Packing them up and dusting them off: RNA helicases and mRNA storage

Cytoplasmic mRNA can be translated, translationally repressed, localized or degraded. Regulation of translation is an important step in control of gene expression and the cell can change whether and to what extent an mRNA is translated. If an mRNA is not translating, it will associate with translation repression factors; the mRNA can be stored in these non-translating states. The movement of mRNA into storage and back to translation is dictated by the recognition of the mRNA by trans factors. So, remodeling the factors that bind mRNA is critical for changing the fate of mRNA. RNA helicases, which have the ability to remodel RNA or RNA–protein complexes, are excellent candidates for facilitating such rearrangements. This review will focus on the RNA helicases implicated in translation repression and/or mRNA storage and how their study has illuminated mechanisms of mRNA regulation. This article is part of a Special Issue entitled: The Biology of RNA helicases — Modulation for life.     

The active form of Xp54 RNA helicase in translational repression is an RNA-mediated oligomer

Previously, we reported that in clam oocytes, cytoplasmic polyadenylation element-binding protein (CPEB) co-immunoprecipitates with p47, a member of the highly conserved RCK family of RNA helicases which includes Drosophila Me31B and Saccharomyces cerevisiae Dhh1. Xp54, the Xenopus homologue, with helicase activity, is a component of stored mRNP. In tethered function assays in Xenopus oocytes, we showed that MS2–Xp54 represses the translation of non-adenylated firefly luciferase mRNAs and that mutations in two core helicase motifs, DEAD and HRIGR, surprisingly, activated translation. Here we show that wild-type MS2–Xp54 tethered to the reporter mRNA 3′-untranslated region (UTR) represses translation in both oocytes and eggs in an RNA-dependent complex with endogenous Xp54. Injection of mutant helicases or adenylated reporter mRNA abrogates this association. Thus Xp54 oligomerization is a hallmark of translational repression. Xp54 complexes, which also contain CPEB and eIF4E in oocytes, change during meiotic maturation. In eggs, CPEB is degraded and, while eIF4E still interacts with Xp54, this interaction becomes RNA dependent. Supporting evidence for RNA-mediated oligomerization of endogenous Xp54, and RNA-independent association with CPEB and eIF4E in oocytes was obtained by gel filtration. Altogether, our data are consistent with a model in which the active form of the Xp54 RNA helicase is an oligomer in vivo which, when tethered, via either MS2 or CPEB to the 3′UTR, represses mRNA translation, possibly by sequestering eIF4E from the translational machinery.     

Multiple binding of repressed mRNAs by the P-body protein Rck/p54

Translational repression is achieved by protein complexes that typically bind 3' UTR mRNA motifs and interfere with the formation of the cap-dependent initiation complex, resulting in mRNPs with a closed-loop conformation. We demonstrate here that the human DEAD-box protein Rck/p54, which is a component of such complexes and central to P-body assembly, is in considerable molecular excess with respect to cellular mRNAs and enriched to a concentration of 0.5 mM in P-bodies, where it is organized in clusters. Accordingly, multiple binding of p54 proteins along mRNA molecules was detected in vivo. Consistently, the purified protein bound RNA with no sequence specificity and high nanomolar affinity. Moreover, bound RNA molecules had a relaxed conformation. While RNA binding was ATP independent, relaxing of bound RNA was dependent on ATP, though not on its hydrolysis. We propose that Rck/p54 recruitment by sequence-specific translational repressors leads to further binding of Rck/p54 along mRNA molecules, resulting in their masking, unwinding, and ultimately recruitment to P-bodies. Rck/p54 proteins located at the 5' extremity of mRNA can then recruit the decapping complex, thus coupling translational repression and mRNA degradation.     

An essential role for the Saccharomyces cerevisiae DEAD-box helicase DHH1 in G1/S DNA-damage checkpoint recovery

The eukaryotic cell cycle displays a degree of plasticity in its regulation; cell cycle progression can be transiently arrested in response to environmental stresses. While the signaling pathways leading to cell cycle arrest are beginning to be well understood, the regulation of the release from arrest has not been well characterized. Here we show that DHH1, encoding a DEAD-box RNA helicase orthologous to the human putative proto-oncogene p54/RCK, is important in release from DNA-damage-induced cell cycle arrest at the G1/S checkpoint. DHH1 mutants are not defective for DNA repair and recover normally from the G2/M and replication checkpoints, suggesting a specific function for Dhh1p in recovery from G1/S checkpoint arrest. Dhh1p has been suggested to play a role in partitioning mRNAs between translatable and nontranslatable pools, and our results implicate this modulation of mRNA metabolism in the recovery from G1/S cell cycle arrest following DNA damage. Furthermore, the high degree of conservation between DHH1 and its human ortholog suggests that this mechanism is conserved among all eukaryotes and potentially important in human disease.     

Multiple Nudix family proteins possess mRNA decapping activity

RNA decapping is an important contributor to gene expression and is a critical determinant of mRNA decay. The recent demonstration that mammalian cells harbor at least two distinct decapping enzymes that preferentially modulate a subset of mRNAs raises the intriguing possibility of whether additional decapping enzymes exist. Because both known decapping proteins, Dcp2 and Nudt16, are members of the Nudix hydrolase family, we set out to determine whether other members of this family of proteins also contain intrinsic RNA decapping activity. Here we demonstrate that six additional mouse Nudix proteins—Nudt2, Nudt3, Nudt12, Nudt15, Nudt17, and Nudt19—have varying degrees of decapping activity in vitro on both monomethylated and unmethylated capped RNAs. The decapping products from Nudt17 and Nudt19 were analogous to Dcp2 and predominantly generated m⁷GDP, while cleavage by Nudt2, Nudt3, Nudt12, and Nudt15 was more pleiotropic and generated both m⁷GMP and m⁷GDP. Interestingly, all six Nudix proteins as well as both Dcp2 and Nudt16 could hydrolyze the cap of an unmethylated capped RNA, indicating that decapping enzymes may be less constrained for the presence of the methyl moiety. Investigation of Saccharomyces cerevisiae Nudix proteins revealed that the yeast homolog of Nudt3, Ddp1p, also possesses decapping activity in vitro. Moreover, the bacterial Nudix pyrophosphohydrolase RppH displayed RNA decapping activity and released m⁷GDP product comparable to Dcp2, indicating that decapping is an evolutionarily conserved activity that preceded mammalian cap formation. These findings demonstrate that multiple Nudix family hydrolases may function in mRNA decapping and mRNA stability.     

Unwinding the functions of the Pif1 family helicases

Helicases are ubiquitous enzymes found in all organisms that are necessary for all (or virtually all) aspects of nucleic acid metabolism. The Pif1 helicase family is a group of 5′ → 3′ directed, ATP-dependent, super family IB helicases found in nearly all eukaryotes. Here, we review the discovery, evolution, and what is currently known about these enzymes in Saccharomyces cerevisiae (ScPif1 and ScRrm3), Schizosaccharomyces pombe (SpPfh1), Trypanosoma brucei (TbPIF1, 2, 5, and 8), mice (mPif1), and humans (hPif1). Pif1 helicases variously affect telomeric, ribosomal, and mitochondrial DNA replication, as well as Okazaki fragment maturation, and in at least some cases affect these processes by using their helicase activity to disrupt stable nucleoprotein complexes. While the functions of these enzymes vary within and between organisms, it is evident that Pif1 family helicases are crucial for both nuclear and mitochondrial genome maintenance.     

Intra- and Intermolecular Regulatory Interactions in Upf1, the RNA Helicase Central to Nonsense-Mediated mRNA Decay in Yeast

RNA helicases are involved in almost every aspect of RNA metabolism, yet very little is known about the regulation of this class of enzymes. In Saccharomyces cerevisiae, the stability and translational fidelity of nonsense-containing mRNAs are controlled by the group I RNA helicase Upf1 and the proteins it interacts with, Upf2 and Upf3. Combining the yeast two-hybrid system with genetic analysis, we show here that the cysteine- and histidine-rich (CH) domain and the RNA helicase domain of yeast Upf1 can engage in two new types of molecular interactions: an intramolecular interaction between these two domains and self-association of each of these domains. Multiple observations indicate that these molecular interactions are crucial for Upf1 regulation. First, coexpression of the CH domain and the RNA helicase domain in trans can reconstitute Upf1 function in both promoting nonsense-mediated mRNA decay (NMD) and preventing nonsense suppression. Second, mutations that disrupt Upf1 intramolecular interaction cause loss of Upf1 function. These mutations weaken Upf2 interaction and, surprisingly, promote Upf1 self-association. Third, the genetic defects resulting from deficiency in Upf1 intramolecular interaction or RNA binding are suppressed by expression of Upf2. Collectively, these data reveal a set of sequential molecular interactions and their roles in regulating Upf1 function during activation of NMD and suggest that cis intramolecular interaction and trans self-association may be general mechanisms for regulation of RNA helicase functions.     

Genetic analysis of the Caenorhabditis elegans GLH family of P-granule proteins

The Vasa DEAD-box helicases are widespread markers of germ cells across species, and in some organisms have been shown to be essential for germ-cell formation and development. In contrast to the single Vasa gene in most systems analyzed, Caenorhabditis elegans has four Vasa family members, the germline helicases GLH-1, GLH-2, GLH-3, and GLH-4. Our analysis of deletion alleles of each glh gene demonstrates that GLH-1 is the key member of the family: loss of GLH-1 function causes sterility that is mainly maternal effect, is manifested predominantly at elevated temperature, and is due to reduced germ-cell proliferation and impaired formation of both sperm and oocytes. The other GLHs are not essential. However, GLH-4 serves redundant roles with GLH-1: loss of both genes' function causes glh-1-like sterility at all temperatures. Molecular epistasis analysis demonstrates that GLH-1 and GLH-4 are required for proper association of the PGL family of proteins with P granules, suggesting a pathway of P-granule assembly in which the GLHs are upstream of the PGL proteins and the mRNA cap-binding protein IFE-1. While loss of some P-granule components causes worms to be defective in RNA interference, loss of GLH-1 and GLH-4 does not compromise RNAi. Thus, RNAi likely does not require intact P granules but instead relies on particular P-granule factors. We discuss the evolution of the Vasa/GLH genes and current views of their functions and the assembly and roles of germ granules among species.     

Selective pharmacological targeting of a DEAD box RNA helicase

RNA helicases represent a large family of proteins implicated in many biological processes including ribosome biogenesis, splicing, translation and mRNA degradation. However, these proteins have little substrate specificity, making inhibition of selected helicases a challenging problem. The prototypical DEAD box RNA helicase, eIF4A, works in conjunction with other translation factors to prepare mRNA templates for ribosome recruitment during translation initiation. Herein, we provide insight into the selectivity of a small molecule inhibitor of eIF4A, hippuristanol. This coral-derived natural product binds to amino acids adjacent to, and overlapping with, two conserved motifs present in the carboxy-terminal domain of eIF4A. Mutagenesis of amino acids within this region allowed us to alter the hippuristanol-sensitivity of eIF4A and undertake structure/function studies. Our results provide an understanding into how selective targeting of RNA helicases for pharmacological intervention can be achieved.     

RNA解旋酶和应激颗粒

应激颗粒（stress granules, SGs）是细胞在环境压力刺激下停止蛋白质翻译后,mRNA与多种细胞蛋白组装而成的胞质颗粒结构.RNA 解旋酶家族作为生物体内普遍存在的一类高度保守的蛋白质酶类,参与了RNA代谢各个环节,近年来其家族成员被陆续发现是一类新的SG重要组分.本文综述了RNA解旋酶参与应激颗粒形成过程,RNA解旋酶家族蛋白的结构和其参与应激颗粒形成的研究进展.     

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.