Hu proteins are RNA-binding proteins involved in diverse biological processes. The neuronal members of the Hu family, HuB, HuC, and HuD play important roles in neuronal differentiation and plasticity, while the ubiquitously expressed family member, HuR, has numerous functions mostly related to cellular stress response. The pivotal roles of Hu proteins are dictated by their molecular functions affecting a large number of target genes. Hu proteins affect many post-transcriptional aspects of RNA metabolism, from splicing to translation. In this communication, we will focus on these molecular events and review our current understanding of how Hu proteins mediate them. In particular, emphasis will be put on the nuclear functions of these proteins, which were recently discovered. Three examples including calcitonin/calcitonin gene-related peptide, neurofibromatosis type 1, and Ikaros will be discussed in detail. In addition, an intriguing theme of antagonism between Hu proteins and other AU-rich sequence binding proteins will be discussed.

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.Neurofibromatosis type I (NF1), which affects 1 in 3,500 individuals (11), is one of the most common dominantly inherited autosomal disorders. Loss-of-function mutations in the NF1 gene cause several abnormalities, including development of benign peripheral and optic nerve tumors (neurofibromas and gliomas) and abnormal distribution of melanocytes (café-aulait spots). NF1 patients also have increased risk of developing malignant tumors of neuronal origin (11, 13). The tumor suppression function of NF1 was linked to a domain in its encoded protein, neurofibromin, which is structurally similar to the Ras GTPase-activating protein (GAP) family (13). In addition to its widely accepted tumor suppression function, NF1 also plays a significant role in brain development. About 30 to 60% of children with NF1 mutations develop learning disabilities, ranging from mild cognitive impairment to attention deficit disorders (15).Exon 23a is an in-frame exon encoding 21 amino acids in the NF1 GAP region. This exon is alternatively included, producing two NF1 isoforms (5). The type I isoform does not contain this exon, while the type II isoform does. The ratio of the two isoforms varies in different tissues and during development. The type I isoform is predominantly expressed in neurons of the adult central nervous system (21, 25) and shows 10-timeshigher activity in down-regulating Ras activity than the type II isoform (5, 48). In the pheochromocytoma cell line PC12, production of the NF1 type 1 isoform can be induced by nerve growth factor treatment (48). These lines of evidence suggest that a balance of the two isoforms is important during neuronal differentiation. Indeed, when exon 23a was deleted from the NF1 locus by gene targ...

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.histone acetylation | neurofibromatosis type 1 | Fas R ecent genome-wide transcriptome analysis has demonstrated that more than 95% of human genes undergo alternative splicing to produce multiple proteins from one gene (1-4). Most of these alternative splicing events lead to coding differences and occur in a cell type-and/or developmental stage-specific manner (3, 5), underscoring the essential role of alternative splicing in gene expression control. In addition to the well-established role of RNA-binding proteins in the regulation of pre-mRNA alternative splicing (6, 7), recent studies have revealed a role for chromatin-associated proteins and the transcription machinery in splicing regulation (8)(9)(10).A recent study of large human genes demonstrated that premRNA splicing is cotranscriptional and occurs within 5-10 min of synthesis (11). The tight coupling of transcription and splicing predicts cross-talk between chromatin structure and splicing regulation. Indeed, several recent studies have documented a number of interesting links between chromatin features and exon behavior. First, a ChIP analysis indicated that a specific histone modification, trimethylation of lysine 36 of histone H3 (H3K36me3), differentially marks exons (12, 13). Remarkably, this histone mark appears to be associated more significantly with constitutive exons than with alternative exons (13). Second, a genome-wide analysis of nucleosome occupancy showed that nucleosomes are enriched in exons and are depleted in introns, suggesting that nucleosome position helps to distinguish introns from exons (12,(14)(15)(16)(17). Although these studies provide significant evidence for cross-talk between chromatin and splicing, the nature of the cross-talk remains largely unknown. Several studies support a model in which histone marks function to recruit basal spliceosomal factors or splicing regulators to ensure efficient splicing regulation. For example, the histone mark H3K4m...

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 calciumresponsive 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.alternative splicing | histone hyperacetylation | transcriptional elongation rate | calcium | cardiomyocytes A lternative splicing is a robust mechanism that regulates the functional output of a genome. More than 90% of human protein-coding genes undergo alternative splicing, which significantly increases proteomic complexity of the human genome (1, 2). The precise spatial-temporal regulation of alternative splicing plays a crucial role in controlling gene expression. Decades of studies have yielded a wealth of knowledge on how tissue-and developmental stage-specific alternative splicing is regulated (3). However, signaling-controlled alternative splicing in response to environmental cues has attracted far less attention, and regulatory mechanistic insights have only begun to emerge in recent years (4).Calcium is an important intracellular second messenger that regulates many biological processes. Although most studies have focused on how calcium regulates gene expression at the transcriptional level (5, 6), a small number of studies have also explored how changes in intracellular calcium levels can lead to alternative splicing pattern alterations (7-12). For example, in neuronal cells, a splicing-sensitive exon array identified more than 5,000 genes that change their splicing pattern in response to elevated calcium levels (13). However, because only a handful of studies have investigated the underlying mechanisms, it remains largely unknown how calcium-induced alternative splicing changes are regulated. These studies have revealed two distinct mechanisms by which calcium-responsive alternative exons can be regulated: one RNA-binding protein (RBP)-dependent and the other RBP-independent (14, 15).Several studies using depolarized...

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 U2AF65. 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.

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.

Edited by Ronald C. WekMyotonic 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 (⌿) resulted in a modest reduction of MBNL1 binding. Interestingly, ⌿ 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. Myotonic dystrophy type 1 (DM1)3 is a genetic neuromuscular disease caused by expression of expanded CUG repeats in the 3Ј UTR of the dystrophia myotonica protein kinase (DMPK) gene. Similar to DM1, myotonic dystrophy type 2 (DM2) is caused by expression of expanded CCUG repeats in an intron of the CCHC-type zinc finger nucleic acid-binding protein (CNBP) gene. DM1 and DM2 occur when the CUG/CCUG repeats are expanded beyond 100 repeats, and patients can have up to thousands of CUG/CCUG repeats (1, 2). A primary component of the currently accepted DM1 and DM2 disease mechanism is that expanded CUG/CCUG repeats sequester RNAbinding proteins (primarily the Muscleblind-like family), which prevents these proteins from performing their functions in cells (3, 4).The members of the Muscleblind-like family of proteins (MBNL1, MBNL2, and MBNL3) bind RNA and regulate several RNA processing pathways, including alternative splicing, premiRNA biogenesis, mRNA localization, alternative polyadenylation, and circular RNA generation (5-9). MBNL proteins bind to the consensus YGCY RNA sequence (6, 10). CUG and CCUG repeats are composed of YGCY motifs creating hundreds or thousands of perfect MBNL-binding sites resulting in large numbers of MBNL proteins binding to the repeats and forming nuclear foci (11). When MBNL proteins are sequestered, they are unable to regulate RNA processing events, and consequently, many DM1 and DM2 symptoms are caused by misregulated alternative splicing and potentially the loss of other MBNL activities (12). It is therefore important to understand how MBNL proteins bind to their toxic and cellular RNA substrates to develop mechanisms to alleviate MBNL sequestration in DM1 and DM2.MBNL pro...

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 23a⌬/23a⌬ 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 23a⌬/23a⌬ 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.