Current Chemical Biology - Volume 5, Issue 2, 2011

Post-transcriptional control in the cytoplasm is an important arm of gene expression. Regulation of messenger RNAs (mRNAs) largely occurs in specialized structures collectively termed RNA granules. These large ribonucleoproteins are microscopically visible, although hard to purify biochemically, and are believed to serve as the functional units for mRNA transport, storage, repression and decay. It was during the study of mRNA cytoplasmic transport, a few decades ago, that RNA granules were identified by several groups. Current evidence indicates that this level of supramolecular organization is not restricted to complex vertebrate cells, but it is rather a widespread phenomenon. The presence of RNA granules involved in several functions has been reported in highly differentiated cells as well as simple unicellular organisms, ranging from trypanosomatids and yeast, to insect and vertebrate oocytes and neurons. In the latter cell type, both the somatodendritic compartment and the axon are loaded with a plethora of RNA granules of distinct composition. RNA granules are believed to self-assemble to coordinate the translational activation, subcellular localization and silencing of mRNAs with a common fate. More recent is the discovery of RNA granules specific to the stress response, which is a complex cell reaction to ensure survival upon noxious conditions. During acute stress, the assembly of the so-called Stress Granules (SGs) takes place. Evidence has been adduced that SGs help remodeling of the translational apparatus to allow translation of protective molecules, although they do not control the global translational silencing triggered by cell stress. SGs are related to another kind of RNA granules, termed Processing Bodies (PBs). SGs and PBs are referred to as mRNA silencing foci, as they harbor mRNAs that are not being translated. Similar structures exist in unicellular organisms where gene expression is regulated mostly at the post-transcriptional level. This series of mini-reviews on RNA granules in health and disease focus on hot topics that highlight the relevance of RNA granules in distinct cell types and processes. In the chapter entitled “Common themes in RNA subcellular transport, stress granule formation and abnormal protein aggregation”, Bensenor and co-workers summarize the mechanisms for mRNA transport and localization, which are operative in embryos and somatic cells. RNA transport granules contain several RNA binding proteins that are obligate SG components. In addition, SGs share common features with aggresomes and other abnormal protein aggregates associated to neurodegeneration. The presence of aggregates containing the SG components TAR DNA-binding protein (TDP-43) or Fused-in-Sarcoma/Translocated-in-Liposarcoma (FUS/TLS) is a pathological hallmark of frontotemporal lobar degeneration (FTLD), Amyotrophic Lateral Sclerosis (ALS) and Alzheimer's disease. The shared presence of the aforementioned SG components, as well as additional functional similarities between these cytoplasmic aggregates are herein described. Current and future findings will open new avenues of research aimed to understand the relevance of SGs in the pathogenesis of these diseases. In the chapter focused on “RNA Metabolism in Neurodegenerative Diseases”, Volkening and Strong summarize the specific role of RNA granules on RNA regulation in mammalian neurons and discuss in which way recently described mutations in TDP-43 and FUS/TLS, contribute to ALS and FTLD by altering the normal metabolism of specific RNA molecules. The field is flourishing and future research will yield light on the affected cellular machineries, thus helping the design of therapies to handle each particular disease mechanism. Arguing a widespread accepted idea, mRNA translation is also important in the axon, which comprises a relative large fraction of the total neuronal cell volume. Addressing this intriguing aspect of RNA regulation in neurons, Canclini and co-workers focused on “The axonal transcriptome: RNA localization and function”. Their review highlights the fact that regulation of local translation at the axon involves specific RNA granules, and emphasizes the need of motor-mediated transport and the participation of micro RNAs. Evidence is presented that a number of RNAs in peripheral axons are apparently derived from neighboring glial cells, and this is particularly intriguing during axon regeneration after nerve injury. Finally, Casola's section -“RNA Granules Living a Post-transcriptional Life: the Trypanosomes' Case”- is focused on the metabolism of RNA in trypanosomes. These organisms are the etiological agent of Chagas and Sleeping Sickness Diseases, which represent an important jeopardy for human health worldwide. This chapter summarizes how gene expression in trypanosomes is controlled mostly by mRNA degradation, silencing and translation repression. Not surprisingly, these processes involve the participation of a number of macromolecular aggregates related in composition and function to SGs, PBs and RNA granules from higher eukaryotes. However, the speculation is that subtle differences will help to develop rationale therapies to attack the parasites with minimal damage to the host cell.

Control of protein synthesis and quality are critical steps to support eukaryotic cells' maintenance and survival. Two very distinctive mechanisms emerge as key checkpoints of protein synthesis regulation. The first one is the delivery of mRNA molecules, packed into ribonucleoprotein (mRNP) granules, to specific subcellular regions in order to restrict protein synthesis to distinct cytoplasmic domains. In the presence of cellular stress or injury, translation is aborted by sequestering mRNA molecules into a sub-type of RNP particles called stress granules (SGs). The second mechanism deals with the folding state and further processing of synthesized proteins. Misbehavior of a particular protein, affecting its processing, functioning, and/or conformation can cause the formation of protein inclusions called aggresomes. Interestingly, self-aggregation of abnormal proteins is one of the leading causes of neurodegenerative disorders. Recently, intracellular transport directed by microtubule-motors, has emerged as an important step in the assembly and dynamic of SGs and aggresomes. This mechanism allows for a precise temporal and spatial trafficking of RNA and protein complexes. Furthermore, it facilitates the regulation of the RNA silencing domains and targets abnormal protein aggregates for degradation. In this review we will explore the specific and common features of mRNA transport and of SG and aggresome formation, and will provide details on the role of the microtubule network and motors in their movement and dynamics.

RNA metabolism is a vital process through which RNA is produced, transported, regulated, stored, and translated or degraded. Recently, the discoveries of mutations in key RNA binding proteins involved in several human neuronal based diseases have firmly placed the process of RNA metabolism as central to disease etiology. This review first recaps the process of RNA metabolism in the mammalian neuron and describes the roles of RNA granules in this process. Using the recently described alterations in TAR DNA binding protein (TDP-43) and fused in sarcoma/translocated in liposarcoma (FUS/TLS) in amyotrophic lateral sclerosis (ALS) and frontal temporal lobar degeneration (FTLD), we discuss how RNA binding protein abnormalities can affect RNA metabolism. We then discuss two additional RNA based mechanisms distinct from alterations in RNA binding protein function that impact RNA metabolism and result in disease. Cumulatively, these observations provide strong support for the hypothesis that alterations in RNA metabolism can lead to neurodegenerative disease, including ALS.

Neurons are highly polarized cells, often with very long processes, which can comprise up to 95% of the cytoplasm. This raises the issue of transporting and turnover of proteins transport. In addition to fast axonal transport, axonal translation has been proposed to be responsible for producing at least some axonal proteins. In multiple experimental models, local synthesis of several proteins has been demonstrated in axons, growth cones, and nerve terminals. Multiple lines of evidence are consistent with the existence of axonal rRNAs, mRNAs, tRNAs, and micro RNAs and micro RNA regulatory proteins (RISC complex). Multiple signalling pathways, including mTOR, may regulate axonal translation. The origin of axonal RNAs is often attributed to transport from the neuronal soma, but evidence supporting cell-to-cell transport from Schwann cells to axons as a source of at least some axonal RNAs is emerging. This intercellular transport will be discussed in the context of normal and regenerative conditions.

Trypanosomes are protozoan parasites responsible for recalcitrant infectious diseases such as Sleeping sickness and Chagas disease in Africa and America, respectively. Their complex life-cycles are accompanied by alternation of forms specific of the insect vectors and vertebrate hosts, each with different metabolic and structural requirements. Unlike most other eukaryotes, these single-cell microorganisms seem to control the expression of protein-coding genes mostly by mRNA degradation, silencing and translation efficiency. Recent evidence showed that genuine cytoplasmic Stress Granules are formed as a response to heat stress in Trypanosoma brucei, basically formed by stalled translation initiation complexes on mRNA. On the other hand, Processing bodies (P bodies) are constitutive components of cytoplasmic mRNA metabolism in trypanosomes, which could have an important role in translational repression. During physiological starvation conditions in trypanosomes, components from P bodies fuse with other ribonucleoprotein complexes to form mRNA granules, where transcripts are stored and protected from degradation in a quiescent state. Other novel types of foci with unknown function that are related to RNA metabolism can be found in these parasites, namely heat-induced granules containing the 5' to 3' exoribonuclease XRNA, and starvation-induced granules containing transfer RNA halves. Thus, trypanosomes make use of non-membranous structures as a strategy to compartmentalize ribonucleoprotein complexes in the cytoplasm, aiding to cope with stressful situations avoiding mRNA translation or degradation. The relevance of stressinduced foci in trypanosomes has yet to be scored, although recent evidence suggests that these cytoplasmic organelles are required for survival under adverse growing conditions.

Although membrane proteins constitute more than 20% of the total proteins, the structures of only a few are known in detail. An important group of integral membrane proteins are ion-transporting ATPases of the P-type family, which share the formation of an acid-stable phosphorylated intermediate as part of their reaction cycle. There are several crystal structures of the sarcoplasmic reticulum Ca2+ pump (SERCA) revealing different conformations, and recently, crystal structures of the H+-ATPase and the Na+/K+-ATPase were reported as well. However, there are no atomic resolution structures for other P-type ATPases including the plasma membrane calcium pump (PMCA), which is integral to cellular Ca2+ signaling. Crystallization of these proteins is challenging because there is often no natural source from which the protein can be obtained in large quantities, and the presence of multiple isoforms in the same tissue further complicates efforts to obtain homogeneous samples suitable for crystallization. Alternative techniques to study structural aspects and conformational transitions in the PMCAs (and other P-type ATPases) have therefore been developed. Specifically, information about the structure and assembly of the transmembrane domain of an integral membrane protein can be obtained from an analysis of the lipid-protein interactions. Here, we review recent efforts using different hydrophobic photo-labeling methods to study the non-covalent interactions between the PMCA and surrounding phospholipids under different experimental conditions, and discuss how the use of these lipid probes can reveal valuable information on the membrane organization and conformational state transitions in the PMCA, Na+/K+-ATPase, and other P-type ATPases.

Neurabin 1 and neurabin 2/spinophilin were discovered in late 1990s on the basis of their F-actin-binding and protein phosphatase 1 catalytic subunit (PP1c) modulation activities. The neurabins are proteins with modular domains, such as one F-actin-, a receptor- (not found in neurabin 1) and a PP1c-binding domains, a PSD95/DLG/zo-1, three coiledcoil domains and a sterile alpha motif (not found in spinophilin) that govern protein-protein interactions. In the past years, the roles of neurabins have evolved from PP1c-regulatory subunits to important scaffolds binding to a rapidly growing list of cellular proteins. Among the spinophilin and neurabin 1 interactomes, some partner proteins are involved in G-proteincoupled receptor (GPCR) signalling. The most significant difference is that spinophilin, but not neurabin 1, interacts with and plays a specific and direct role in the regulation of at least the α-adrenergic (AR), muscarinic-acetylcholine (m- AChR), dopamine D2 and mu opioid receptors. In contrast, the two scaffolding proteins bind to different members of the regulator of G-protein signalling (RGS) familly. Spinophilin antagonizes multiple functions of arrestin and G-proteincoupled receptor kinase 2 in α-AR signalling. Moreover, spinophilin and neurabin 1 form a functional pair of opposing regulators that reciprocally regulate signalling intensity by the α1-AR. To date, the data on the regulation of GPCR signalling by neurabin 1 are very sparse and the reciprocal regulation seems not to be a general phenomenon. Several studies make on the α1-AR and the m-AChR suggest that spinophilin may selectively regulate Gq-coupled receptor signalling. This review highlights information regarding spinophilin and neurabin 1 function in GPCR signalling.

Anionic antimicrobial peptides (AAMPs) are important components of the innate immune system and here, we review recent research into these peptides. As examples, we refer to two major families of AAMPs: those that adopt cysteine stabilized β-sheet structure, such as plant cyclotides, and those that are encrypted in larger proteins, such as bovine kappacins. This review shows that AAMPs use a diverse range of antimicrobial mechanisms, which in some cases, such as ovine SAAPs, involves translocation across the membrane to utilize intracellular sites of antimicrobial action. In other cases, the membrane itself is the major site of action for AAMPs as in the case of cyclotides, which permeabilize membranes via pore formation, and human β-defensins, which induce the disintegration of membranes via carpet-type mechanisms prior to action against intracellular targets. These AAMPs show the potential for development as antiviral agents, insecticides, topical biocides, fertility control agents, therapeutically useful antibiotics, decontaminants food preservatives and agents against dental and periodontal diseases.