Alternative Splicing of a Novel Inducible Exon Diversifies the CASK Guanylate Kinase Domain.

Alternative pre-mRNA splicing has a major impact on cellular functions and development with the potential to fine-tune cellular localization, posttranslational modification, interaction properties, and expression levels of cognate proteins. The plasticity of regulation sets the stage for cells to adjust the relative levels of spliced mRNA isoforms in response to stress or stimulation. As part of an exon profiling analysis of mouse cortical neurons stimulated with high KCl to induce membrane depolarization, we detected a previously unrecognized exon (E24a) of the CASK gene, which encodes for a conserved peptide insertion in the guanylate kinase interaction domain.

The CUGBP2 splicing factor regulates an ensemble of branchpoints from perimeter binding sites with implications for autoregulation.

Alternative pre-mRNA splicing adjusts the transcriptional output of the genome by generating related mRNAs from a single primary transcript, thereby expanding protein diversity. A fundamental unanswered question is how splicing factors achieve specificity in the selection of target substrates despite the recognition of information-poor sequence motifs. The CUGBP2 splicing regulator plays a key role in the brain region-specific silencing of the NI exon of the NMDA R1 receptor.

Meayamycin inhibits pre-messenger RNA splicing and exhibits picomolar activity against multidrug-resistant cells.

FR901464 is a potent antitumor natural product that binds to the splicing factor 3b complex and inhibits pre-mRNA splicing. Its analogue, meayamycin, is two orders of magnitude more potent as an antiproliferative agent against human breast cancer MCF-7 cells. Here, we report the picomolar antiproliferative activity of meayamycin against various cancer cell lines and multidrug-resistant cells.

Exon silencing by UAGG motifs in response to neuronal excitation.

Alternative pre-mRNA splicing plays fundamental roles in neurons by generating functional diversity in proteins associated with the communication and connectivity of the synapse. The CI cassette of the NMDA R1 receptor is one of a variety of exons that show an increase in exon skipping in response to cell excitation, but the molecular nature of this splicing responsiveness is not yet understood. Here we investigate the molecular basis for the induced changes in splicing of the CI cassette exon in primary rat cortical cultures in response to KCl-induced depolarization using an expression assay with a tight neuron-specific readout.

Splicing-active nuclear extracts from rat brain.

In the nervous system, alternative pre-mRNA splicing generates the diverse protein machineries needed for cell excitation and synaptic communication. Yet, many questions remain about how these mechanisms are regulated by RNA binding proteins in the environment of differentiated cells and tissues. Here, we describe the preparation and use of splicing active nuclear extracts derived from the cerebellum and cerebral cortex regions of rat brain as a resource for in vitro studies.

Evidence for direct roles of two additional factors, SECp43 and soluble liver antigen, in the selenoprotein synthesis machinery.

Selenocysteine (Sec) is inserted into selenoproteins co-translationally with the help of various cis- and trans-acting factors. The specific mechanisms of Sec biosynthesis and insertion into protein in eukaryotic cells, however, are not known. Two proteins, SECp43 and the soluble liver antigen (SLA), were previously reported to interact with tRNA([Ser]Sec), but their functions remained elusive.

A combinatorial code for splicing silencing: UAGG and GGGG motifs.

Alternative pre-mRNA splicing is widely used to regulate gene expression by tuning the levels of tissue-specific mRNA isoforms. Few regulatory mechanisms are understood at the level of combinatorial control despite numerous sequences, distinct from splice sites, that have been shown to play roles in splicing enhancement or silencing. Here we use molecular approaches to identify a ternary combination of exonic UAGG and 5'-splice-site-proximal GGGG motifs that functions cooperatively to silence the brain-region-specific CI cassette exon (exon 19) of the glutamate NMDA R1 receptor (GRIN1) transcript.

Region-specific alternative splicing in the nervous system: implications for regulation by the RNA-binding protein NAPOR.

Alternative RNA splicing generates extensive proteomic diversity in the nervous system, yet few neural-specific RNA binding proteins have been implicated in splicing control. Here we show that the biochemical properties and spatial expression of mouse neuroblastoma apoptosis-related RNA-binding protein (NAPOR; also called NAPOR-1) are consistent with its roles in the regulation of the exon 5 and exon 21 splicing events of the N-methyl-D-aspartate (NMDA) receptor R1 transcript. NAPOR, which is closely related to CUG binding protein 2 (CUG-BP2), promotes exon 21 and represses exon 5 splicing in functional coexpression assays.

Function of quaking in myelination: regulation of alternative splicing.

Proteomic diversity is frequently achieved by alternative RNA-splicing events that can be fine-tuned in tissue-specific and developmentally regulated ways. Understanding this type of genetic regulation is compelling because of the extensive complexity of alternative splicing found in the nervous system. quaking (qk), one of the classical mouse dysmyelination mutants, is defective for the expression of myelin-associated glycoprotein (MAG), and the misregulation of MAG pre-mRNA alternative splicing is implicated as a causal factor.

Mutations in RRM4 uncouple the splicing repression and RNA-binding activities of polypyrimidine tract binding protein.

The polypyrimidine tract binding protein (PTB, or hnRNP I) contains four RNA-binding domains of the ribonucleoprotein fold type (RRMs 1, 2, 3, and 4), and mediates the negative regulation of alternative splicing through sequence-specific binding to intronic splicing repressor elements. To assess the roles of individual RRM domains in splicing repression, a neural-specific splicing extract was used to screen for loss-of-function mutations that fail to switch splicing from the neural to nonneural pathway. These results show that three RRMs are sufficient for wild-type RNA binding and splicing repression activity, provided that RRM4 is intact.