Endoribonuclease Dotaz Zobrazit nápovědu
BACKGROUND: Many prokaryotic genomes comprise Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) offering defense against foreign nucleic acids. These immune systems are conditioned by the production of small CRISPR-derived RNAs matured from long RNA precursors. This often requires a Csy4 endoribonuclease cleaving the RNA 3'-end. METHODS: We report extended explicit solvent molecular dynamic (MD) simulations of Csy4/RNA complex in precursor and product states, based on X-ray structures of product and inactivated precursor (55 simulations; ~3.7μs in total). RESULTS: The simulations identify double-protonated His29 and deprotonated terminal phosphate as the likely dominant protonation states consistent with the product structure. We revealed potential substates consistent with Ser148 and His29 acting as the general base and acid, respectively. The Ser148 could be straightforwardly deprotonated through solvent and could without further structural rearrangements deprotonate the nucleophile, contrasting similar studies investigating the general base role of nucleobases in ribozymes. We could not locate geometries consistent with His29 acting as general base. However, we caution that the X-ray structures do not always capture the catalytically active geometries and then the reactive structures may be unreachable by the simulation technique. CONCLUSIONS: We identified potential catalytic arrangement of the Csy4/RNA complex but we also report limitations of the simulation technique. Even for the dominant protonation state we could not achieve full agreement between the simulations and the structural data. GENERAL SIGNIFICANCE: Potential catalytic arrangement of the Csy4/RNA complex is found. Further, we provide unique insights into limitations of simulations of protein/RNA complexes, namely, the influence of the starting experimental structures and force field limitations. This article is part of a Special Issue entitled Recent developments of molecular dynamics.
- MeSH
- Cas proteiny chemie metabolismus MeSH
- časové faktory MeSH
- CRISPR-Cas systémy * MeSH
- endoribonukleasy chemie metabolismus MeSH
- katalytická doména MeSH
- krystalografie rentgenová MeSH
- sekvence CRISPR * MeSH
- simulace molekulární dynamiky * MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
We have synthesized two different groups of oligonucleotides containing chiral isopolar nonisosteric phosphonate internucleotide linkages, and studied their properties in combination with natural phosphodiester ones. The improved synthetic procedures for the monomers preparation are also reported.
Background: RNase L is known as a terminal component of antiviral and Interferon (IFN) pathways in mammalian cells. On the other hand, the human miR-29 family of microRNAs (miRs) has three mature members, miR-29a, miR-29b, and miR-29c. miR-29 is encoded by two gene clusters and the family members have multifunctional roles in various biological processes. Objectives: To determine the potential role of miR-29 in the regulation of RNASEL gene expression by designing inhibitors against its targeting miRNA, miR-29a-3p and evaluate the RNase L expression. Material and Methods: After selecting miR-29a-3p as a main regulating miRNA for RNASEL in silico, two inhibitors were designed against it and synthesized. Synthesized strands were made double-stranded DNA oligos, treated with T4 polynucleotide kinase (PNK), cloned into the pCDH-CMV-MCS-EF1-cGFP-T2A-Puro vector and transformed into DH5α. Colony PCR and sequencing was done for affirmation. Then the miR-29a-3p inhibitors were transfected into HEK-293T cell line and RNASEL gene expression was analyzed. Results: The miR-29a-3p inhibitors decreased miR-29a-3p expression in vitro. In addition, miR-29a-3p expression reduction resulted in an increase of RNASEL gene expression. Conclusions: miR-29a-3p inhibitors could increase in RNASEL gene expression which potentially affects the antiviral/IFN pathway. The inhibitors could be considered as drug candidates in different diseases especially viral infections.
- MeSH
- endoribonukleasy * genetika MeSH
- exprese genu MeSH
- HEK293 buňky MeSH
- lidé MeSH
- mikro RNA * genetika MeSH
- Check Tag
- lidé MeSH
The oligoadenylate synthetase-ribonuclease L pathway is a major player in the interferon-induced antiviral defense mechanism of cells. Upon sensing viral dsRNA, 5'-phosphorylated 2',5'-oligoadenylates are synthesized, and subsequently activate latent RNase L. To determine the influence of 5'-phosphate end on the activation of human RNase L, four sets of 5'-phosphonate modified oligoadenylates were prepared on solid-phase. The ability of these 5'-modified oligoadenylates bearing shortened, isosteric and prolonged phosphonate linkages to activate RNase L was explored. We found that isosteric linkages and linkages prolonged by one atom were in general well tolerated by the enzyme with the EC50 values comparable to that of the natural activator. In contrast, linkages shortened by one atom or prolonged by two atoms exhibited decrease in the activity.
- MeSH
- adeninnukleotidy chemická syntéza chemie farmakologie MeSH
- endoribonukleasy metabolismus MeSH
- konformace nukleové kyseliny MeSH
- lidé MeSH
- oligoribonukleotidy chemická syntéza chemie farmakologie MeSH
- organofosfonáty chemická syntéza chemie farmakologie MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
RNase L, a key enzyme in the host defense system, is activated by the binding of 2'-5'-linked oligoadenylates (2-5A) to the N-terminal ankyrin repeat domain, which causes the inactive monomer to form a catalytically active homodimer. We focused on the structural changes of human RNase L as a result of interactions with four different activators: natural 2-5 pA(4) and three tetramers with 3'-end AMP units replaced with ribo-, arabino- and xylo-configured phosphonate analogs of AMP (pA(3)X). The extent of the RNase L dimerization and its cleavage activity upon binding of all these activators were similar. A drop-coating deposition Raman (DCDR) spectroscopy possessed uniform spectral changes upon binding of all of the tetramers, which verified the same binding mechanism. The estimated secondary structural composition of monomeric RNase L is 44% α-helix, 28% β-sheet, 17% β-turns and 11% of unordered structures, whereas dimerization causes a slight decrease in α-helix and increase in β-sheet (ca. 2%) content. The dimerization affects at least three Tyr, five Phe and two Trp residues. The α-β structural switch may fix domain positions in the hinge region (residues ca. 336-363) during homodimer formation.
Removal of the mRNA 5' cap primes transcripts for degradation and is central for regulating gene expression in eukaryotes. The canonical decapping enzyme Dcp2 is stringently controlled by assembly into a dynamic multi-protein complex together with the 5'-3'exoribonuclease Xrn1. Kinetoplastida lack Dcp2 orthologues but instead rely on the ApaH-like phosphatase ALPH1 for decapping. ALPH1 is composed of a catalytic domain flanked by C- and N-terminal extensions. We show that T. brucei ALPH1 is dimeric in vitro and functions within a complex composed of the trypanosome Xrn1 ortholog XRNA and four proteins unique to Kinetoplastida, including two RNA-binding proteins and a CMGC-family protein kinase. All ALPH1-associated proteins share a unique and dynamic localization to a structure at the posterior pole of the cell, anterior to the microtubule plus ends. XRNA affinity capture in T. cruzi recapitulates this interaction network. The ALPH1 N-terminus is not required for viability in culture, but essential for posterior pole localization. The C-terminus, in contrast, is required for localization to all RNA granule types, as well as for dimerization and interactions with XRNA and the CMGC kinase, suggesting possible regulatory mechanisms. Most significantly, the trypanosome decapping complex has a unique composition, differentiating the process from opisthokonts.
The capability of current MD simulations to be used as a tool in rational design of agonists of medically interesting enzyme RNase L was tested. Dimerization and enzymatic activity of RNase L is stimulated by 2',5'-linked oligoadenylates (pA₂₅A₂₅A; 2-5A). First, it was necessary to ensure that a complex of monomeric human RNase L and 25A was stable in MD simulations. It turned out that Glu131 had to be protonated. The non-protonated Glu131 caused dissociation of 2-5A from RNase L. Because of the atypical 2'-5' internucleotide linkages and a specific spatial arrangement of the 25A trimer, when a single molecule carries all possible conformers of the glycosidic torsion angle, several versions of the AMBER force field were tested. One that best maintained functionally important interactions of 25A and RNase L was selected for subsequent MD simulations. Furthermore, we wonder whether powerful GPUs are able to produce MD trajectories long enough to convincingly demonstrate effects of subtle perturbations of interactions between 25A and RNase L. Detrimental impacts of various point mutations of RNase L (R155A, F126A, W60A, K89A) on 2-5A binding were observed on a time scale of 200 ns. Finally, 2-5A analogues with a bridged 3'--O,4'--C-alkylene linkage (B) introduced into the adenosine units (A) were used to assess ability of MD simulations to distinguish on the time scale of hundreds of nanoseconds between agonists of RNase L (pA₂₅A₂₅B, pB₂₅A₂₅A, pB₂₅A₂₅B) and inactive analogs (pA₂₅B₂₅A, pA₂₅B₂₅B, pB₂₅B₂₅A, pB₂₅B₂₅B). Agonists were potently bound to RNase L during 200 ns MD runs. For inactive 2-5A analogs, by contrast, significant disruptions of their interactions with RNase L already within 100 ns MD runs were found.
- MeSH
- adeninnukleotidy chemie metabolismus MeSH
- aktivace enzymů MeSH
- endoribonukleasy chemie metabolismus MeSH
- lidé MeSH
- molekulární konformace MeSH
- multimerizace proteinu MeSH
- oligoribonukleotidy chemie metabolismus MeSH
- simulace molekulární dynamiky * MeSH
- simulace molekulového dockingu MeSH
- vazba proteinů MeSH
- vodíková vazba MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
