DHX8 | |||||||||||||||||||||||||||||||||||||||||||||||||||
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Aliases | DHX8 , DDX8, HRH1, PRP22, PRPF22, DEAH-box helicase 8, Dhr2 | ||||||||||||||||||||||||||||||||||||||||||||||||||
External IDs | OMIM: 600396; MGI: 1306823; HomoloGene: 3628; GeneCards: DHX8; OMA:DHX8 - orthologs | ||||||||||||||||||||||||||||||||||||||||||||||||||
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Wikidata | |||||||||||||||||||||||||||||||||||||||||||||||||||
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DEAH-box helicase 8, is a protein that in humans is encoded by the DHX8 gene. This protein is member of the DEAH box polypeptide family. The main characteristic of this group is their conserved motif DEAH (Asp- Glu- Ala- His). [5] A wide range of RNA helicases belongs to this family. Specifically, DHX8 acts as an ATP-dependent RNA helicase involved in splicing and the regulation of the releasing of spliced mRNAs from spliceosomes out of the nucleus. [6] Published studies have shown the consequences of DHX8 mutations, some of them are critical for biological processes such as hematopoiesis and are related to some diseases. [7] [8]
Protein DHX8 is part of a protein complex called spliceosome, which is in charge of pre-mRNA splicing. The spliceosome has eight major functional states, each with distinct composition and structure; five of the eight states have been structurally characterized. [10]
DHX8 have different domains: a S1 RNA binding domain (DEAD/DEAH box), an helicase conserved C-terminal domain, helicase associated domain (HA2), and an oligonucleotide/oligosaccharide-binding (OB)-fold, each joined by intrinsically disordered regions. [11]
There are some regions of the protein which are very important for its activity, like R620 and the hook-loop and hook-turn regions. Also, DHX8Δ547 is the catalytically active core of the protein DHX8. It is made of two RecA domains and the C-terminal WH, ratchet-like and OB-fold domains and the N-terminal region. [9]
The total weight of the DHX8 structure is 156580.13 Da.
Secondary structure:
DHX8 is localized in the cellular nucleus and stimulated upon RNA presence. This protein is a component of the spliceosome, so it takes part in pre-mRNA splicing. [9] Splicing is the process of joining exons from primary transcripts of messenger RNA and the elimination of intron sequences, by means of a spliceosomal mechanism, so that the mRNA produced is the one without introns, consisting exclusively of the joined exons. [12] Splicing finishes with the spliceosomal complex disassembly and the ATP-dependent liberation of the resulting mature RNAs to the outer of the nucleus. [13] Spliceosome requires conformational changes to be able to catalyze splicing reactions and the later mature mRNA releasing to the outer of the nucleus. One of the ATP-dependent helicase needed for these conformational changes is DHX8. Furthermore, DHX8 plays a key role in the releasing, facilitating the nuclear export of spliced mRNA. Protein characterization has shown that DHX8 has a binding preference for adenine-rich RNA. This binding is followed by ATP hydrolysis and thus, ADP release. [9]
DHX8 has multiple molecular functions like ATP-binding by, selective and non-covalent interactions with the coenzyme and enzyme regulator adenosine 5’ triphosphate. [14] Also identical protein binding (creates a similar type of interactions as described above but with other proteins), RNA binding and RNA helicase activity, based on catalysis of the reaction that unwinds an RNA helix: [15]
ATP + H2O = ADP + phosphate [16]
DHX8 protein has its main focuses of expression in the brain, especially in the cerebellum where it can be found mostly in Purkinje cells. Other important are the prostate and the gallbladder, which are zones where the polypetide is found also highly expressed.
DHX8 protein is not expressed in all tissues and organs significantly, some clear examples are the bone marrow or the soft tissue (Peripheral nerve), where we do not have enough quantity of the protein to be representative. [17]
Assays in enzymology for the biochemical characterization of proteins need high concentrations of the protein of interest and its protocols should be efficient, simple and cost-efficient to ensure a successful purification. One example of a purification approach for DHX8 is via a protein tag called GST-His grafted onto DHX8 protein is used.
The N-terminal Glutathione Sepharose TAG (GST) and C-termianl His-tag, also known as GST-His is a 29 kDa tag which allows small-scale affinity purification for recombinant proteins. This method is based on two different tags flanking the two extremes of the protein. However, it might influence physiological properties of the protein and thus, empirical testing is required for each case. [18]
DHX8 constructs are generated by PCR cloning using restriction enzymes. To generate His6GST-DHX8Δ54 (hexahistidine-GST), the coding sequence for residues A548 to R1220 is inserted into a version of "pFastBac", a specific vector kit of Thermo Fisher Scientific, [19] modified to encode an N-terminal His6GST-tag followed by an HRV 3C protease (recombinant restriction-grade protease) cleavage site. HRV 3C is a highly purified recombinant 6XHis-fusion protein, that recognizes the same cleavage site as the native enzyme. Both, vector and insert, are digested with NdeI and EcoRI restriction enzymes.
To create full-length DHX8 (fl-DHX8-His6) and DHX8Δ547-His6, PCR primers are designed, so that a His6-tag is fused to the C-terminus of DHX8. The resultant PCR amplicons encoding fl-DHX8 (M1 to R1220) or DHX8Δ547 (A548 to R1220) are inserted into the "pFBDM" vector [20] downstream of the polyhedrin promoter. Both, vector and insert, are digested with BamHI and NotI restriction enzymes. [9]
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The U4 small nuclear Ribo-Nucleic Acid is a non-coding RNA component of the major or U2-dependent spliceosome – a eukaryotic molecular machine involved in the splicing of pre-messenger RNA (pre-mRNA). It forms a duplex with U6, and with each splicing round, it is displaced from the U6 snRNA in an ATP-dependent manner, allowing U6 to re-fold and create the active site for splicing catalysis. A recycling process involving protein Brr2 releases U4 from U6, while protein Prp24 re-anneals U4 and U6. The crystal structure of a 5′ stem-loop of U4 in complex with a binding protein has been solved.
DEAD box proteins are involved in an assortment of metabolic processes that typically involve RNAs, but in some cases also other nucleic acids. They are highly conserved in nine motifs and can be found in most prokaryotes and eukaryotes, but not all. Many organisms, including humans, contain DEAD-box (SF2) helicases, which are involved in RNA metabolism.
Splicing factor 3 subunit 1 is a protein that in humans is encoded by the SF3A1 gene.
Probable ATP-dependent RNA helicase DDX17 (p72) is an enzyme that in humans is encoded by the DDX17 gene.
Splicing factor 3A subunit 2 is a protein that in humans is encoded by the SF3A2 gene.
Splicing factor 3B subunit 2 is a protein that in humans is encoded by the SF3B2 gene.
Aly/REF export factor, also known as THO complex subunit 4 is a protein that in humans is encoded by the ALYREF gene.
Pre-mRNA-splicing factor ATP-dependent RNA helicase PRP16 is an enzyme that in humans is encoded by the DHX38 gene.
Chromodomain-helicase-DNA-binding protein 8 is an enzyme that in humans is encoded by the CHD8 gene.
ATP-dependent RNA helicase DDX19B is an enzyme that in humans is encoded by the DDX19B gene.
Probable ATP-dependent RNA helicase DDX23 is an enzyme that in humans is encoded by the DDX23 gene.
Probable ATP-dependent RNA helicase DHX36 also known as DEAH box protein 36 (DHX36) or MLE-like protein 1 (MLEL1) or G4 resolvase 1 (G4R1) or RNA helicase associated with AU-rich elements (RHAU) is an enzyme that in humans is encoded by the DHX36 gene.
Putative pre-mRNA-splicing factor ATP-dependent RNA helicase DHX15 is an enzyme that in humans is encoded by the DHX15 gene.
Putative pre-mRNA-splicing factor ATP-dependent RNA helicase DHX32 is an enzyme that in humans is encoded by the DHX32 gene.
Putative pre-mRNA-splicing factor ATP-dependent RNA helicase DHX16 is an enzyme that in humans is encoded by the DHX16 gene.
PIF1 5'-to-3' DNA helicase is a protein that in humans is encoded by the PIF1 gene.
MIF4GD, or MIF4G domain-containing protein, is a protein which in humans is encoded by the MIF4GD gene. It is also known as SLIP1, SLBP -interacting protein 1, AD023, and MIFD. MIF4GD is expressed ubiquitously in humans, and has been found to be involved in activating proteins for histone mRNA translation, alternative splicing and translation of mRNAs, and is a factor in the regulation of cell proliferation.