Sampling native-like structures of RNA-protein complexes through Rosetta folding and docking

AbstractRNA-protein complexes underlie numerous cellular processes including translation, splicing, and posttranscriptional regulation of gene expression. The structures of these complexes are crucial to their functions but often elude high-resolution structure determination. Computational methods are needed that can integrate low-resolution data for RNA-protein complexes while modeling de novo the large conformational changes of RNA components upon complex formation. To address this challenge, we describe a Rosetta method called RNP-denovo to simultaneously fold and dock RNA to a protein surface. On a benchmark set of structurally diverse RNA-protein complexes that are not solvable with prior strategies, this fold-and-dock method consistently sampled native-like structures with better than nucleotide resolution. We revisited three past blind modeling challenges in which previous methods gave poor results: human telomerase, an RNA methyltransferase with a ribosomal RNA domain, and the spliceosome. When coupled with the same sparse FRET, cross-linking, and functional data used in previous work, RNP-denovo gave models with significantly improved accuracy. These results open a route to computationally modeling global folds of RNA-protein complexes from low-resolution data.

Download Full-text

On the Use of low-resolution Data to Improve Structure Prediction of Proteins and Protein Complexes

Journal of Chemical Theory and Computation ◽

10.1021/ct900305m ◽

2009 ◽

Vol 5 (11) ◽

pp. 3129-3137 ◽

Cited By ~ 7

Author(s):

Marco D’Abramo ◽

Tim Meyer ◽

Pau Bernadó ◽

Carles Pons ◽

Juan Fernández Recio ◽

...

Keyword(s):

Structure Prediction ◽

Protein Complexes ◽

Low Resolution ◽

Resolution Data

Download Full-text

Distinct RNA-unwinding mechanisms of DEAD-box and DEAH-box RNA helicase proteins in remodeling structured RNAs and RNPs

Biochemical Society Transactions ◽

10.1042/bst20170095 ◽

2017 ◽

Vol 45 (6) ◽

pp. 1313-1321 ◽

Cited By ~ 27

Author(s):

Benjamin Gilman ◽

Pilar Tijerina ◽

Rick Russell

Keyword(s):

Conformational Changes ◽

Protein Complexes ◽

Rna Helicase ◽

Conformational Transitions ◽

Messenger Rnas ◽

Base Pairs ◽

Dead Box ◽

Evolutionarily Conserved ◽

Partially Unfolded ◽

Rna Protein Complexes

Structured RNAs and RNA–protein complexes (RNPs) fold through complex pathways that are replete with misfolded traps, and many RNAs and RNPs undergo extensive conformational changes during their functional cycles. These folding steps and conformational transitions are frequently promoted by RNA chaperone proteins, notably by superfamily 2 (SF2) RNA helicase proteins. The two largest families of SF2 helicases, DEAD-box and DEAH-box proteins, share evolutionarily conserved helicase cores, but unwind RNA helices through distinct mechanisms. Recent studies have advanced our understanding of how their distinct mechanisms enable DEAD-box proteins to disrupt RNA base pairs on the surfaces of structured RNAs and RNPs, while some DEAH-box proteins are adept at disrupting base pairs in the interior of RNPs. Proteins from these families use these mechanisms to chaperone folding and promote rearrangements of structured RNAs and RNPs, including the spliceosome, and may use related mechanisms to maintain cellular messenger RNAs in unfolded or partially unfolded conformations.

Download Full-text

Crystal structure of a Y-box binding protein 1 (YB-1)–RNA complex reveals key features and residues interacting with RNA

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.007545 ◽

2019 ◽

Vol 294 (28) ◽

pp. 10998-11010 ◽

Cited By ~ 15

Author(s):

Xiao-Juan Yang ◽

Hong Zhu ◽

Shi-Rong Mu ◽

Wen-Juan Wei ◽

Xun Yuan ◽

...

Keyword(s):

Gene Expression ◽

Posttranscriptional Regulation ◽

Rna Binding ◽

Binding Protein ◽

Consensus Sequence ◽

Regulation Of Gene Expression ◽

Binding Activity ◽

Core Motif ◽

Variable Exon ◽

Nucleotide Resolution

The Y-box binding protein 1 (YB-1) is a member of the cold shock domain (CSD) protein family and is recognized as an oncogenic factor in several solid tumors. By binding to RNA, YB-1 participates in several steps of posttranscriptional regulation of gene expression, including mRNA splicing, stability, and translation; microRNA processing; and stress granule assembly. However, the mechanisms in YB-1–mediated regulation of RNAs are unclear. Previously, we used both systematic evolution of ligands by exponential enrichment (SELEX) and individual-nucleotide resolution UV cross-linking and immunoprecipitation coupled RNA-Seq (iCLIP-Seq) analyses, which defined the RNA-binding consensus sequence of YB-1 as CA(U/C)C. We also reported that through binding to its core motif CAUC in primary transcripts, YB-1 regulates the alternative splicing of a CD44 variable exon and the biogenesis of miR-29b-2 during both Drosha and Dicer steps. To elucidate the molecular basis of the YB-1–RNA interactions, we report high-resolution crystal structures of the YB-1 CSD in complex with different RNA oligos at 1.7 Å resolution. The structure revealed that CSD interacts with RNA mainly through π–π stacking interactions assembled by four highly conserved aromatic residues. Interestingly, YB-1 CSD forms a homodimer in solution, and we observed that two residues, Tyr-99 and Asp-105, at the dimer interface are important for YB-1 CSD dimerization. Substituting these two residues with Ala reduced CSD's RNA-binding activity and abrogated the splicing activation of YB-1 targets. The YB-1 CSD–RNA structures presented here at atomic resolution provide mechanistic insights into gene expression regulated by CSD-containing proteins.

Download Full-text

Fast and unbiased purification of RNA-protein complexes after UV cross-linking

10.1101/597575 ◽

2019 ◽

Author(s):

Erika C Urdaneta ◽

Benedikt M Beckmann

Keyword(s):

Protein Interactions ◽

Protein Complexes ◽

Regulation Of Gene Expression ◽

Source Material ◽

Regulatory Rna ◽

Polyadenylated Rna ◽

Simple Protocol ◽

The Individual ◽

Post Transcriptional Regulation ◽

Rna Protein Complexes

Post-transcriptional regulation of gene expression in cells is facilitated by formation of RNA-protein complexes (RNPs). While many methods to study eukaryotic (m)RNPs rely on purification of polyadenylated RNA, other important regulatory RNA classes or bacterial mRNA could not be investigated at the same depth. To overcome this limitation, we developed Phenol Toluol extraction (PTex), a novel and unbiased method for the purification of UV cross-linked RNPs in living cells. PTex is a fast (2-3 hrs) and simple protocol. The purification principle is solely based on physicochemical properties of cross-linked RNPs, enabling us to interrogate RNA-protein interactions system-wide and beyond poly(A) RNA from a variety of species and source material. Here, we are presenting an introduction of the underlying separation principles and give a detailed discussion of the individual steps as well as incorporation of PTex in high-throughput pipelines.

Download Full-text

Plant Individual Nucleotide Resolution Cross-Linking and Immunoprecipitation to Characterize RNA-Protein Complexes

Methods in Molecular Biology - RNA Tagging ◽

10.1007/978-1-0716-0712-1_15 ◽

2020 ◽

pp. 255-267

Author(s):

Tino Köster ◽

Dorothee Staiger

Keyword(s):

Protein Complexes ◽

Cross Linking ◽

Nucleotide Resolution ◽

Rna Protein Complexes

Download Full-text

De novo computational RNA modeling into cryoEM maps of large ribonucleoprotein complexes

10.1101/332791 ◽

2018 ◽

Cited By ~ 1

Author(s):

Kalli Kappel ◽

Shiheng Liu ◽

Kevin P. Larsen ◽

Georgios Skiniotis ◽

Elisabetta Viani Puglisi ◽

...

Keyword(s):

Rna Splicing ◽

Structure Prediction ◽

Model Building ◽

De Novo ◽

Protein Complexes ◽

Real Space ◽

Model Interpretation ◽

Ribonucleoprotein Complexes ◽

Mitochondrial Ribosome ◽

Rna Protein Complexes

AbstractRNA-protein assemblies carry out many critical biological functions including translation, RNA splicing, and telomere extension. Increasingly, cryo-electron microscopy (cryoEM) is used to determine the structures of these complexes, but nearly all maps determined with this method have regions in which the local resolution does not permit manual coordinate tracing. Because RNA coordinates typically cannot be determined by docking crystal structures of separate components and existing structure prediction algorithms cannot yet model RNA-protein complexes, RNA coordinates are frequently omitted from final models despite their biological importance. To address these omissions, we have developed a new framework for De novo Ribonucleoprotein modeling in Real-space through Assembly of Fragments Together with Electron density in Rosetta (DRRAFTER). We show that DRRAFTER recovers near-native models for a diverse benchmark set of small RNA-protein complexes, as well as for large RNA-protein machines, including the spliceosome, mitochondrial ribosome, and CRISPR-Cas9-sgRNA complexes where the availability of both high and low resolution maps enable rigorous tests. Blind tests on yeast U1 snRNP and spliceosomal P complex maps demonstrate that the method can successfully build RNA coordinates in real-world modeling scenarios. Additionally, to aid in final model interpretation, we present a method for reliable in situ estimation of DRRAFTER model accuracy. Finally, we apply this method to recently determined maps of telomerase, the HIV-1 reverse transcriptase initiation complex, and the packaged MS2 genome, demonstrating that DRRAFTER can be used to accelerate accurate model building in challenging cases.

Download Full-text

Proteins and direct methods

Zeitschrift für Kristallographie - Crystalline Materials ◽

10.1524/zkri.1991.197.3-4.197 ◽

1991 ◽

Vol 197 (3-4) ◽

Cited By ~ 7

Author(s):

Fan Hai-fu ◽

Hao Quan ◽

M. M. Woolfson

Keyword(s):

First Principles ◽

Figure Of Merit ◽

Direct Methods ◽

Large Structure ◽

Low Resolution ◽

Figures Of Merit ◽

Large Structures ◽

Resolution Data

AbstractConventional direct methods, which work so well for small structures, are less successful for macromolecules. Where it has been demonstrated that a solution might be found using direct methods it is then found that the usual figures of merit are unable to distinguish the few good sets of phases from the large number of sets generated. The reasons for the difficulties with very large structures are considered from a first-principles approach taking into account both the factors of having a large number of atoms and low resolution data. A proposal is made for trying to recognize good phase sets by taking a large structure as a sum of a number of smaller structures for each of which a conventional figure of merit can be applied.

Download Full-text

Faculty Opinions recommendation of Super-resolution biomolecular crystallography with low-resolution data.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.3066960.2795059 ◽

2010 ◽

Author(s):

Fred Dyda

Keyword(s):

Super Resolution ◽

Low Resolution ◽

Biomolecular Crystallography ◽

Resolution Data

Download Full-text

STAU2 protein level is controlled by caspases and the CHK1 pathway and regulates cell cycle progression in the non-transformed hTERT-RPE1 cells

BMC Molecular and Cell Biology ◽

10.1186/s12860-021-00352-y ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Lionel Condé ◽

Yulemi Gonzalez Quesada ◽

Florence Bonnet-Magnaval ◽

Rémy Beaujois ◽

Luc DesGroseillers

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Cell Proliferation ◽

Dna Damage ◽

Steady State ◽

Posttranscriptional Regulation ◽

Cell Cycle Progression ◽

Rna Binding ◽

Regulation Of Gene Expression ◽

Cycle Progression

AbstractBackgroundStaufen2 (STAU2) is an RNA binding protein involved in the posttranscriptional regulation of gene expression. In neurons, STAU2 is required to maintain the balance between differentiation and proliferation of neural stem cells through asymmetric cell division. However, the importance of controlling STAU2 expression for cell cycle progression is not clear in non-neuronal dividing cells. We recently showed that STAU2 transcription is inhibited in response to DNA-damage due to E2F1 displacement from theSTAU2gene promoter. We now study the regulation of STAU2 steady-state levels in unstressed cells and its consequence for cell proliferation.ResultsCRISPR/Cas9-mediated and RNAi-dependent STAU2 depletion in the non-transformed hTERT-RPE1 cells both facilitate cell proliferation suggesting that STAU2 expression influences pathway(s) linked to cell cycle controls. Such effects are not observed in the CRISPR STAU2-KO cancer HCT116 cells nor in the STAU2-RNAi-depleted HeLa cells. Interestingly, a physiological decrease in the steady-state level of STAU2 is controlled by caspases. This effect of peptidases is counterbalanced by the activity of the CHK1 pathway suggesting that STAU2 partial degradation/stabilization fines tune cell cycle progression in unstressed cells. A large-scale proteomic analysis using STAU2/biotinylase fusion protein identifies known STAU2 interactors involved in RNA translation, localization, splicing, or decay confirming the role of STAU2 in the posttranscriptional regulation of gene expression. In addition, several proteins found in the nucleolus, including proteins of the ribosome biogenesis pathway and of the DNA damage response, are found in close proximity to STAU2. Strikingly, many of these proteins are linked to the kinase CHK1 pathway, reinforcing the link between STAU2 functions and the CHK1 pathway. Indeed, inhibition of the CHK1 pathway for 4 h dissociates STAU2 from proteins involved in translation and RNA metabolism.ConclusionsThese results indicate that STAU2 is involved in pathway(s) that control(s) cell proliferation, likely via mechanisms of posttranscriptional regulation, ribonucleoprotein complex assembly, genome integrity and/or checkpoint controls. The mechanism by which STAU2 regulates cell growth likely involves caspases and the kinase CHK1 pathway.

Download Full-text