Methods for comprehensive experimental identification of RNA-protein interactions

The importance of RNA-protein interactions in controlling mRNA regulation and non-coding RNA function is increasingly appreciated. A variety of methods exist to comprehensively define RNA-protein interactions. We describe these methods and the considerations required for designing and interpreting these experiments

Fragment-based identification of determinants of conformational and spectroscopic change at the ricin active site

<p>Abstract</p> <p>Background</p> <p>Ricin is a potent toxin and known bioterrorism threat with no available antidote. The ricin A-chain (RTA) acts enzymatically to cleave a specific adenine base from ribosomal RNA, thereby blocking translation. To understand better the relationship between ligand binding and RTA active site conformational change, we used a fragment-based approach to find a minimal set of bonding interactions able to induce rearrangements in critical side-chain positions.</p> <p>Results</p> <p>We found that the smallest ligand stabilizing an open conformer of the RTA active site pocket was an amide group, bound weakly by only a few hydrogen bonds to the protein. Complexes with small amide-containing molecules also revealed a switch in geometry from a parallel towards a splayed arrangement of an arginine-tryptophan cation-pi interaction that was associated with an increase and red-shift in tryptophan fluorescence upon ligand binding. Using the observed fluorescence signal, we determined the thermodynamic changes of adenine binding to the RTA active site, as well as the site-specific binding of urea. Urea binding had a favorable enthalpy change and unfavorable entropy change, with a ΔH of -13 ± 2 kJ/mol and a ΔS of -0.04 ± 0.01 kJ/(K*mol). The side-chain position of residue Tyr80 in a complex with adenine was found not to involve as large an overlap of rings with the purine as previously considered, suggesting a smaller role for aromatic stacking at the RTA active site.</p> <p>Conclusion</p> <p>We found that amide ligands can bind weakly but specifically to the ricin active site, producing significant shifts in positions of the critical active site residues Arg180 and Tyr80. These results indicate that fragment-based drug discovery methods are capable of identifying minimal bonding determinants of active-site side-chain rearrangements and the mechanistic origins of spectroscopic shifts. Our results suggest that tryptophan fluorescence provides a sensitive probe for the geometric relationship of arginine-tryptophan pairs, which often have significant roles in protein function. Using the unusual characteristics of the RTA system, we measured the still controversial thermodynamic changes of site-specific urea binding to a protein, results that are relevant to understanding the physical mechanisms of protein denaturation.</p

The Xist lncRNA interacts directly with SHARP to silence transcription through HDAC3

Many long non-coding RNAs (lncRNAs) affect gene expression, but the mechanisms by which they act are still largely unknown. One of the best-studied lncRNAs is Xist, which is required for transcriptional silencing of one X chromosome during development in female mammals. Despite extensive efforts to define the mechanism of Xist-mediated transcriptional silencing, we still do not know any proteins required for this role. The main challenge is that there are currently no methods to comprehensively define the proteins that directly interact with a lncRNA in the cell. Here we develop a method to purify a lncRNA from cells and identify proteins interacting with it directly using quantitative mass spectrometry. We identify ten proteins that specifically associate with Xist, three of these proteins—SHARP, SAF-A and LBR—are required for Xist-mediated transcriptional silencing. We show that SHARP, which interacts with the SMRT co-repressor that activates HDAC3, is not only essential for silencing, but is also required for the exclusion of RNA polymerase II (Pol II) from the inactive X. Both SMRT and HDAC3 are also required for silencing and Pol II exclusion. In addition to silencing transcription, SHARP and HDAC3 are required for Xist-mediated recruitment of the polycomb repressive complex 2 (PRC2) across the X chromosome. Our results suggest that Xist silences transcription by directly interacting with SHARP, recruiting SMRT, activating HDAC3, and deacetylating histones to exclude Pol II across the X chromosome

Physician and nurse well-being, patient safety and recommendations for interventions: cross-sectional survey in hospitals in six European countries.

OBJECTIVES: To determine the well-being of physicians and nurses in hospital practice in Europe, and to identify interventions that hold promise for reducing adverse clinician outcomes and improving patient safety. DESIGN: Baseline cross-sectional survey of 2187 physicians and 6643 nurses practicing in 64 hospitals in six European countries participating in the EU-funded Magnet4Europe intervention to improve clinicians' well-being. SETTING: Acute general hospitals with 150 or more beds in six European countries: Belgium, England, Germany, Ireland, Sweden and Norway. PARTICIPANTS: Physicians and nurses with direct patient contact working in adult medical and surgical inpatient units, including intensive care and emergency departments. MAIN OUTCOME MEASURES: Burnout, job dissatisfaction, physical and mental health, intent to leave job, quality of care and patient safety and interventions clinicians believe would improve their well-being. RESULTS: Poor work/life balance (57% physicians, 40% nurses), intent to leave (29% physicians, 33% nurses) and high burnout (25% physicians, 26% nurses) were prevalent. Rates varied by hospitals within countries and between countries. Better work environments and staffing were associated with lower percentages of clinicians reporting unfavourable health indicators, quality of care and patient safety. The effect of a 1 IQR improvement in work environments was associated with 7.2% fewer physicians and 5.3% fewer nurses reporting high burnout, and 14.2% fewer physicians and 8.6% fewer nurses giving their hospital an unfavourable rating of quality of care. Improving nurse staffing levels (79% nurses) and reducing bureaucracy and red tape (44% physicians) were interventions clinicians reported would be most effective in improving their own well-being, whereas individual mental health interventions were less frequently prioritised. CONCLUSIONS: Burnout, mental health morbidities, job dissatisfaction and concerns about patient safety and care quality are prevalent among European hospital physicians and nurses. Interventions to improve hospital work environments and staffing are more important to clinicians than mental health interventions to improve personal resilience

RAP-MS: A Method to Identify Proteins that Interact Directly with a Specific RNA Molecule in Cells

RNA molecules interact with proteins to perform a variety of functions in living cells. The binding partners of many RNAs, in particular the newly discovered class of long noncoding RNAs (lncRNAs), remain largely unknown. RNA antisense purification coupled with mass spectrometry (RAP-MS) is a method that enables the identification of direct and specific protein interaction partners of a specific RNA molecule. Because RAP-MS uses direct RNA–protein cross-linking methods coupled along with highly denaturing purification conditions, RAP-MS provides a short list of high confidence protein interactors

RAP-MS: A Method to Identify Proteins that Interact Directly with a Specific RNA Molecule in Cells

RNA molecules interact with proteins to perform a variety of functions in living cells. The binding partners of many RNAs, in particular the newly discovered class of long noncoding RNAs (lncRNAs), remain largely unknown. RNA antisense purification coupled with mass spectrometry (RAP-MS) is a method that enables the identification of direct and specific protein interaction partners of a specific RNA molecule. Because RAP-MS uses direct RNA–protein cross-linking methods coupled along with highly denaturing purification conditions, RAP-MS provides a short list of high confidence protein interactors

Examination of the Cell Cycle Dependence of Cytosine and Adenine Base Editors

Base editors (BEs) are genome editing agents that install point mutations with high efficiency and specificity. Due to their reliance on uracil and inosine DNA damage intermediates (rather than double-strand DNA breaks, or DSBs), it has been hypothesized that BEs rely on more ubiquitous DNA repair pathways than DSB-reliant genome editing methods, which require processes that are only active during certain phases of the cell cycle. We report here the first systematic study of the cell cycle-dependence of base editing using cell synchronization experiments. We find that nickase-derived BEs (which introduce DNA backbone nicks opposite the uracil or inosine base) function independently of the cell cycle, while non-nicking BEs are highly dependent on S-phase (DNA synthesis phase). We found that synchronization in G1 (growth phase) during the process of cytosine base editing causes significant increases in C•G to A•T "byproduct" introduction rates, which can be leveraged to discover new strategies for precise C•G to A•T base editing. We observe that endogenous expression levels of DNA damage repair pathways are sufficient to process base editing intermediates into desired editing outcomes, and the process of base editing does not significantly perturb transcription levels. Overall, our study provides mechanistic data demonstrating the robustness of nickase-derived BEs for performing genome editing across the cell cycle

Fragment-based identification of determinants of conformational and spectroscopic change at the ricin active site-2

<p><b>Copyright information:</b></p><p>Taken from "Fragment-based identification of determinants of conformational and spectroscopic change at the ricin active site"</p><p>http://www.biomedcentral.com/1472-6807/7/72</p><p>BMC Structural Biology 2007;7():72-72.</p><p>Published online 6 Nov 2007</p><p>PMCID:PMC2194779.</p><p></p>ne. The position of Tyr80 in structure 1IFS is shown in cyan, and in 2P8N in yellow. () Closer view of the effect of binding adenine on the active site. Tyr80 in structure 1IFT (without adenine) is in green, 1IFS in cyan, and 2P8N in yellow. The 2mFo-DFc electron density of Tyr80 in 2P8N is contoured at 1.5σ. () RTA-NMU complex. A single water molecule is shown in light blue. () RTA-urea complex