Many molecular mechanisms for neural adaptation to stress remain unknown. Expression of alternative splice variants of Slo, a gene encoding calcium- and voltage-activated potassium channels, was measured in rat adrenal chromaffin tissue from normal and hypophysectomized animals. Hypophysectomy triggered an abrupt decrease in the proportion of Slo transcripts containing a "STREX" exon. The decrease was prevented by adrenocorticotropic hormone injections. In Xenopus oocytes, STREX variants produced channels with functional properties associated with enhanced repetitive firing. Thus, the hormonal stress axis is likely to control the excitable properties of epinephrine-secreting cells by regulating alternative splicing of Slo messenger RNA.

Calcium regulation of gene expression is critical for the long-lasting activity-dependent changes in cellular electrical properties that underlie important physiological functions such as learning and memory. Cellular electrical properties are diversified through the extensive alternative splicing of ion channel pre-messenger RNAs; however, the regulation of splicing by cell signalling pathways has not been well explored. Here we show that depolarization of GH3 pituitary cells represses splicing of the STREX exon in BK potassium channel transcripts through the action of Ca2+/calmodulin-dependent protein kinases (CaMKs). Overexpressing constitutively active CaMK IV, but not CaMK I or II, specifically decreases STREX inclusion in the mRNA. This decrease is prevented by mutations in particular RNA repressor sequences. Transferring 54 nucleotides from the 3' splice site upstream of STREX to a heterologous gene is sufficient to confer CaMK IV repression on an otherwise constitutive exon. These experiments define a CaMK IV-responsive RNA element (CaRRE), which mediates the alternative splicing of ion channel pre-mRNAs. The CaRRE presents a unique molecular target for inducing long-term adaptive changes in cellular electrical properties. It also provides a model system for dissecting the effect of signal transduction pathways on alternative splicing.

The heterogeneous nuclear ribonucleoprotein particle (hnRNP) proteins play important roles in mRNA processing in eukaryotes, but little is known about how they are regulated by cellular signaling pathways. The polypyrimidine-tract binding protein (PTB, or hnRNP I) is an important regulator of alternative pre-mRNA splicing, of viral RNA translation, and of mRNA localization. Here we show that the nucleocytoplasmic transport of PTB is regulated by the 3,5-cAMP-dependent protein kinase (PKA). PKA directly phosphorylates PTB on conserved Ser-16, and PKA activation in PC12 cells induces Ser-16 phosphorylation. PTB carrying a Ser-16 to alanine mutation accumulates normally in the nucleus. However, export of this mutant protein from the nucleus is greatly reduced in heterokaryon shuttling assays. Conversely, hyperphosphorylation of PTB by coexpression with the catalytic subunit of PKA results in the accumulation of PTB in the cytoplasm. This accumulation is again specifically blocked by the S16A mutation. Similarly, in Xenopus oocytes, the phospho-Ser-16-PTB is restricted to the cytoplasm, whereas the non-Ser-16-phosphorylated PTB is nuclear. Thus, direct PKA phosphorylation of PTB at Ser-16 modulates the nucleo-cytoplasmic distribution of PTB. This phosphorylation likely plays a role in the cytoplasmic function of PTB. T he heterogeneous nuclear ribonucleoprotein particle (hnRNP) proteins are involved in a variety of processes in mRNA metabolism including pre-mRNA splicing, mRNA transport, and translation (1). These processes are often regulated by cellular signaling pathways, but how this dynamic control is achieved is mostly unknown. Some hnRNP proteins are known to be phosphorylated, but in most cases the particular kinase that modifies the protein is not known. Moreover, it is generally not clear how such modifications affect protein function. HnRNP proteins localize primarily in the nucleus at steady state but some of them engage in nucleo-cytoplasmic shuttling (2, 3). There are examples of hnRNP localization being altered by specific signaling pathways (4, 5).The polypyrimidine tract-binding protein (PTB, or hnRNP I) has both nuclear and cytoplasmic functions. In the nucleus, it is a splicing repressor of a number of alternative exons (6, 7). In the cytoplasm, PTB plays a role in viral RNA translation through internal ribosome entry sites (8-11). Also in the cytoplasm, PTB is implicated in mRNA localization in Xenopus oocytes (12). The protein contains four RNA-recognition-motif type RNA binding domains (RRMs) and a conserved N-terminal domain. The N-terminal 55-aa segment of PTB contains both nuclear import and export signals and is sufficient to allow some nucleocytoplasmic shuttling in heterokaryon assays (13-17). There is an additional sequence within RRM2 that enhances nuclear export (17). At steady state, PTB is highly enriched in the nucleus, but its distribution must be regulated because the protein also has cytoplasmic functions. However, little is known about this regulation and what cellular signaling...

Alternative splicing controls the activity of many proteins important for neuronal excitation, but the signal-transduction pathways that affect spliced isoform expression are not well understood. One particularly interesting system of alternative splicing is exon 21 (E21) of the NMDA receptor 1 (NMDAR1 E21), which controls the trafficking of NMDA receptors to the plasma membrane and is repressed by Ca++/calmodulin-dependent protein kinase (CaMK) IV signaling. Here, we characterize the splicing of NMDAR1 E21. We find that E21 splicing is reversibly repressed by neuronal depolarization, and we identify two RNA elements within the exon that function together to mediate the inducible repression. One of these exonic elements is similar to an intronic CaMK IV–responsive RNA element (CaRRE) originally identified in the 3′ splice site of the BK channel STREX exon, but not previously observed within an exon. The other element is a new RNA motif. Introduction of either of these two motifs, called CaRRE type 1 and CaRRE type 2, into a heterologous constitutive exon can confer CaMK IV–dependent repression on the new exon. Thus, either exonic CaRRE can be sufficient for CaMK IV–induced repression. Single nucleotide scanning mutagenesis defined consensus sequences for these two CaRRE motifs. A genome-wide motif search and subsequent RT-PCR validation identified a group of depolarization-regulated alternative exons carrying CaRRE consensus sequences. Many of these exons are likely to alter neuronal function. Thus, these two RNA elements define a group of co-regulated splicing events that respond to a common stimulus in neurons to alter their activity.

The regulation of gene expression through alternative pre-mRNA splicing is common in metazoans and is often controlled by intracellular signaling pathways that are important in cell physiology. We have shown that the alternative splicing of a number of genes is controlled by membrane depolarization and Ca 2؉ /calmodulin-dependent protein kinase IV (CaMKIV) through CaMKIV-responsive RNA elements (CaRRE1 and CaRRE2); however, the trans-acting factors remain unknown. Here we show that the heterogeneous nuclear ribonucleoprotein (hnRNP) L is a CaRRE1 binding factor in nuclear extracts. An hnRNP L high affinity CA (cytidine-adenosine) repeat element is sufficient to mediate CaMKIV and hnRNP L repression of splicing in a location (3-splice site proximity)-dependent way. Depletion of hnRNP L by RNA interference followed by rescue with coexpressed exogenous hnRNP L demonstrates that hnRNP L mediates the CaMKIV-regulated splicing through CA repeats in heterologous contexts. Depletion of hnRNP L also led to increased inclusion of the stress axis-regulated exon and a CA repeat-harboring exon under depolarization or with activated CaMKIV. Moreover, hnRNP L binding to CaRRE1 was increased by CaMKIV and, conversely, was reduced by pretreatments with protein phosphatases. Therefore, hnRNP L is an essential component of CaMKIV-regulated alternative splicing through CA repeats, with its phosphorylation likely playing a critical role.Alternative pre-mRNA splicing allows the generation of multiple protein isoforms from a single transcript, contributing greatly to proteomic complexity (1-6). It is essential for normal cell function, and aberrant splicing is implicated in the development of human genetic diseases (7,8). Many alternative splicing events can be regulated by cell signals and may play important roles in cell physiology (9 -11). However, little is known of the essential molecular components between cell signals and the splicing machinery.Alternative splicing in mammalian systems is often controlled by multiple cis-acting regulatory elements in introns and exons (1, 12). These elements are mostly bound by transacting protein factors and in some cases by small nucleolar RNA (13) or form RNA secondary structures (14). The transacting factors are thought to control the assembly of constitutive splicing components to the target splice sites. For cell signal control of alternative splicing, RNA elements and trans-acting factors have been identified in several systems (9,10,(15)(16)(17)(18)(19)(20)(21)(22)(23).CA repeats are the most abundant and highly polymorphic dinucleotide repeats in the human genome. They are widely used in genetic linkage analyses (24), and their instability is characteristic of the mutator phenotype of cells defective in DNA repair genes (25). Recent studies indicate that long (Ն19 repeats) or short clustered CA repeats in downstream introns are constitutive enhancers or repressors depending on their proximity to the 5Ј-splice site (26,27). These effects are mediated by the heterogeneous nuclear ribonuc...

Background: Excitable cells show activity-dependent alternative splicing of ion channels. Results: CaMKIV phosphorylates hnRNP L at Ser-513, which is essential for depolarization-repression of a Slo1 potassium channel exon and splicing factor U2AF65. Conclusion: Depolarization controls alternative splicing of Slo1 channels through Ser-513 phosphorylation and inhibition of U2AF65.Significance: This provides the first direct link between depolarization/CaMKIV and the constitutive spliceosome.

Neurons make extensive use of alternative pre-mRNA splicing to regulate gene expression and diversify physiological responses. We showed previously in a pituitary cell line that the Ca ++ /calmodulin-dependent protein kinase CaMK IV specifically repressed splicing of the BK channel STREX exon. This repression is dependent on a CaMK IV-responsive RNA element (CaRRE) within the STREX 3 0 splice site. Here, we report that similar Ca ++ regulation of splicing, mediated by L-type calcium channels and CaM kinase IV, occurs in cultured neurons and in the brain. We identify a critical CaRRE motif (CACATNRTTAT) that is essential for conferring CaMK IV repression on an otherwise constitutive exon. Additional Ca ++ -regulated exons that carry this consensus sequence are also identified in the human genome. Thus, the Ca ++ /CaMK IV pathway in neurons controls the alternative splicing of a group of exons through this short CaRRE consensus sequence. The functions of some of these exons imply that splicing control through the CaMK IV pathway will alter neuronal activity.

Alternative splicing contributes greatly to the diversification of mammalian proteomes, but the molecular basis for the evolutionary emergence of splice variants remains poorly understood. We have recently found a novel class of splicing regulatory elements between the polypyrimidine tract (Py) and 3= AG (REPA) at intron ends in many human genes, including the multifunctional PRMT5 (for protein arginine methyltransferase 5) gene. The PRMT5 element is comprised of two G tracts that arise in most mammals and accompany significant exon skipping in human transcripts. The G tracts inhibit splicing by recruiting heterogeneous nuclear ribonucleoprotein (hnRNP) H and F (H/F) to reduce U2AF65 binding to the Py, causing exon skipping. The resulting novel shorter variant PRMT5S exhibits a histone H4R3 methylation effect similar to that seen with the original longer PRMT5L isoform but exhibits a distinct localization and preferential control of critical genes for cell cycle arrest at interphase in comparison to PRMT5L. This report thus provides a molecular mechanism for the evolutionary emergence of a novel splice variant with an opposite function in a fundamental cell process. The presence of REPA elements in a large group of genes implies their wider impact on different cellular processes for increased protein diversity in humans.A lternative precursor mRNA (pre-mRNA) splicing greatly increases the proteomic diversity in metazoans (1-3). In particular, splice variants have reached the highest complexity in humans and other primates (4, 5), with about 90% of human genes alternatively spliced (6, 7). Aberrant splicing causes a large fraction of human genetic diseases (8, 9). However, the molecular basis for the evolutionary emergence of alternative exons that impact protein functions and cellular processes remains largely unknown, although several models have been proposed (10).The 3= end of introns between the polypyrimidine tract (Py) and 3=AG is highly constrained in sequence and length, with a consensus sequence of PyNYAG (Y, pyrimidine; N, any nucleotide) (11). However, we have found a CA-rich splicing regulatory element called CaRRE1 at this location (12-15), suggesting relaxation of the constraint in some transcripts and the potential existence of other, similar elements. In particular, a purine-rich (Grich or A-rich) element such as a G tract at this location is expected to strongly disrupt the 3= splice site (3=SS).G tracts with a minimal functional GGG motif are splicing regulatory elements bound by heterogeneous nuclear ribonucleoprotein (hnRNP) H (H1) or its paralogues, including hnRNP F (16-24). They are enhancers or silencers of splicing, depending on their location in the pre-mRNA (17, 22, 24-28). We have identified G tracts between the Py and 3= AG in more than a thousand human genes, including PRMT5 (for protein arginine methyltransferase 5). We call elements at this location REPA (regulatory elements between the Py and 3=AG) (29). These REPA G tracts appear to have mostly emerged in mammalian ancestors to ...