an-optimized-quantitative-pull-down-analysis-rna-binding-proteins

An Optimized Quantitative Pull-Down Analysis of RNA-Binding Proteins Using Short Biotinylated RNA

Protein-RNA interactions regulate gene expression and cellular functions at transcriptional and post-transcriptional levels. For this reason, identifying ...

Italian Institute of Technology (IIT)

Sapienza University of Rome

Condensation, or liquid-like phase separation, is a phenomenon indispensable for the spatiotemporal regulation of molecules within the cell. Recent studies indicate that the composition and molecular organization of phase-separated organelles such as Stress Granules (SGs) and Processing Bodies (PBs) are highly variable and dynamic. A dense contact network involving both RNAs and proteins controls the formation of SGs and PBs and an intricate molecular architecture, at present poorly understood, guarantees that these assemblies sense and adapt to different stresses and environmental changes. Here, we investigated the physico-chemical properties of SGs and PBs components and studied the architecture of their interaction networks. We found that proteins and RNAs establishing the largest amount of contacts in SGs and PBs have distinct properties and intrinsic disorder is enriched in all protein-RNA, protein-protein and RNA-RNA interaction networks. The increase of disorder in proteins is accompanied by an enrichment in single-stranded regions of RNA binding partners. Our results suggest that SGs and PBs quickly assemble and disassemble through dynamic contacts modulated by unfolded domains of their components.

The Interplay Between Disordered Regions in RNAs and Proteins Modulates Interactions Within Stress Granules and Processing Bodies.

The new flow of high-throughput RNA secondary structure data coming from different techniques allowed the further development of machine learning approaches. We developed CROSS and CROSSalive, two algorithms trained on experimental data able to predict the RNA secondary structure propensity both in vitro and in vivo. Since the in vivo folding of RNA molecules depends on multiple factors due to the cellular crowded environment, prediction is a complex problem that needs additional calculations for the interaction with proteins and other molecules. In the following chapter, we will describe the differences in predicting RNA secondary structure propensity using experimental data as input for an Artificial Neural Network (ANN) in vitro and in vivo.

Predicting RNA Secondary Structure Using In Vitro and In Vivo Data.

Phase separation drives X-chromosome inactivation.

Aptamers are artificial oligonucleotides binding to specific molecular targets. They have a promising role in therapeutics and diagnostics but are often difficult to design. Here, we exploited the catRAPID algorithm to generate aptamers targeting TAR DNA-binding protein 43 (TDP-43), whose aggregation is associated with Amyotrophic Lateral Sclerosis. On the pathway to forming insoluble inclusions, TDP-43 adopts a heterogeneous population of assemblies, many smaller than the diffraction-limit of light. We demonstrated that our aptamers bind TDP-43 and used the tightest interactor, Apt-1, as a probe to visualize TDP-43 condensates with super-resolution microscopy. At a resolution of 10 nanometers, we tracked TDP-43 oligomers undetectable by standard approaches. In cells, Apt-1 interacts with both diffuse and condensed forms of TDP-43, indicating that Apt-1 can be exploited to follow TDP-43 phase transition. The de novo generation of aptamers and their use for microscopy opens a new page to study protein condensation.

Article	Year
The Interplay Between Disordered Regions in RNAs and Proteins Modulates Interactions Within Stress Granules and Processing Bodies. Journal of molecular biology\| PubMed ID: 34274326	2022
Predicting RNA Secondary Structure Using In Vitro and In Vivo Data. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 34694602	2022
Phase separation drives X-chromosome inactivation. Nature structural & molecular biology\| PubMed ID: 35301494	2022
Probing TDP-43 condensation using an in silico designed aptamer. Nature communications\| PubMed ID: 35739092	2022

Gian Gaetano Tartaglia

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