Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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In brain, mRNAs are transported from the cell body to the processes, allowing for local protein translation at sites distant from the nucleus. Using subcellular fractionation, we isolated a fraction from rat embryonic day 18 brains enriched for structures that resemble amorphous collections of ribosomes. This fraction was enriched for the mRNA encoding beta-actin, an mRNA that is transported in dendrites and axons of developing neurons. Abundant protein components of this fraction, determined by tandem mass spectrometry, include ribosomal proteins, RNA-binding proteins, microtubule-associated proteins (including the motor protein dynein), and several proteins described only as potential open reading frames. The conjunction of RNA-binding proteins, transported mRNA, ribosomal machinery, and transporting motor proteins defines these structures as RNA granules. Expression of a subset of the identified proteins in cultured hippocampal neurons confirmed that proteins identified in the proteomics were present in neurites associated with ribosomes and mRNAs. Moreover many of the expressed proteins co-localized together. Time lapse video microscopy indicated that complexes containing one of these proteins, the DEAD box 3 helicase, migrated in dendrites of hippocampal neurons at the same speed as that reported for RNA granules. Although the speed of the granules was unchanged by activity or the neurotrophin brain-derived neurotrophic factor, brain-derived neurotrophic factor, but not activity, increased the proportion of moving granules. These studies define the isolation and composition of RNA granules expressed in developing brain.
Mol Cell Proteomics 2006 Apr
PMID:Characterization of an RNA granule from developing brain. 1635 23

The causative agent for the most fatal form of malaria, Plasmodium falciparum, has developed insecticide and drug resistance with time. Therefore combating this disease is becoming increasingly difficult and this calls for finding alternate ways to control malaria. One of the feasible ways could be to find out inhibitors/drugs specific for the indispensable enzymes of malaria parasite such as helicases. These helicases, which contain intrinsic nucleic acid-dependent ATPase activity, are capable of enzymatically unwinding energetically stable duplex nucleic acids into single-stranded templates and are required for all the nucleic acid transactions. Most of the helicases contain a set of nine extremely conserved amino acid sequences, which are called 'helicase motifs'. Due to the presence of the DEAD (Asp-Glu-Ala-Asp) in one of the conserved motifs, this family is also known as the 'DEAD-box' family. In this review, using bioinformatic approach, we describe the 'DEAD-box' helicases of malaria parasite P. falciparum. An in depth analysis shows that the parasite contains 22 full-length genes, some of which are homologues of well-characterized helicases of this family from other organisms. Recently we have cloned and characterized the first member of this family, which is a homologue of p68 and is expressed during the schizont stage of the development of the parasite [Pradhan, A., Chauhan, V.S., Tuteja, R., 2005a. A novel 'DEAD-box' DNA helicase from Plasmodium falciparum is homologous to p68. Mol. Biochem. Parasitol. 140, 55-60.; Pradhan A., Chauhan V.S., Tuteja R., 2005b. Plasmodium falciparum DNA helicase 60 is a schizont stage specific, bipolar and dual helicase stimulated by PKC phosphorylation. Mol. Biochem. Parasitol. 144, 133-141.]. It will be really interesting to clone and characterize other members of the 'DEAD-box' family and understand their role in the replication and transmission of the parasite. These detailed studies may help to identify a parasite-specific enzyme, which could be a potential drug target to treat malaria. The various steps at which this probable drug can act are also discussed.
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PMID:Unraveling the 'DEAD-box' helicases of Plasmodium falciparum. 1671 33

Here, we develop computational methods to assess and consolidate large, diverse protein interaction data sets, with the objective of identifying proteins involved in the coupling of multicomponent complexes within the yeast gene expression pathway. From among approximately 43 000 total interactions and 2100 proteins, our methods identify known structural complexes, such as the spliceosome and SAGA, and functional modules, such as the DEAD-box helicases, within the interaction network of proteins involved in gene expression. Our process identifies and ranks instances of three distinct, biologically motivated motifs, or patterns of coupling among distinct machineries involved in different subprocesses of gene expression. Our results confirm known coupling among transcription, RNA processing, and export, and predict further coupling with translation and nonsense-mediated decay. We systematically corroborate our analysis with two independent, comprehensive experimental data sets. The methods presented here may be generalized to other biological processes and organisms to generate principled, systems-level network models that provide experimentally testable hypotheses for coupling among biological machines.
Mol Syst Biol 2006
PMID:Systems-level analyses identify extensive coupling among gene expression machines. 1673 50

DEAD box RNA helicases use the energy of ATP hydrolysis to unwind double-stranded RNA regions or to disrupt RNA/protein complexes. A minimal RNA helicase comprises nine conserved motifs distributed over two RecA-like domains. The N-terminal domain contains all motifs involved in nucleotide binding, namely the Q-motif, the DEAD box, and the P-loop, as well as the SAT motif, which has been implicated in the coordination of ATP hydrolysis and RNA unwinding. We present here the crystal structure of the N-terminal domain of the Thermus thermophilus RNA helicase Hera in complex with adenosine monophosphate (AMP). Upon binding of AMP the P-loop adopts a partially collapsed or half-open conformation that is still connected to the DEAD box motif, and the DEAD box in turn is linked to the SAT motif via hydrogen bonds. This network of interactions communicates changes in the P-loop conformation to distant parts of the helicase. The affinity of AMP is comparable to that of ADP and ATP, substantiating that the binding energy from additional phosphate moieties is directly converted into conformational changes of the entire helicase. Importantly, the N-terminal Hera domain forms a dimer in the crystal similar to that seen in another thermophilic prokaryote. It is possible that this mode of dimerization represents the prototypic architecture in RNA helicases of thermophilic origin.
J Mol Biol 2006 Aug 25
PMID:Crystal structure and nucleotide binding of the Thermus thermophilus RNA helicase Hera N-terminal domain. 1689 Feb 41

Synthesis of rRNA in eukaryotes involves the action of a large population of snoRNA-protein complexes (snoRNPs), which create modified nucleotides and participate in cleavage of pre-rRNA. The snoRNPs mediate these functions through direct base pairing, in many cases through long complementary sequences. This feature suggests that RNA helicases may be involved in the binding and release of snoRNPs from pre-rRNA. In this study, we determined that the DEAD box helicase Has1p, a nucleolar protein required for the production of 18S rRNA, copurifies with the snR30/U17 processing snoRNP but is also present with other snoRNPs. Blocking Has1p expression causes a substantial increase in snoRNPs associated with 60S-90S preribosomal RNP complexes, including the U3 and U14 processing snoRNPs and several modifying snoRNPs examined. Cosedimentation persisted even after deproteinization. This effect was not observed with depletion of two nonhelicase proteins, Esf1p and Dim2p, that are also required for 18S rRNA production. Point mutations in ATPase and helicase motifs of Has1p block U14 release from pre-rRNA. Surprisingly, depletion of Has1p causes a reduction in the level of free U6 snRNP. The results indicate that the Has1p helicase is required for snoRNA release from pre-rRNA and production of the U6 snRNP.
Mol Cell Biol 2006 Oct
PMID:The helicase Has1p is required for snoRNA release from pre-rRNA. 1690 38

The vasa gene encodes an ATP-dependent RNA helicase belonging to the DEAD-box family that, in many organisms, is specifically expressed in germline cells throughout the life cycle. In this study we first cloned Pacific white shrimp (Litopenaeus vannamei) partial cDNAs of two members of the DEAD-box family, one belonging to the vasa subfamily (Lv-Vasa) and the other to the PL10 subfamily (Lv-PL10). Examination of their spatial expression pattern in adult tissues revealed that Lv-Vasa is restricted to the gonads, whereas Lv-PL10 is found in gonads as well as in somatic tissues. Next, we cloned the full-length shrimp vasa cDNA and found that Lv-Vasa encoded a protein with a DEAD-like helicase domain followed by a helicase superfamily C-terminal domain. In addition, Lv-Vasa encoded N-terminal three repeats of the C2HC-type zinc finger domain, a motif encoded by vasa genes of several crustaceans and several other invertebrate organisms. In situ hybridization of ovarian sections showed that the Lv-Vasa transcript is localized to the cytoplasm of the oocyte throughout oogenesis. The abundance of Lv-Vasa mRNA in mature oocytes suggests a maternal contribution for the developing embryo. It is demonstrated that the vasa homolog from L. vannamei is a gonad specific germline cell marker that could be exploited to enhance our understanding of developmental and reproductive processes in the germline of this economically important shrimp.
Mol Reprod Dev 2007 Feb
PMID:Characterization of a vasa-like gene from the pacific white shrimp Litopenaeus vannamei and its expression during oogenesis. 1695 7

Helicases unwind RNA or DNA duplexes and displace proteins from nucleic acids in an ATP-dependent fashion. To unwind duplexes, helicases typically load onto one of the two nucleic acid strands, usually at a single-stranded region, and then translocate on this strand in a unidirectional fashion, thereby displacing the complementary DNA or RNA. Here we show that the DEAD-box RNA helicase Ded1 unwinds duplexes in a different manner. Ded1 uses the single-stranded region to gain access to the duplex. Strand separation is directly initiated from the duplex region and no covalent connection between the single strand and the duplex region is required. This new type of helicase activity explains observations with other DEAD-box proteins and may be the prototype for duplex-unwinding reactions in RNA metabolism.
Nat Struct Mol Biol 2006 Nov
PMID:The DEAD-box protein Ded1 unwinds RNA duplexes by a mode distinct from translocating helicases. 1708 89

The RNA-catalyzed splicing of group I and group II introns is facilitated by proteins that stabilize the active RNA structure or act as RNA chaperones to disrupt stable inactive structures that are kinetic traps in RNA folding. In Neurospora crassa and Saccharomyces cerevisiae, the latter function is fulfilled by specific DEAD-box proteins, denoted CYT-19 and Mss116p, respectively. Previous studies showed that purified CYT-19 stimulates the in vitro splicing of structurally diverse group I and group II introns, and uses the energy of ATP binding or hydrolysis to resolve kinetic traps. Here, we purified Mss116p and show that it has RNA-dependent ATPase activity, unwinds RNA duplexes in a non-polar fashion, and promotes ATP-independent strand-annealing. Further, we show that Mss116p binds RNA non-specifically and promotes in vitro splicing of both group I and group II intron RNAs, as well as RNA cleavage by the aI5gamma-derived D135 ribozyme. However, Mss116p also has ATP hydrolysis-independent effects on some of these reactions, which are not shared by CYT-19 and may reflect differences in its RNA-binding properties. We also show that a non-mitochondrial DEAD-box protein, yeast Ded1p, can function almost as efficiently as CYT-19 and Mss116p in splicing the yeast aI5gamma group II intron and less efficiently in splicing the bI1 group II intron. Together, our results show that Mss116p, like CYT-19, can act broadly as an RNA chaperone to stimulate the splicing of diverse group I and group II introns, and that Ded1p also has an RNA chaperone activity that can be assayed by its effect on splicing mitochondrial introns. Nevertheless, these DEAD-box protein RNA chaperones are not completely interchangeable and appear to function in somewhat different ways, using biochemical activities that have likely been tuned by coevolution to function optimally on specific RNA substrates.
J Mol Biol 2007 Jan 19
PMID:Involvement of DEAD-box proteins in group I and group II intron splicing. Biochemical characterization of Mss116p, ATP hydrolysis-dependent and -independent mechanisms, and general RNA chaperone activity. 1708 64

The group II intron ai5gamma from S. cerevisiae requires high temperature and salt to self-splice in vitro, but it is assisted by the protein Mss116 in vivo. Here we show that Mss116 can stimulate splicing of ai5gamma under near-physiological conditions in vitro, which represents one of the first cases in which a DExH/D protein is shown to act on its natural target. Importantly, we demonstrate that a small subset of DEAD-box proteins can also stimulate ai5gamma splicing in vitro and may represent a distinct subfamily of DEAD-box proteins that functions in RNA tertiary structure assembly. Mutational analysis shows that while ATPase activity is required for stimulation of splicing by Mss116, helicase activity is not. This finding indicates that Mss116 is unlikely to promote intron splicing through the unwinding of kinetic traps. Rather, we propose that Mss116 promotes the ordered assembly of large RNA molecules through stabilization of on-pathway intermediates.
Mol Cell 2006 Nov 17
PMID:A DEAD protein that activates intron self-splicing without unwinding RNA. 1718 36

SecA is the preprotein translocase ATPase subunit and a superfamily 2 (SF2) RNA helicase. Here we present the 2 A crystal structures of the Escherichia coli SecA homodimer in the apo form and in complex with ATP, ADP and adenosine 5'-[beta,gamma-imido]triphosphate (AMP-PNP). Each monomer contains the SF2 ATPase core (DEAD motor) built of two domains (nucleotide binding domain, NBD and intramolecular regulator of ATPase 2, IRA2), the preprotein binding domain (PBD), which is inserted in NBD and a carboxy-terminal domain (C-domain) linked to IRA2. The structures of the nucleotide complexes of SecA identify an interfacial nucleotide-binding cleft located between the two DEAD motor domains and residues critical for ATP catalysis. The dimer comprises two virtually identical protomers associating in an antiparallel fashion. Dimerization is mediated solely through extensive contacts of the DEAD motor domains leaving the C-domain facing outwards from the dimerization core. This dimerization mode explains the effect of functionally important mutations and is completely different from the dimerization models proposed for other SecA structures. The repercussion of these findings on translocase assembly and catalysis is discussed.
J Mol Biol 2007 Mar 09
PMID:Structure of dimeric SecA, the Escherichia coli preprotein translocase motor. 1722 38


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