of DM-associated MBNL loss of function through mutation of the three zebrafish mbnl genes. In contrast to mouse models, zebrafish double and triple homozygous mbnl mutants were viable to adulthood. Zebrafish mbnl mutants displayed disease-relevant physical phenotypes including decreased body size and impaired movement. They also exhibited widespread alternative splicing changes, including the misregulation of many DM-relevant exons. Physical and molecular phenotypes were more severe in compound mbnl mutants than in single mbnl mutants, suggesting partially redundant functions of Mbnl proteins. The high fecundity and larval optical transparency of this complete series of zebrafish mbnl mutants will make them useful for studying DM-related phenotypes and how individual Mbnl proteins contribute to them, and for testing potential therapeutics. This article has an associated First Person interview with the first author of the paper.

Identification and Characterization of Zebrafish Tlr4 Coreceptor Md-2.

The zebrafish (Danio rerio) is a powerful model organism for studies of the innate immune system. One apparent difference between human and zebrafish innate immunity is the cellular machinery for LPS sensing. In amniotes, the protein complex formed by TLR4 and myeloid differentiation factor 2 (Tlr4/Md-2) recognizes the bacterial molecule LPS and triggers an inflammatory response. It is believed that zebrafish have neither Md-2 nor Tlr4; Md-2 has not been identified outside of amniotes, whereas the zebrafish tlr4 genes appear to be paralogs, not orthologs, of amniote TLR4s We revisited these conclusions. We identified a zebrafish gene encoding Md-2, ly96 Using single-cell RNA sequencing, we found that ly96 is transcribed in cells that also transcribe genes diagnostic for innate immune cells, including the zebrafish tlr4-like genes. In larval zebrafish, ly96 is expressed in a small number of macrophage-like cells. In a functional assay, zebrafish Md-2 and Tlr4ba form a complex that activates NF-κB signaling in response to LPS. In larval zebrafish ly96 loss-of-function mutations perturbed LPS-induced cytokine production but gave little protection against LPS toxicity. Finally, by analyzing the genomic context of tlr4 genes in 11 jawed vertebrates, we found that tlr4 arose prior to the divergence of teleosts and tetrapods. Thus, an LPS-sensitive Tlr4/Md-2 complex is likely an ancestral feature shared by mammals and zebrafish, rather than a de novo invention on the tetrapod lineage. We hypothesize that zebrafish retain an ancestral, low-sensitivity Tlr4/Md-2 complex that confers LPS responsiveness to a specific subset of innate immune cells.

Neurofibromatosis type 1 alternative splicing is a key regulator of Ras/ERK signaling and learning behaviors in mice.

Appropriate activation of the Ras/extracellular signal-regulated kinase (ERK) protein signaling cascade within the brain is crucial for optimal learning and memory. One key regulator of this cascade is the Nf1 Ras GTPase activating protein (RasGAP), which attenuates Ras/ERK signaling by converting active Ras is bound to guanosine triphosphate, activating Ras into inactive Ras is bound to guanosine diphosphate, inactivating Ras. A previous study using embryonic stem cells and embryonic stem cell-derived neurons indicated that Nf1 RasGAP activity is modulated by the highly regulated alternative splicing of Nf1 exon 23a. In this study, we generated Nf123aIN/23aIN mice, in which the splicing signals surrounding Nf1 exon 23a were manipulated to increase exon inclusion. Nf123aIN/23aIN mice are viable and exon 23a inclusion approaches 100% in all tissues, including the brain, where the exon is normally almost completely skipped. Ras activation and phosphorylation of ERK1/2 downstream of Ras are both greatly increased in Nf123aIN/23aIN mouse brain lysates, confirming that exon 23a inclusion inhibits Nf1 RasGAP activity in vivo as it does in cultured cells. Consistent with the finding of altered Ras/ERK signaling in the brain, Nf123aIN/23aIN mice showed specific deficits in learning and memory compared with Nf1+/+ mice. Nf123aIN/23aIN mice performed poorly on the T-maze and Morris water maze tests, which measure short- and long-term spatial memory, respectively. In addition, Nf123aIN/23aIN mice showed abnormally elevated context-dependent fear and a diminished ability to extinguish a cued fear response, indicating defective associative fear learning. Therefore, the regulated alternative splicing of Nf1 is an important mechanism for fine-tuning Ras/ERK signaling as well as learning and memory in mice.

Pseudouridine Modification Inhibits Muscleblind-like 1 (MBNL1) Binding to CCUG Repeats and Minimally Structured RNA through Reduced RNA Flexibility.

Myotonic dystrophy type 2 is a genetic neuromuscular disease caused by the expression of expanded CCUG repeat RNAs from the non-coding region of the CCHC-type zinc finger nucleic acid-binding protein (CNBP) gene. These CCUG repeats bind and sequester a family of RNA-binding proteins known as Muscleblind-like 1, 2, and 3 (MBNL1, MBNL2, and MBNL3), and sequestration plays a significant role in pathogenicity. MBNL proteins are alternative splicing regulators that bind to the consensus RNA sequence YGCY (Y = pyrimidine). This consensus sequence is found in the toxic RNAs (CCUG repeats) and in cellular RNA substrates that MBNL proteins have been shown to bind. Replacing the uridine in CCUG repeats with pseudouridine (Ψ) resulted in a modest reduction of MBNL1 binding. Interestingly, Ψ modification of a minimally structured RNA containing YGCY motifs resulted in more robust inhibition of MBNL1 binding. The different levels of inhibition between CCUG repeat and minimally structured RNA binding appear to be due to the ability to modify both pyrimidines in the YGCY motif, which is not possible in the CCUG repeats. Molecular dynamic studies of unmodified and pseudouridylated minimally structured RNAs suggest that reducing the flexibility of the minimally structured RNA leads to reduced binding by MBNL1.

Calcium-mediated histone modifications regulate alternative splicing in cardiomyocytes.

In cardiomyocytes, calcium is known to control gene expression at the level of transcription, whereas its role in regulating alternative splicing has not been explored. Here we report that, in mouse primary or embryonic stem cell-derived cardiomyocytes, increased calcium levels induce robust and reversible skipping of several alternative exons from endogenously expressed genes. Interestingly, we demonstrate a calcium-mediated splicing regulatory mechanism that depends on changes of histone modifications. Specifically, the regulation occurs through changes in calcium-responsive kinase activities that lead to alterations in histone modifications and subsequent changes in the transcriptional elongation rate and exon skipping. We demonstrate that increased intracellular calcium levels lead to histone hyperacetylation along the body of the genes containing calcium-responsive alternative exons by disrupting the histone deacetylase-to-histone acetyltransferase balance in the nucleus. Consequently, the RNA polymerase II elongation rate increases significantly on those genes, resulting in skipping of the alternative exons. These studies reveal a mechanism by which calcium-level changes in cardiomyocytes impact on the output of gene expression through altering alternative pre-mRNA splicing patterns.

Neurofibromatosis type 1 alternative splicing is a key regulator of Ras signaling in neurons.

Neurofibromatosis type I (Nf1) is a GTPase-activating protein (GAP) that inactivates the oncoprotein Ras and plays important roles in nervous system development and learning. Alternative exon 23a falls within the Nf1 GAP domain coding sequence and is tightly regulated in favor of skipping in neurons; however, its biological function is not fully understood. Here we generated mouse embryonic stem (ES) cells with a constitutive endogenous Nf1 exon 23a inclusion, termed Nf1 23aIN/23aIN cells, by mutating the splicing signals surrounding the exon to better match consensus sequences. We also made Nf1 23aΔ/23aΔ cells lacking the exon. Active Ras levels are high in wild-type (WT) and Nf1 23aIN/23aIN ES cells, where the Nf1 exon 23a inclusion level is high, and low in Nf1 23aΔ/23aΔ cells. Upon neuronal differentiation, active Ras levels are high in Nf1 23aIN/23aIN cells, where the exon inclusion level remains high, but Ras activation is low in the other two genotypes, where the exon is skipped. Signaling downstream of Ras is significantly elevated in Nf1 23aIN/23aIN neurons. These results suggest that exon 23a suppresses the Ras-GAP activity of Nf1. Therefore, regulation of Nf1 exon 23a inclusion serves as a mechanism for providing appropriate levels of Ras signaling and may be important in modulating Ras-related neuronal functions.

All three RNA recognition motifs and the hinge region of HuC play distinct roles in the regulation of alternative splicing.

The four Hu [embryonic lethal abnormal vision-like (ELAVL)] protein family members regulate alternative splicing by binding to U-rich sequences surrounding target exons and affecting the interaction of the splicing machinery and/or local chromatin modifications. Each of the Hu proteins contains a divergent N-terminus, three highly conserved RNA recognition motifs (RRM1, RRM2 and RRM3) and a hinge region separating RRM2 and RRM3. The roles of each domain in splicing regulation are not well understood. Here, we investigate how HuC, a relatively poorly characterized family member, regulates three target pre-mRNAs: neurofibromatosis type I, Fas and HuD. We find that the HuC N-terminus is dispensable for splicing regulation, and the three RRMs are required for splicing regulation of each target, whereas the hinge region contributes to regulation of only some targets. Interestingly, the regions of the hinge and RRM3 required for regulating different targets only partially overlap, implying substrate-specific mechanisms of HuC-mediated splicing regulation. We show that RRM1 and RRM2 are required for binding to target pre-mRNAs, whereas the hinge and RRM3 are required for HuC-HuC self-interaction. Finally, we find that the portions of RRM3 required for HuC-HuC interaction overlap with those required for splicing regulation of all three targets, suggesting a role of HuC-HuC interaction in splicing regulation.

Hu proteins regulate alternative splicing by inducing localized histone hyperacetylation in an RNA-dependent manner.

Recent studies have provided strong evidence for a regulatory link among chromatin structure, histone modification, and splicing regulation. However, it is largely unknown how local histone modification patterns surrounding alternative exons are connected to differential alternative splicing outcomes. Here we show that splicing regulator Hu proteins can induce local histone hyperacetylation by association with their target sequences on the pre-mRNA surrounding alternative exons of two different genes. In both primary and mouse embryonic stem cell-derived neurons, histone hyperacetylation leads to an increased local transcriptional elongation rate and decreased inclusion of these exons. Furthermore, we demonstrate that Hu proteins interact with histone deacetylase 2 and inhibit its deacetylation activity. We propose that splicing regulators may actively modulate chromatin structure when recruited to their target RNA sequences cotranscriptionally. This "reaching back" interaction with chromatin provides a means to ensure accurate and efficient regulation of alternative splicing.

The neurofibromatosis type I pre-mRNA is a novel target of CELF protein-mediated splicing regulation.

The CUG-BP and ETR-3 like factors (CELF) are a family of six highly conserved RNA-binding proteins that preferentially bind to UG-rich sequences. One of the key functions of these proteins is to mediate alternative splicing in a number of tissues, including brain, heart and muscle. To fully understand the function of CELF proteins, it is important to identify downstream targets of CELF proteins. In this communication, we report that neurofibromatosis type I (NF1) exon 23a is a novel target of CELF protein-mediated splicing regulation in neuron-like cells. NF1 regulates Ras signaling, and the isoform that excludes exon 23a shows 10 times greater ability to down-regulate Ras signaling than the isoform that includes exon 23a. Five of the six CELF proteins strongly suppress the inclusion of NF1 exon 23a. Over-expression or siRNA knockdown of these proteins in cell transfection experiments altered the levels of NF1 exon 23a inclusion. In vitro binding and splicing analyses demonstrate that CELF proteins block splicing through interfering with binding of U2AF(65). These studies, combined with our previous investigations demonstrating a role for Hu proteins and TIA-1/TIAR in controlling NF1 exon 23a inclusion, highlight the complex nature of regulation of this important alternative splicing event.

Regulation of neuron-specific alternative splicing of neurofibromatosis type 1 pre-mRNA.

Neurofibromatosis type 1 (NF1) is one of the most common heritable autosomal dominant disorders. Alternative splicing modulates the function of neurofibromin, the NF1 gene product, by inserting the in-frame exon 23a into the region of NF1 mRNA that encodes the GTPase-activating protein-related domain. This insertion, which is predominantly skipped in neurons, reduces the ability of neurofibromin to regulate Ras by 10-fold. Here, we report that the neuron-specific Hu proteins control the production of the short protein isoform by suppressing inclusion of NF1 exon 23a, while TIA-1/TIAR proteins promote inclusion of this exon. We identify two binding sites for Hu proteins, located upstream and downstream of the regulated exon, and provide biochemical evidence that Hu proteins specifically block exon definition by preventing binding of essential splicing factors. In vitro analyses using nuclear extracts show that at the downstream site, Hu proteins prevent binding of U1 and U6 snRNPs to the 5' splice site, while TIAR increases binding. Hu proteins also decrease U2AF binding at the 3' splice site located upstream of exon 23a. In addition to providing the first mechanistic insight into tissue-specific control of NF1 splicing, these studies establish a novel strategy whereby Hu proteins regulate RNA processing.