| 1. |
- Banijamali, Elnaz, et al.
(author)
-
RNA:RNA interaction in ternary complexes resolved by chemical probing
- 2022
-
In: RNA. - : Cold Spring Harbor Laboratory Press (CSHL). - 1355-8382 .- 1469-9001. ; 29:3, s. 317-329
-
Journal article (peer-reviewed)abstract
- RNA regulation can be performed by a second targeting RNA molecule, such as in the microRNA regulation mechanism. Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) probes the structure of RNA molecules and can resolve RNA:protein interactions, but RNA:RNA interactions have not yet been addressed with this technique. Here, we apply SHAPE to investigate RNA-mediated binding processes in RNA:RNA and RNA:RNA-RBP complexes. We use RNA:RNA binding by SHAPE (RABS) to investigate microRNA-34a (miR-34a) binding its mRNA target, the silent information regulator 1 (mSIRT1), both with and without the Argonaute protein, constituting the RNA-induced silencing complex (RISC). We show that the seed of the mRNA target must be bound to the microRNA loaded into RISC to enable further binding of the compensatory region by RISC, while the naked miR-34a is able to bind the compensatory region without seed interaction. The method presented here provides complementary structural evidence for the commonly performed luciferase-assay-based evaluation of microRNA binding-site efficiency and specificity on the mRNA target site and could therefore be used in conjunction with it. The method can be applied to any nucleic acid-mediated RNA- or RBP-binding process, such as splicing, antisense RNA binding, or regulation by RISC, providing important insight into the targeted RNA structure.
|
|
| 2. |
- Grätz, Lukas, et al.
(author)
-
NanoBiT‐ and NanoBiT/BRET‐based assays allow the analysis of binding kinetics of Wnt‐3a to endogenous Frizzled 7 in a colorectal cancer model
- 2023
-
In: British Journal of Pharmacology. - : John Wiley & Sons. - 0007-1188 .- 1476-5381.
-
Journal article (peer-reviewed)abstract
- Background and PurposeWnt binding to Frizzleds (FZD) is a crucial step that leads to the initiation of signalling cascades governing multiple processes during embryonic development, stem cell regulation and adult tissue homeostasis. Recent efforts have enabled us to shed light on Wnt–FZD pharmacology using overexpressed HEK293 cells. However, assessing ligand binding at endogenous receptor expression levels is important due to differential binding behaviour in a native environment. Here, we study FZD paralogue, FZD7, and analyse its interactions with Wnt-3a in live CRISPR-Cas9-edited SW480 cells typifying colorectal cancer.Experimental ApproachSW480 cells were CRISPR-Cas9-edited to insert a HiBiT tag on the N-terminus of FZD7, preserving the native signal peptide. These cells were used to study eGFP-Wnt-3a association with endogenous and overexpressed HiBiT-FZD7 using NanoBiT/bioluminescence resonance energy transfer (BRET) and NanoBiT to measure ligand binding and receptor internalization.Key ResultsWith this new assay the binding of eGFP-Wnt-3a to endogenous HiBiT-FZD7 was compared with overexpressed receptors. Receptor overexpression results in increased membrane dynamics, leading to an apparent decrease in binding on-rate and consequently in higher, up to 10 times, calculated Kd. Thus, measurements of binding affinities to FZD7 obtained in overexpressed cells are suboptimal compared with the measurements from endogenously expressing cells.Conclusions and ImplicationsBinding affinity measurements in the overexpressing cells fail to replicate ligand binding affinities assessed in a (patho)physiologically relevant context where receptor expression is lower. Therefore, future studies on Wnt–FZD7 binding should be performed using receptors expressed under endogenous promotion.
|
|
| 3. |
- Kosek, David M, et al.
(author)
-
Efficient 3′-pairing renders microRNA targeting less sensitive to mRNA seed accessibility
- 2023
-
In: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 51:20, s. 11162-11177
-
Journal article (peer-reviewed)abstract
- MicroRNAs (miRNAs) are short RNAs that post-transcriptionally regulate gene expression by binding to specific sites in mRNAs. Site recognition is primarily mediated by the seed region (nucleotides g2–g8 in the miRNA), but pairing beyond the seed (3′-pairing) is important for some miRNA:target interactions. Here, we use SHAPE, luciferase reporter assays and transcriptomics analyses to study the combined effect of 3′-pairing and secondary structures in mRNAs on repression efficiency. Using the interaction between miR-34a and its SIRT1 binding site as a model, we provide structural and functional evidence that 3′-pairing can compensate for low seed-binding site accessibility, enabling repression of sites that would otherwise be ineffective. We show that miRNA 3′-pairing regions can productively base-pair with nucleotides far upstream of the seed-binding site and that both hairpins and unstructured bulges within the target site are tolerated. We use SHAPE to show that sequences that overcome inaccessible seed-binding sites by strong 3′-pairing adopt the predicted structures and corroborate the model using luciferase assays and high-throughput modelling of 8177 3′-UTR targets for six miRNAs. Finally, we demonstrate that PHB2, a target of miR-141, is an inaccessible target rescued by efficient 3′-pairing. We propose that these results could refine predictions of effective target sites.
|
|
| 4. |
|
|
| 5. |
- Petzold, Katja, 1981-, et al.
(author)
-
Conserved nucleotides in an RNA essential for hepatitis B virus replication show distinct mobility patterns.
- 2007
-
In: Nucleic Acids Research. - : Oxford University Press (OUP). - 1362-4962 .- 0305-1048. ; 35:20, s. 6854-6861
-
Journal article (peer-reviewed)abstract
- The number of regulatory RNAs with identified non-canonical structures is increasing, and structural transitions often play a role in their biological function. This stimulates interest in internal motions of RNA, which can underlie structural transitions. Heteronuclear NMR relaxation measurements, which are commonly used to study internal motion, only report on local motions of few sites within the molecule. Here we have studied a 27-nt segment of the human hepatitis B virus (HBV) pregenomic RNA, which is essential for viral replication. We combined heteronuclear relaxation with the new off-resonance ROESY technique, which reports on internal motions of H,H contacts. Using off-resonance ROESY, we could for the first time detect motion of through-space H,H contacts, such as in intra-residue base-ribose contacts or inter-nucleotide contacts, both essential for NMR structure determination. Motions in non-canonical structure elements were found primarily on the sub-nanosecond timescale. Different patterns of mobility were observed among several mobile nucleotides. The most mobile nucleotides are highly conserved among different HBV strains, suggesting that their mobility patterns may be necessary for the RNA’s biological function.
|
|
| 6. |
- Petzold, Katja, 1981-
(author)
-
NMR studies of host-pathogen interactions
- 2009
-
Doctoral thesis (other academic/artistic)abstract
- This thesis describes the use of Nuclear Magnetic Resonance (NMR) for characterizing two host-pathogen interactions: The behavior of a regulatory RNA of the Hepatitis B virus (HBV) and the attachment of Helicobacter pylori (H. pylori) to the gastric mucosa. NMR is a powerful tool in biomedicine, because molecules ranging from small ligands to biomacromolecules can be studied with atomic resolution. Different NMR experiments are designed to determine structures, or to monitor interactions, folding, stability or motion. Paper I describes the analysis of the motions of a regulatory RNA of HBV. The NMR structure of the RNA had revealed before that several well-conserved nucleotides adopt multiple conformations. Therefore an analysis of possible underlying motions was undertaken using two different NMR techniques, one of which (off-resonance ROESY) was applied to nucleic acids for the first time. The observed motions suggest an explanation why the structurally poorly defined nucleotides are highly conserved. In paper II we improved the ROESY NMR experiment, which is used to measure internuclear distances for structure determination of medium-sized molecules. Using a small protein and an organometallic complex as examples, we demonstrated that the new EASY ROESY experiment yields clean spectra that can directly be integrated to derive interatomic distances. H. pylori, the bacterium involved in peptic ulcer disease and gastric cancer, survives in the harsh acidic environment of the stomach. It possesses many membrane proteins which mediate adherence, raising the question, if their activity is related to membrane composition. In paper III & IV we analyzed therefore the phospholipid composition of H. pylori membranes. In paper III, an advanced method for the analysis of the phospholipid composition of biological membranes was developed. The two-dimensional semi-constant-time 31P,1H-COSY experiment combines information from phosphorus and hydrogen atoms of phospholipids for their unambiguous identification. Furthermore, the high resolution of the two-dimensional experiment allows the quantification of phospholipids where conventional methods fail. In paper IV we applied the new experiment to analyze the lipid composition of whole H. pylori cells, their inner and outer membranes, and of vesicles shed by the bacterium. The goal of this study was to characterize the vesicles which are suggested to play a role in the inflammation process. We established that the outer membrane and the vesicles have similar phospholipid compositions, suggesting that the vesicles are largely derived from the outer membrane. The NMR results presented here elucidate details of molecular systems engaged in pathogenicity, as basis for therapeutic strategies against these pathogens.
|
|
| 7. |
- Thiele, Christina Marie, et al.
(author)
-
EASY ROESY : reliable cross-peak integration in adiabatic symmetrized ROESY
- 2009
-
In: Chemistry - A European Journal. - : Wiley. - 0947-6539 .- 1521-3765. ; 15:3, s. 585-588
-
Journal article (peer-reviewed)abstract
- Estimates of intramolecular distances are essential for structure determination. For medium-sized molecules, ROESY NMR is the method of choice for obtaining distances. However, the integration of ROESY cross-peaks is problematic due to the offset dependence of theintegrals and/or TOCSY artefacts. We here present EASY ROESY (rEliable Adiabatic SYmmetrized ROESY), which yields reliable intramolecular distances without sample-specific setup.
|
|
