Control of alternative pre-mRNA splicing by Ca(++) signals

Alternative pre-mRNA splicing is a common way of gene expression regulation in metazoans. The selective use of specific exons can be modulated in response to various manipulations that alter Ca(++) signals, particularly in neurons. A number of splicing factors have also been found to be controlled by Ca(++) signals. Moreover, pre-mRNA elements have been identified that are essential and sufficient to mediate Ca(++)-regulated splicing, providing model systems for dissecting the involved molecular components. In neurons, this regulation likely contributes to the fine-tuning of neuronal properties.     

Regulation of apoptosis by alternative pre-mRNA splicing

Apoptosis, a phenomenon that allows the regulated destruction and disposal of damaged or unwanted cells, is common to many cellular processes in multicellular organisms. In humans more than 200 proteins are involved in apoptosis, many of which are dysregulated or defective in human diseases including cancer. A large number of apoptotic factors are regulated via alternative splicing, a process that allows for the production of discrete protein isoforms with often distinct functions from a common mRNA precursor. The abundance of apoptosis genes that are alternatively spliced and the often antagonistic roles of the generated protein isoforms strongly imply that alternative splicing is a crucial mechanism for regulating life and death decisions. Importantly, modulation of isoform production of cell death proteins via pharmaceutical manipulation of alternative splicing may open up new therapeutic avenues for the treatment of disease.     

RNA elongation by RNA polymerase II is not inhibited by N-ethylmaleimide or iodoacetamide.

The elongation of RNA by RNA polymerase II is not inhibited by N-ethylmaleimide or iodoacetamide. Three systems were studied: a soluble chromatin, purified RNA polymerase II with DNA, and a HeLa cell extract with an adenovirus 2 promoter sequence.     

Modulation of alternative pre-mRNA splicing in vivo by pinin

Pre-mRNA splicing occurs in a large macromolecular RNA-protein complex called the spliceosome. The major components of the spliceosome include snRNP and SR proteins. We have previously identified an SR-like protein, pinin (pnn), which is localized not only in nuclear speckles but also at desmosomes. The nuclear localization of pnn is a dynamic process because pnn can be found not only with SR proteins in nuclear speckles but also in enlarged speckles following treatment of cells with RNA polymerase II inhibitors, DRB, and alpha-amanitin. Using adenovirus E1A and chimeric calcitonin/dhfr construct as a splicing reporter minigene in combination with cellular cotransfection, we found that pnn regulates alternative 5(') and 3(') splicing by decreasing the use of distal splice sites. Regulation of 5(') splice site choice was also observed for RNPS1, a general splicing activator that interacts with pnn in nuclear speckles. The regulatory ability of pnn in alternative 5(') splicing, however, was not dependent on RNPS1 and a pnn mutant, lacking the N-terminal 167 amino acids, behaved like a dominant negative species, inhibiting E1A splicing when applied in splicing assays. These results provide direct evidence that pnn functions as a splicing regulator which participates itself directly in splicing reaction or indirectly via other components of splicing machinery.     

Bigger is not always better: when brains get smaller

Many studies assume that an increase in brain size is beneficial. However, the costs of producing and maintaining a brain are high, and we argue that brain size should be secondarily reduced by natural selection whenever the costs outweigh the benefits. Our results confirm this by showing that brain size is subject to bidirectional selection. Relative to the ancestral state, brain size in bats has been reduced in fast flyers, while it has increased in manoeuvrable flyers adapted to flight in complex habitats. This study emphasizes that brain reduction and enlargement are equally important, and they should both be considered when investigating brain size evolution.     

More is not always better: the genetic constraints of polyploidy

Polyploid cells are a characteristic feature of certain human tissues, and notably many cancers. In a systematic genomic screen in yeast, Storchová and co-workers identified the genetic requirements of tetraploidy. Surprisingly, they showed that only three connected pathways are essential for the viability of tetraploid yeast cells. These data provide exciting new targets that might be essential specifically in polyploid cancer cells.     

The apoptosis-promoting factor TIA-1 is a regulator of alternative pre-mRNA splicing

We report here that the apoptosis-promoting protein TIA-1 regulates alternative pre-mRNA splicing of the Drosophila melanogaster gene male-specific-lethal 2 and of the human apoptotic gene Fas. TIA-1 associates selectively with pre-mRNAs that contain 5' splice sites followed by U-rich sequences. TIA-1 binding to the U-rich stretches facilitates 5' splice site recognition by U1 snRNP. This activity is critical for activation of the weak 5' splice site of msl-2 and for modulating the choice of splice site partner in Fas. Structural and functional similarities with the Saccharomyces cerevisiae splicing factor Nam8 suggest striking evolutionary conservation of a mechanism of pre-mRNA splicing regulation that controls biological processes as diverse as meiosis in yeast, dosage compensation in fruit flies, or programmed cell death in humans.     

Regulation of alternative pre-mRNA splicing by the ERK MAP-kinase pathway

Differential gene expression through alternative pre-mRNA splicing is crucial to various physiological and pathological conditions. Upon activation of B and T lymphocytes during an immune response, variant isoforms of the cell surface molecule CD44 are generated by alternative pre-mRNA splicing. We show here that in primary mouse T cells as well as in the murine LB-17 T-cell line upregulation of variant CD44 mRNA species upon T-cell activation requires activation of the MEK-ERK pathway. By employing mutant signaling molecules and a novel luciferase-based splice reporter system we demonstrate that the Ras-Raf-MEK-ERK signaling cascade, but not the p38 MAP-kinase pathway, activates a mechanism that retains variant CD44 exon v5 sequence in mature mRNA. The findings demonstrate that a highly conserved pleiotropic signaling pathway links extracellular cues to splice regulation, providing an avenue for tissue-specific, developmental or pathology-associated splicing decisions.     

Blood coagulation and alternative pre-mRNA splicing: an overview

Throughout the 20th century, great advances were made in understanding of how blood coagulation occurs, what physiological and biochemical mechanisms are responsible for its regulation, and what genes and their protein products comprise the essential components of the hemostatic network. Recently, complete sequencing of the human genome revealed that the structural diversity of higher eukaryotes cannot be solely attributed to the number of protein-encoding genes, whereas tools of molecular biology helped establish that pre-mRNAs produced by most protein-encoding genes undergo alternative splicing, a mechanism that enables production of multiple protein isoforms by a single gene. Research in the field of thrombosis and hemostasis revealed that the genes encoding several critical proteins at various junctures of the coagulation cascade produce alternatively spliced protein isoforms with distinct structural and biochemical characteristics, revealing a principally novel dimension in the regulation of blood clotting and, possibly, a few novel therapeutic approaches to treatment of abnormal hemostasis. This review summarizes recently published data pertaining to biosynthesis of the alternatively spliced isoforms of tissue factor (TF, or coagulation factor III), tissue factor pathway inhibitor (TFPI), and coagulation factor XI (FXI), and discusses future directions of this continuously evolving area of biomedical research, with an emphasis on molecular mechanics responsible for regulation of constitutive as well as alternative pre-mRNA splicing.     

Selenotrisulfide inhibits initiation by RNA polymerase II but not elongation

We previously reported that RNA polymerase II (purified from wheat germ) is inhibited by selenotrisulfides, the products of the reaction of selenite with sulfhydryl compounds [Frenkel, Walcott, and Middleton, Molecular Pharmacology 31, 112 (1987)]. We have now found that the initiation stage of the reaction is inhibited by selenotrisulfide but the elongation stage of the reaction is not. The actual start of the RNA chain is not inhibited by the selenotrisulfide, but rather the formation of the enzyme-DNA binary complex. Selenotrisulfide has a similar differential effect on initiation and elongation by RNA polymerase II from HeLa cells; in contrast, with E. coli RNA polymerase, it inhibits elongation as well.