RNA-splicing endonuclease structure and function

The RNA-splicing endonuclease is an evolutionarily conserved enzyme responsible for the excision of introns from nuclear transfer RNA (tRNA) and all archaeal RNAs. Since its first identification from yeast in the late 1970s, significant progress has been made toward understanding the biochemical mechanisms of this enzyme. Four families of the splicing endonucleases possessing the same active sites and overall architecture but with different subunit compositions have been identified. Two related consensus structures of the precursor RNA splice sites and the critical elements required for intron excision have been established. More recently, a glimpse was obtained of the structural mechanism by which the endonuclease recognizes the consensus RNA structures and cleaves at the splice sites. This review summarizes these findings and discusses their implications in the evolution of intron removal processes. Received 24 August 2007; received after revision 24 November 2007; accepted 27 November 2007     

Structural organization and cellular localization of tuftelin-interacting protein 11 (TFIP11)

Tuftelin-interacting protein (TFIP11) was first identified in a yeast two-hybrid screening as a protein interacting with tuftelin. The ubiquitous expression of TFIP11 suggested that it might have other functions in non-dental tissues. TFIP11 contains a G-patch, a protein domain believed to be involved in RNA binding. Using a green fluorescence protein tag, TFIP11 was found to locate in a novel subnuclear structure that we refer to as the TFIP body. An in vivo splicing assay demonstrated that TFIP11 is a novel splicing factor. TFIP11 diffuses from the TFIP body following RNase A treatment, suggesting that the retention of TFIP11 is RNA dependent. RNA polymerase II inhibitor (-amanitin and actinomycin D) treatment causes enlargement in size and decrease in number of TFIP bodies, suggesting that TFIP bodies perform a storage function rather than an active splicing function. The TFIP body may therefore represent a new subnuclear storage compartment for splicing components.Received 8 December 2004; received after revision 27 January 2005; accepted 8 March 2004The nucleotide sequence for the cDNA to mouse TFIP11 (previously known as TIP39 and TIP39kDa) has been submitted to Gen- Bank^TM/ EBI Data Bank with accession numbers AF290474 and NM_018783. The accession number for the human TFIP11 homologueis NM_012143.     

The family of iron responsive RNA structures regulated by changes in cellular iron and oxygen

The life of aerobes is dependent on iron and oxygen for efficient bioenergetics. Due to potential risks associated with iron/oxygen chemistry, iron acquisition, concentration, storage, utilization, and efflux are tightly regulated in the cell. A central role in regulating iron/oxygen chemistry in animals is played by mRNA translation or turnover via the iron responsive element (IRE)/iron regulatory protein (IRP) system. The IRE family is composed of three-dimensional RNA structures located in 3′ or 5′ untranslated regions of mRNA. To date, there are 11 different IRE mRNAs in the family, regulated through translation initiation or mRNA stability. Iron or oxidant stimuli induce a set of graded responses related to mRNA-specific IRE substructures, indicated by differential responses to iron in vivo and binding IRPs in vitro. Molecular effects of phosphorylation, iron and oxygen remain to be added to the structural information of the IRE-RNA and IRP repressor in the regulatory complex. Received 21 April 2007; received after revision 13 July 2007; accepted 2 August 2007     

Functional aspects of animal microRNAs

MicroRNAs (miRNAs) are a recently discovered family of small regulatory molecules that function by modulating protein production. There are approximately 500 known mammalian miRNA genes, and each miRNA may regulate hundreds of different protein-coding genes. Mature miRNAs bind to target mRNAs in a protein complex known as the miRNA-induced silencing complex (miRISC), sometimes referred to as the miRNP (miRNA-containing ribonucleoprotein particles), where mRNA translation is inhibited or mRNA is degraded. These actions of miRNAs have been shown to regulate several developmental and physiological processes including stem cell differentiation, haematopoiesis, cardiac and skeletal muscle development, neurogenesis, insulin secretion, cholesterol metabolism and the immune response. Furthermore, aberrant expression has been implicated in a number of diseases including cancer and heart disease. The role of miRNAs in these developmental, physiological and pathological processes will be reviewed. Received 3 August 2007; received after revision 3 October 2007; accepted 5 October 2007     

The interface of protein-protein complexes: Analysis of contacts and prediction of interactions

Specific protein-protein interactions are essential for cellular functions. Experimentally determined three-dimensional structures of protein-protein complexes offer the possibility to characterize binding interfaces in terms of size, shape and packing density. Comparison with crystal-packing interfaces representing nonspecific protein-protein contacts gives insight into how specific binding differs from nonspecific low-affinity binding. An overview is given on empirical structural rules for specific protein-protein recognition derived from known complex structures. Although single parameters such as interface size, shape or surface complementary show clear trends for different interface types, each parameter alone is insufficient to fully distinguish between specific versus crystal-packing contacts. A combination of interface parameters is, however, well suited to characterize a specific interface. This knowledge provides us with the essential ingredients that make up a specific protein recognition site. It is also of great value for the prediction of protein binding sites and for the evaluation of predicted complex structures. Received 1 October 2007; received after revision 9 November 2007; accepted 9 November 2007     

Emerging mechanisms and consequences of calcium regulation of alternative splicing in neurons and endocrine cells

Alternative splicing contributes greatly to proteomic complexity. How it is regulated by external stimuli to sculpt cellular properties, particularly the highly diverse and malleable neuronal properties, is an underdeveloped area of emerging interest. A number of recent studies in neurons and endocrine cells have begun to shed light on its regulation by calcium signals. Some mechanisms include changes in the trans-acting splicing factors by phosphorylation, protein level, alternative pre-mRNA splicing, and nucleocytoplasmic redistribution of proteins to alter protein–RNA or protein–protein interactions, as well as modulation of chromatin states. Importantly, functional analyses of the control of specific exons/splicing factors in the brain point to a crucial role of this regulation in synaptic maturation, maintenance, and transmission. Furthermore, its deregulation has been implicated in the pathogenesis of neurological disorders, particularly epilepsy/seizure. Together, these studies have not only provided mechanistic insights into the regulation of alternative splicing by calcium signaling but also demonstrated its impact on neuron differentiation, function, and disease. This may also help our understanding of similar regulations in other types of cells.     

Alternative splicing of Bcl-2-related genes: functional consequences and potential therapeutic applications

Apoptosis is a morphologically distinct form of cell death. It is executed and regulated by several groups of proteins. Bcl-2 family proteins are the main regulators of the apoptotic process acting either to inhibit or promote it. More than 20 members of the family have been identified so far and most have two or more isoforms. Alternative splicing is one of the major mechanisms providing proteomic complexity and functional diversification of the Bcl-2 family proteins. Pro- and anti-apoptotic Bcl-2 family members should function in harmony for the regulation of the apoptosis machinery, and their relative levels are critical for cell fate. Any mechanism breaking down this harmony by changing the relative levels of these antagonistic proteins could contribute to many diseases, including cancer and neurodegenerative disorders. Recent studies have shown that manipulation of the alternative splicing mechanisms could provide an opportunity to restore the proper balance of these regulator proteins. This review summarises current knowledge on the alternative splicing products of Bcl-2-related genes and modulation of splicing mechanisms as a potential therapeutic approach.Received 5 January 2004; received after revision 31 March 2004; accepted 6 April 2004     

Role of Sam68 as an adaptor protein in signal transduction

Sam68, the substrate of Src in mitosis, belongs to the family of RNA binding proteins. Sam68 contains consensus sequences to interact with other proteins via specific domains. Thus, Sam68 has various proline-rich sequences to interact with SH3 domain-containing proteins. Moreover, Sam68 also has a C-terminal domain rich in tyrosine residues that is a substrate for tyrosine kinases. Tyrosine phosphorylation of Sam68 promotes its interaction with SH2 containing proteins. The association of Sam68 with SH3 domain-containing proteins, and its tyrosine phosphorylation may negatively regulate its RNA binding activity. The presence of these consensus sequences to interact with different domains allows this protein to participate in signal transduction pathways triggered by tyrosine kinases. Thus, Sam68 participates in the signaling of T cell receptors, leptin and insulin receptors. In these systems Sam68 is tyrosine phosphorylated and recruited to specific signaling complexes. The participation of Sam68 in signaling suggests that it may function as an adaptor molecule, working as a dock to recruit other signaling molecules. Finally, the connection between this role of Sam68 in protein-protein interaction with RNA binding activity may connect signal transduction of tyrosine kinases with the regulation of RNA metabolism.Received 16 July 2004; received after revision 12 August 2004; accepted 18 August 2004     

Differential protein expression in pancreatic islets after treatment with an imidazoline compound

The effects of an imidazoline compound (BL11282) on protein expression in rat pancreatic islets were investigated with a proteomic approach. The compound increases insulin release selectively at high glucose concentrations and is therefore of interest in type 2 diabetes. Whole cell extracts from isolated drug-treated and native pancreatic rat islets were compared after separation by 2-D gel electrophoresis. Differentially expressed proteins were identified by mass spectrometry; 15 proteins were selectively up-regulated and 7 selectively down-regulated in drug-treated islets. Of special interest among the differentially expressed proteins are those involved in protein folding (Hsp60, protein disulfide isomerase, calreticulin), Ca²⁺ binding (calgizzarin, calcyclin and annexin I) and metabolism or signalling (pyruvate kinase, alpha enolase and protein kinase C inhibitor 1). Received 19 March 2007; received after revision 11 April 2007; accepted 11 April 2007     

Hcc-1 is a novel component of the nuclear matrix with growth inhibitory function

Hcc-1 is a novel nuclear protein containing the SAF-box DNA-binding domain. It binds to both double-stranded and single-stranded DNA with higher affinity for the single-stranded form. In addition, it also binds specifically to scaffold/matrix attachment region DNA. These nucleic acid-binding characteristics suggest a potential function for Hcc-1 as a component of the heterogeneous ribonucleoprotein complex. Using a yeast two-hybrid screen, two DEAD-box RNA helicases, BAT1 and DDX39, were identified as proteins that interact with Hcc-1. Interactions with these RNA helicases suggested a role for Hcc-1 in nucleic acid biogenesis. Expression of Hcc-1 in the HEK293 cell line resulted in a slower growth rate compared to controls (p = 0.0173) and an accumulation of cells at the G2/M phase (p = 0.0276 compared to control HEK293 cells). Taken together, these results suggest a role for Hcc-1 in growth regulation and nucleic acids metabolism.Received 13 May 2004; received after revision 30 June 2004; accepted 6 July 2004     

A structural view of translation initiation in bacteria

The assembly of the protein synthesis machinery occurs during translation initiation. In bacteria, this process involves the binding of messenger RNA(mRNA) start site and fMet-tRNA^fMet to the ribosome, which results in the formation of the first codon-anticodon interaction and sets the reading frame for the decoding of the mRNA. This interaction takes place in the peptidyl site of the 30S ribosomal subunit and is controlled by the initiation factors IF1, IF2 and IF3 to form the 30S initiation complex. The binding of the 50S subunit and the ejection of the IFs mark the irreversible transition to the elongation phase. Visualization of these ligands on the ribosome has been achieved by cryo-electron microscopy and X-ray crystallography studies, which has helped to understand the mechanism of translation initiation at the molecular level. Conformational changes associated with different functional states provide a dynamic view of the initiation process and of its regulation. Received 16 July 2008; received after revision 31 August 2008; accepted 10 September 2008 A. Simonetti, S. Marzid: These authors contributed equally to this work.     

Functional interaction between calsequestrin and ryanodine receptor in the heart

Evidence obtained in the last two decades indicates that calsequestrin (CSQ2), as the major Ca²⁺-binding protein in the sarcoplasmic reticulum of cardiac myocytes, communicates changes in the luminal Ca²⁺ concentration to the cardiac ryanodine receptor (RYR2) channel. This review summarizes the major aspects in the interaction between CSQ2 and the RYR2 channel. The single channel properties of RYR2 channels, discussed here in the context of structural changes in CSQ2 after Ca²⁺ binding, are particularly important. We focus on five important questions concerning: (1) the method for reliable detection of CSQ2 on the reconstituted RYR2 channel complex; (2) the power of the procedure to strip CSQ2 from the RYR2 channel complex; (3) structural changes in CSQ2 upon binding of Ca²⁺ which cause CSQ2 dissociation; (4) the potential role of CSQ2-independent regulation of the RYR2 activity by luminal Ca²⁺; and (5) the vizualization of CSQ2 dissociation from the RYR2 channel complex on the single channel level. We discuss the potential sources of the conflicting experimental results which may aid detailed understanding of the CSQ2 regulatory role. Although we mainly focus on the cardiac isoform of the proteins, some aspects of more extensive work carried out on the skeletal isoform are also discussed.     

The regulation of ion channels and transporters by glycolytically derived ATP

Glycolysis is an evolutionary conserved metabolic pathway that provides small amounts of energy in the form of ATP when compared to other pathways such as oxidative phosphorylation or fatty acid oxidation. The ATP levels inside metabolically active cells are not constant and the local ATP level will depend on the site of production as well as the respective rates of ATP production, diffusion and consumption. Membrane ion transporters (pumps, exchangers and channels) are located at sites distal to the major sources of ATP formation (the mitochondria). We review evidence that the glycolytic complex is associated with membranes; both at the plasmalemma and with membranes of the endo/sarcoplasmic reticular network. We examine the evidence for the concept that many of the ion transporters are regulated preferentially by the glycolytic process. These include the Na⁺/K⁺-ATPase, the H⁺-ATPase, various types of Ca²⁺-ATPases, the Na⁺/H⁺ exchanger, the ATP-sensitive K⁺ channel, cation channels, Na⁺ channels, Ca²⁺ channels and other channels involved in intracellular Ca²⁺ homeostasis. Regulation of these pumps, exchangers and ion channels by the glycolytic process has important consequences in a variety of physiological and pathophysiological processes, and a better understanding of this mode of regulation may have important consequences for developing future strategies in combating disease and developing novel therapeutic approaches. Received 20 July 2007; received after revision 30 July 2007; accepted 17 August 2007     

Profiling of the secreted proteins during 3T3-L1 adipocyte differentiation leads to the identification of novel adipokines

Adipose tissue is an endocrine organ capable of secreting a number of adipokines with a role in the regulation of adipose tissue and whole-body metabolism. We used two-dimensional gel electrophoresis combined with mass spectrometry to profile the secreted proteins from (pre)adipocytes. The culture medium of 3T3-L1 cells during adipocyte differentiation was screened, and 41 proteins that responded to blocking of secretion by 20°C treatment and/or brefeldin A treatment were identified. Prohibitin, stress-70 protein, and adhesion-regulating molecule 1 are reported for the first time as secreted proteins. In addition, procollagen C-proteinase enhancer protein, galectin-1, cyclophilin A and C, and SF20/IL-25 are newly identified as adipocyte secreted factors. Secretion profiles indicated a dynamic environment including an actively remodeling extracellular matrix and several factors involved in growth regulation.Received 15 June 2004; received after revision 26 July 2004; accepted 2 August 2004