Dbp5 — From nuclear export to translation

The DEAD-box RNA helicase Dbp5 is an essential and conserved mRNA export factor which functions in the ATP dependent remodeling of RNA/protein complexes. As such it displaces mRNA bound proteins at the cytoplasmic site of the nuclear pore complex. For the regulation of its RNA-dependent ATPase activity during late steps of nuclear transport, Dbp5 requires the nucleoporin Nup159 and its cofactors Gle1 and IP₆. In addition to its role in mRNA export, a second important function of Dbp5 was identified in translation termination, where it acts together with eRF1 once the translation machinery has reached the stop codon. Similar to mRNA export, this function also requires Gle1–IP₆, however, the counterpart of Nup159 is still missing. Potential other functions of the nucleo-cytoplasmic protein Dbp5 are discussed as well as its substrate specificity and details in its regulatory cycle that are based on recent biochemical and structural characterization. This article is part of a Special Issue entitled: The Biology of RNA helicases — Modulation for life.     

RNA templating of molecular assembly and covalent modification patterning in early molecular evolution and modern biosystems

The Direct RNA Template (DRT) hypothesis proposes that an early stage of genetic code evolution involved RNA molecules acting as stereochemical recognition templates for assembly of specific amino acids in sequence-ordered arrays, providing a framework for directed covalent peptide bond formation. It is hypothesized here that modern biological precedents may exist for RNA-based structural templating with functional analogies to hypothetical DRT systems. Beyond covalent molecular assembly, an extension of the DRT concept can include RNA molecules acting as dynamic structural template guides for the specific non-covalent assembly of multi-subunit complexes, equivalent to structural assembly chaperones. However, despite numerous precedents for RNA molecules acting as scaffolds for protein complexes, true RNA-mediated assembly chaperoning appears to be absent in modern biosystems. Another level of function with parallels to a DRT system is possible if RNA structural motifs dynamically guided specific patterns of catalytic modifications within multiple target sites in a pre-formed polymer or macromolecular complex. It is suggested that this type of structural RNA templating could logically play a functional role in certain areas of biology, one of which is the glycome of complex organisms. If any such RNA templating processes are shown to exist, they would share no necessary evolutionary relationships with events during early molecular evolution, but may promote understanding of the practical limits of biological RNA functions now and in the ancient RNA World. Awareness of these formal possibilities may also assist in the current search for functions of extensive non-coding RNAs in complex organisms, or for efforts towards artificial rendering of DRT systems.     

Determinants of RNA Binding and Translational Repression by the Bicaudal-C Regulatory Protein

Bicaudal-C (Bic-C) RNA binding proteins function as important translational repressors in multiple biological contexts within metazoans. However, their RNA binding sites are unknown. We recently demonstrated that Bic-C functions in spatially regulated translational repression of the xCR1 mRNA during Xenopus development. This repression contributes to normal development by confining the xCR1 protein, a regulator of key signaling pathways, to specific cells of the embryo. In this report, we combined biochemical approaches with in vivo mRNA reporter assays to define the minimal Bic-C target site within the xCR1 mRNA. This 32-nucleotide Bic-C target site is predicted to fold into a stem-loop secondary structure. Mutational analyses provided evidence that this stem-loop structure is important for Bic-C binding. The Bic-C target site was sufficient for Bic-C mediated repression in vivo. Thus, we describe the first RNA binding site for a Bic-C protein. This identification provides an important step toward understanding the mechanisms by which evolutionarily conserved Bic-C proteins control cellular function in metazoans.     

RNA secondary structure and in vitro translation efficiency

Cell-free protein synthesis is a promising technology featuring many advantages compared to in vivo expression techniques. However, most proteins are still synthesized in vivo due to relatively low protein yields commonly achieved in vitro, especially in the batch mode of reaction. In Escherichia coli S30 extract-based cell-free systems protein yields are supposed to be partially limited by a secondary structure formation of the mRNA. In this study we checked promising members of various classes of RNA chaperones and several different RNA helicases on their ability to enhance in vitro translation. The data clearly show that the addition of none of these factors provides a general solution to the problem. However, protein yields can be increased in presence of a microRNA hybridizing with the 5′ untranslated region of mRNAs, possibly by inducing structural changes improving accessibility of the Shine Dalgarno sequence for the ribosomes.     

P‐bodies and their functions during mRNA cell cycle: Mini‐review

P‐bodies (processing bodies) are observed in different organisms such as yeast, Caenorhabditis elegans and mammals. A typical eukaryotic cell contains several types of spatially formed granules, such as P‐bodies, stress granules and a variety of ribonucleoprotein bodies. These microdomains play important role in mRNA processing, including RNA interference, repression of translation and mRNA decay. The P‐bodies components as well as stress granules may play an important role in host defense against viral infection. The complete set of P‐bodies protein elements is still poor known. They contain conserved protein core limited to different organisms or to stress status of the cell. P‐bodies are related also to some neuronal mRNA granules as well as to maternal RNA granules or male germ cell granules. In this mini‐review, we focus on the structure of P‐bodies and their function in the mRNA utilization and processing because of the high mRNA's dynamics between different cellular compartments and its key role in modulation of gene expression. Copyright © 2012 John Wiley & Sons, Ltd.     

Proteins that interact with bacterial small RNA regulators

Small regulatory RNAs have been identified in a wide range of organisms, where they modify mRNA stability, translation or protein function. Small RNA regulators (sRNAs) either pair with mRNA targets or modify protein activities. Here we discuss current knowledge of the various proteins that interact with RNA regulators and review the physiologic implications of sRNA-protein complexes in DNA, RNA and protein metabolism, as well as in RNA and protein quality control in prokaryotes. Proteins that interact with the sRNAs can possess catalytic activity, induce conformational changes of the sRNA, or be sequestered by the sRNA to prevent the action of the protein.     

The DHH1/RCKp54 family of helicases: An ancient family of proteins that promote translational silencing

Translational control is a vital aspect of gene expression. Message specific translational repressors have been known for decades. Recent evidence, however, suggests that a general machinery exists that dampens the translational capacity of the majority of mRNAs. This activity has been best ascribed to a conserved family of RNA helicases called the DHH1/RCKp54 family. The function of these helicases is to promote translational silencing. By transitioning mRNA into quiescence, DHH1/RCKp54 helicases promote either mRNA destruction or storage. In this review we describe the known roles of these helicases and propose a mechanistic model to explain their mode of action. This article is part of a Special Issue entitled: The Biology of RNA helicases — Modulation for life.     

1H, 13C and 15N resonance assignments of RNA pyrophosphohydrolase RppH from Escherichia coli

The mRNA degradation is an important regulatory mechanism which controls gene expression by limiting the number of translation times. Previous studies demonstrated that this process is essential for organisms. Escherichia coli RNA pyrophosphohydrolase (RppH) is an enzyme that triggers mRNA degradation by removing the 5′ pyrophosphate, which is a rate-determining step. In order to understand the molecular mechanism of the biological function, the structural information of RppH is required. Herein, we report the resonance assignments of ¹H, ¹⁵N, ¹³C atoms of the E. coli RppH.     

RNA delivery by heat shock protein-70 into mammalian cells: a preliminary study

Heat shock proteins (Hsps) are ubiquitous molecular chaperones with indispensable roles in assisting protein folding and giving protection from proteotoxic environmental harm. Members of the 70-kDa heat shock protein family have been demonstrated to recognize and bind with distinguished RNA sequences, which function as determinants of eukaryotic mRNA stability. We have earlier identified the molecular domains involved in RNA-binding and characterized in detail the specificity, affinity and some regulatory aspects of this molecular interaction using various deletion mutants and homologues of Hsp70. We have shown that wild type, but not any of the tested truncated mutants of Hsp70, is efficiently taken up by P388 mouse macrophage cells. Here we addressed the question of whether Hsp70 is capable of delivering bound RNA into mammalian cells. Employing fluorescence and confocal microscopy, we demonstrated that full length Hsp70 facilitates the uptake of RNA molecules into the cytoplasm of mammalian cells. We propose that further optimization of this system might enable the development of a valuable tool to deliver RNA molecules, such as siRNA, dsRNA or other regulatory RNA sequences to probe or influence various regulatory processes in eukaryotic cells.     

Phosphorylation of the spinach chloroplast 24 kDa RNA-binding protein (24RNP) increases its binding to petD and psbA 3' untranslated regions

The chloroplast 24 kDa RNA binding protein (24RNP) from Spinacea oleracea is a nuclear encoded protein that binds the 3' untranslated region (3'UTR) of some chloroplast mRNAs and seems to be involved in some processes of mRNA metabolism, such as 3'UTR processing, maturation and stabilization. The 24RNP is similar to the 28RNP which is involved in the correct maturation of petD and psbA 3'UTRs, and when phosphorylated, decreases its binding affinity for RNA. In the present work, we determined that the recombinant 24RNP was phosphorylated in vitro either by an animal protein kinase C, a plant Ca(2+)-dependent protein kinase, or a chloroplastic kinase activity present in a protein extract with 3'-end processing activity in which the 24RNP is also present. Phosphorylation of 24RNP increased the binding capacity (B(max)) 0.25 time for petD 3'UTR, and three times for psbA 3'UTR; the affinity for P-24RNP only increased when the interaction with petD was tested. Competition experiments suggested that B(max), not K(d), might be a more important factor in the P-24RNP-3'UTR interaction. The data suggested that the 24RNP role in chloroplast mRNA metabolism may be regulated in vivo by changes in its phosphorylation status carried out by a chloroplastic kinase.     

When one is better than two: RNA with dual functions

The central dogma of biology, until not long ago, held that genetic information stored on DNA molecules was translated into the final protein products through RNA as intermediate molecules. Then, an additional level of complexity in the regulation of genome expression was added, implicating new classes of RNA molecules called non-coding RNA (ncRNA). These ncRNA are also often referred to as functional RNA in that, although they do not contain the capacity to encode proteins, do have a function as RNA molecules. They have been thus far considered as truly non-coding RNA since no ORF long enough to be considered, nor protein, have been associated with them. However, the recent identification and characterization of bifunctional RNA, i.e. RNA for which both coding capacity and activity as functional RNA have been reported, suggests that a definite categorization of some RNA molecules is far from being straightforward.Indeed, several RNA primarily classified as non-protein-coding RNA has been showed to hold coding capacities and associated peptides. Conversely, mRNA, usually regarded as strictly protein-coding, may act as functional RNA molecules. Here, we describe several examples of these bifunctional RNA that have been already characterized from bacteria to mammals. We also extend this concept to fortuitous acquisition of dual function in pathological conditions and to the recently highlighted duality between information carried by a gene and its pseudogenes counterparts.     

Protein domain definition should allow for conditional disorder

Abstract: Proteins are often classified in a binary fashion as either structured or disordered. However this approach has several deficits. Firstly, protein folding is always conditional on the physiochemical environment. A protein which is structured in some circumstances will be disordered in others. Secondly, it hides a fundamental asymmetry in behavior. While all structured proteins can be unfolded through a change in environment, not all disordered proteins have the capacity for folding. Failure to accommodate these complexities confuses the definition of both protein structural domains and intrinsically disordered regions. We illustrate these points with an experimental study of a family of small binding domains, drawn from the RNA polymerase of mumps virus and its closest relatives. Assessed at face value the domains fall on a structural continuum, with folded, partially folded, and near unstructured members. Yet the disorder present in the family is conditional, and these closely related polypeptides can access the same folded state under appropriate conditions. Any heuristic definition of the protein domain emphasizing conformational stability divides this domain family in two, in a way that makes no biological sense. Structural domains would be better defined by their ability to adopt a specific tertiary structure: a structure that may or may not be realized, dependent on the circumstances. This explicitly allows for the conditional nature of protein folding, and more clearly demarcates structural domains from intrinsically disordered regions that may function without folding.     

RNA干扰机制研究进展

RNAi是多种生物体内由dsRNA介导的同源mRNA降解现象。这是一个高度特异化的过程,涉及多种蛋白质的共同参与。在这一过程中,siRNA的结构影响其两条链装配到RISC中去的能力。除了与RISC结合外,siRNA还引导了RITS复合物结合到同源染色质,介导异染色质化过程。干扰效应的扩散,即系统性沉默可能依赖于跨膜蛋白的转运,并且很可能是在多因素调控下完成的。Abstract: RNA interference (RNAi) is a phenomenon that the double-stranded RNA (dsRNA) intermediates the degradation of complementary mRNA found in many organisms. This is a specifically mechanism involved in kinds of proteins to complete the interference function. Structure of siRNA affects which strand will be assembled into RISC. Another role of siRNA is directing RITS complex to bind with homologue chromosome, and then induces heterochromatinization. Although systemic silence induced by dsRNA is observed in Caenorhabditis elegans and plants, this progress is probably transmembrane protein-dependent, and mostly, the systemic silencing is controlled by multi-factors.