Database accession: MF7000829
Name: Human decapping scavenger enzyme (hDcpS) with m7G(5'S)ppSp(5'S)G mRNA 5' cap analog
PDB ID: 5osy
Experimental method: X-ray (2.06 Å)
Assembly: Homodimer
Source organism: Homo sapiens
Primary publication of the structure:
Wojtczak BA, Sikorski PJ, Fac-Dabrowska K, Nowicka A, Warminski M, Kubacka D, Nowak E, Nowotny M, Kowalska J, Jemielity J
5'-Phosphorothiolate Dinucleotide Cap Analogues: Reagents for Messenger RNA Modification and Potent Small-Molecular Inhibitors of Decapping Enzymes.
(2018) J. Am. Chem. Soc. 140: 5987-5999
PMID: 29676910
Abstract:
The 5' cap consists of 7-methylguanosine (m7G) linked by a 5'-5'-triphosphate bridge to messenger RNA (mRNA) and acts as the master regulator of mRNA turnover and translation initiation in eukaryotes. Cap analogues that influence mRNA translation and turnover (either as small molecules or as part of an RNA transcript) are valuable tools for studying gene expression, which is often also of therapeutic relevance. Here, we synthesized a series of 15 dinucleotide cap (m7GpppG) analogues containing a 5'-phosphorothiolate (5'-PSL) moiety (i.e., an O-to-S substitution within the 5'-phosphoester) and studied their biological properties in the context of three major cap-binding proteins: translation initiation factor 4E (eIF4E) and two decapping enzymes, DcpS and Dcp2. While the 5'-PSL moiety was neutral or slightly stabilizing for cap interactions with eIF4E, it significantly influenced susceptibility to decapping. Replacing the γ-phosphoester with the 5'-PSL moiety (γ-PSL) prevented β-γ-pyrophosphate bond cleavage by DcpS and conferred strong inhibitory properties. Combining the γ-PSL moiety with α-PSL and β-phosphorothioate (PS) moiety afforded first cap-derived hDcpS inhibitor with low nanomolar potency. Susceptibility to Dcp2 and translational properties were studied after incorporation of the new analogues into mRNA transcripts by RNA polymerase. Transcripts containing the γ-PSL moiety were resistant to cleavage by Dcp2. Surprisingly, superior translational properties were observed for mRNAs containing the α-PSL moiety, which were Dcp2-susceptible. The overall protein expression measured in HeLa cells for this mRNA was comparable to mRNA capped with the translation augmenting β-PS analogue reported previously. Overall, our study highlights 5'-PSL as a synthetically accessible cap modification, which, depending on the substitution site, can either reduce susceptibility to decapping or confer superior translational properties on the mRNA. The 5'-PSL-analogues may find application as reagents for the preparation of efficiently expressed mRNA or for investigation of the role of decapping enzymes in mRNA processing or neuromuscular disorders associated with decapping.
Annotations from the GeneOntology database. Only terms that fit at least two of the interacting proteins are shown. Molecular function:
5'-(N(7)-methyl 5'-triphosphoguanosine)-[mRNA] diphosphatase activity 5'-(N(7)-methyl 5'-triphosphoguanosine)-[mRNA] diphosphatase activity
identical protein binding identical protein binding
RNA 7-methylguanosine cap binding RNA 7-methylguanosine cap binding
RNA exonuclease activity RNA exonuclease activity
Biological process:
deadenylation-dependent decapping of nuclear-transcribed mRNA deadenylation-dependent decapping of nuclear-transcribed mRNA
mRNA cis splicing, via spliceosome mRNA cis splicing, via spliceosome
mRNA methylguanosine-cap decapping mRNA methylguanosine-cap decapping
nuclear-transcribed mRNA catabolic process, deadenylation-dependent decay nuclear-transcribed mRNA catabolic process, deadenylation-dependent decay
Cellular component:
cytoplasm cytoplasm
cytosol cytosol
mitochondrion mitochondrion
nucleoplasm nucleoplasm
nucleus nucleus
P-body P-body
Structural annotations of the participating protein chains.Entry contents: 2 distinct polypeptide molecules
Chains: A, B
Notes: All chains according to the most probable oligomerization state stored in PDBe were considered.
Number of unique protein segments: 1
Name: m7GpppX diphosphatase
Source organism: Homo sapiens
Length: 337 residues
Sequence:Sequence according to the corresponding UniProt protein segmentMADAAPQLGKRKRELDVEEAHAASTEEKEAGVGNGTCAPVRLPFSGFRLQKVLRESARDKIIFLHGKVNEASGDGDGEDAVVILEKTPFQVEQVAQLLTGSPELQLQFSNDIYSTYHLFPPRQLNDVKTTVVYPATEKHLQKYLRQDLRLIRETGDDYRNITLPHLESQSLSIQWVYNILDKKAEADRIVFENPDPSDGFVLIPDLKWNQQQLDDLYLIAICHRRGIRSLRDLTPEHLPLLRNILHQGQEAILQRYRMKGDHLRVYLHYLPSYYHLHVHFTALGFEAPGSGVERAHLLAEVIENLECDPRHYQQRTLTFALRADDPLLKLLQEAQQS
UniProtKB AC: Q96C86 (positions: 37-336)
Coverage: 89%
Name: m7GpppX diphosphatase
Source organism: Homo sapiens
Length: 337 residues
Sequence:Sequence according to the corresponding UniProt protein segmentMADAAPQLGKRKRELDVEEAHAASTEEKEAGVGNGTCAPVRLPFSGFRLQKVLRESARDKIIFLHGKVNEASGDGDGEDAVVILEKTPFQVEQVAQLLTGSPELQLQFSNDIYSTYHLFPPRQLNDVKTTVVYPATEKHLQKYLRQDLRLIRETGDDYRNITLPHLESQSLSIQWVYNILDKKAEADRIVFENPDPSDGFVLIPDLKWNQQQLDDLYLIAICHRRGIRSLRDLTPEHLPLLRNILHQGQEAILQRYRMKGDHLRVYLHYLPSYYHLHVHFTALGFEAPGSGVERAHLLAEVIENLECDPRHYQQRTLTFALRADDPLLKLLQEAQQS
UniProtKB AC: Q96C86 (positions: 40-336)
Coverage: 88%
Evidence demonstrating that the participating proteins are unstructured prior to the interaction and their folding is coupled to binding. Representative domain in related structures: Scavenger mRNA decapping enzyme (DcpS)
Evidence level: Indirect evidence
Evidence coverage: Only some parts of the structure participates in mutual synergistic folding.
Complex Evidence:
Multidomain structure, where the domain-swapped N-terminal domain is responsible for dimerization. The extensive interface, symmetry, topology, beta sheet augmentation and buried surface area suggest that the N-terminal dimerization domain behaves as a single rigid domain and the monomers would not be stable on their own. Gel-filtration chromatography also suggested a dimeric form (PMID:15068804).
Chain A:
N/A
Chain B:
N/A
Surface and contacts features:
Structures from the PDB that contain the same number of proteins, and the proteins from the two structures show a sufficient degree of pairwise similarity, i.e. they belong to the same UniRef90 cluster (the full proteins exhibit at least 90% sequence identity) and convey roughly the same region to their respective interactions (the two regions from the two proteins share a minimum of 70% overlap). Download the CIF file (.cif)
Download this entry's XML file (.xml)
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