Discovering protein smartphones

EMBL NEWS

New technique invented by EMBL researchers reveals uncharted docking sites in RNA-binding proteins

Some proteins are less like landlines and more like smartphones: they can do more than just talk to other proteins. One molecular app of particular interest is the ‘RNA-binding domain’, which lets proteins engage with RNA and influence how a cell responds to its environment. Lots of proteins use it – even ones that do not appear to have one. So how do you find an app that is clearly in use but has an invisible launch site? Researchers at EMBL invented a technique to do just that. Called RBDmap, the new method was recently published in Molecular Cell.

“We are one step closer to understanding how RNA and proteins interact,” says Matthias Hentze, who led the study.

Decades of research in the RNA field confirmed that proteins of a certain architecture can bind to RNA. But when the Hentze lab systematically searched for proteins that are able to bind to RNA using next generation methods, they saw something surprising: many of the proteins they discovered did not have any signature that could explain their RNA-binding ability. And yet, these enigmRBPs – as they came to be called – could still bind to RNA. But how? And why?

We are one step closer to understanding how RNA and proteins interact

In order to answer these questions, the EMBL scientists first needed to figure out which part of these enigmatic proteins does the binding in the first place. That’s where RBDmap comes in.

Think of RNA-binding proteins as people holding onto a single rope – a strand of RNA. The hands holding the rope represent the part of the protein that can interact with RNA, while the rest of the body is free to do something else. “RBDmap separates the hands from the rest of the body and identifies what these hands are and to whom they belong,” explains Alfredo Castello, who developed the technique as a staff scientist in Hentze’s lab. “It tells us exactly what part of the protein binds to RNA.”

Using this new approach, Hentze, Castello and colleagues mapped over one thousand previously unrecognised RNA-binding sites within 529 proteins. With this information, the researchers look forward to investigating how these RNA-binding proteins work. “If we can change a very small part of the protein, chances are it can no longer bind to RNA,” Hentze said. “But, the protein can still do its other jobs – which may be vital for the cell’s survival,” Hentze said. From these mutations, the researchers can begin to investigate the role of RNA binding in how cells respond to physiological stresses such as starvation and disease.

A comprehensive atlas of RNA binding domains

A new method, built on RNA interactome capture, reports a comprehensive atlas of RNA-binding domains. This mass spectrometry based approach, referred to as RBDmap, makes use of UV crosslinking, oligo(dT) capture, partial proteolysis and mass spectrometry to identify the protein regions in close contact with RNA. Applied to HeLa and HL-1 cardiomyocytes, this method revealed more than thousand RNA-binding sites in hundreds of RBPs. This sites map not only to classical RNA-binding domains, but also to proteins lacking known RNA-binding architectures. RNA-binding sites overlap with protein-protein interaction domains, enzymatic cores and disordered regions. These sites are enriched in known post-translational modifications and disease-associated mutations and thus the functional implications of these novel RNA-binding domains deserve consideration.

Comprehensive Identification of RNA-Binding Domains in Human Cells. Alfredo Castello, Bernd Fischer, Christian K. Frese, Rastislav Horos, Anne-Marie Alleaume, Sophia Foehr, Tomaz Curk, Jeroen Krijgsveld, Matthias W. Hentze. Mol Cell. DOI: http://dx.doi.org/10.1016/j.molcel.2016.06.029

The Cardiomyocyte RNA-Binding Proteome: Links to Intermediary Metabolism and Heart Disease. Yalin Liao, Alfredo Castello, Bernd Fischer, Stefan Leicht, Sophia Föehr, Christian K. Frese, Chikako Ragan, Sebastian Kurscheid, Eloisa Pagler, Hao Yang, Jeroen Krijgsveld5, Matthias W. Hentze, Thomas Preiss. Cell Reports. DOI: http://dx.doi.org/10.1016/j.celrep.2016.06.084

RNA interactome capture provides new insights into embryo development

Two recent reports have determined the RNA interactome of Drosophila melanogaster embryos, revealing hundreds of novel RBPs. One of this works study the plasticity of the RBPome during development by comparing RNA-binding activities in early and late embryo. This analysis reveal the high adaptation capacity of the RBPome to physiological changes.

Nat Commun. 2016 Jul 5;7:12128. doi: 10.1038/ncomms12128.

Global changes of the RNA-bound proteome during the maternal-to-zygotic transition in Drosophila.

The maternal-to-zygotic transition (MZT) is a process that occurs in animal embryos at the earliest developmental stages, during which maternally deposited mRNAs and other molecules are degraded and replaced by products of the zygotic genome. The zygotic genome is not activated immediately upon fertilization, and in the pre-MZT embryo post-transcriptional control by RNA-binding proteins (RBPs) orchestrates the first steps of development. To identify relevant Drosophila RBPs organism-wide, we refined the RNA interactome capture method for comparative analysis of the pre- and post-MZT embryos. We determine 523 proteins as high-confidence RBPs, half of which were not previously reported to bind RNA. Comparison of the RNA interactomes of pre- and post-MZT embryos reveals high dynamicity of the RNA-bound proteome during early development, and suggests active regulation of RNA binding of some RBPs. This resource provides unprecedented insight into the system of RBPs that govern the earliest steps of Drosophila development.

Genome Res. 2016 Jul;26(7):1000-9. doi: 10.1101/gr.200386.115. Epub 2016 Apr 28.

The mRNA-bound proteome of the early fly embryo.

Early embryogenesis is characterized by the maternal to zygotic transition (MZT), in which maternally deposited messenger RNAs are degraded while zygotic transcription begins. Before the MZT, post-transcriptional gene regulation by RNA-binding proteins (RBPs) is the dominant force in embryo patterning. We used two mRNA interactome capture methods to identify RBPs bound to polyadenylated transcripts within the first 2 h of Drosophila melanogaster embryogenesis. We identified a high-confidence set of 476 putative RBPs and confirmed RNA-binding activities for most of 24 tested candidates. Most proteins in the interactome are known RBPs or harbor canonical RBP features, but 99 exhibited previously uncharacterized RNA-binding activity. mRNA-bound RBPs and TFs exhibit distinct expression dynamics, in which the newly identified RBPs dominate the first 2 h of embryonic development. Integrating our resource with in situ hybridization data from existing databases showed that mRNAs encoding RBPs are enriched in posterior regions of the early embryo, suggesting their general importance in posterior patterning and germ cell maturation.

Postdoctoral position in RNA biology and metabolism – DKFZ Heidelberg

The German Cancer Research Center (DKFZ) invites application for a highly motivated and creative postdoctoral researcher to explore the molecular bases of metabolic and genetic regulations. The DKFZ is located in the picturesque city of Heidelberg in the South of Germany and benefits from an exceptionally strong research environment, with Germany’s oldest university and several other leading life science research institutions in Europe.  The  successful  candidate  will  join  the  lab  of  Dr.  Bruno  Galy  in  the  „virus-­‐ associated carcinogenesis unit“.

Metabolic remodeling is emerging as a key process of physiology and disease. Although posttranscriptional regulations play a key role in shaping proteomes, genetic control of metabolism has so far been mostly investigated at the level of transcription. The interconnection between metabolism and the complex life of a mRNA, from its synthesis to its translation and decay, thus remains poorly understood. Iron metabolism is ideally suited   to   study   the   posttranscriptional   regulation   of   metabolism.   Indeed,   iron homeostasis is maintained by two RNA binding factors called Iron Regulatory Proteins (IRPs), which modulate the translation or turnover of target mRNAs. In this postdoctoral project, we will use high-­‐throughput technologies developed recently to define the regulatory scope of the IRP network on a system wide scale. This integrative approach will be carried out in vivo, using state-­‐of-­‐the-­‐art  IRP mouse models. With this work we wish to unveil new facets of a central homeostatic circuit in the cell, and constitute a basis for the study of other gene regulatory networks with similar properties.

The applicant holds a Ph.D. (or equivalent degree) in biology, biochemistry or related field  and  an  excellent  publication  record.  He/She  is  expected  to  have  a  strong background  in  RNA  biology.  A  solid  experience  with  cell  and  molecular  biology techniques and in particular high throughput approaches will be essential; familiarity with standard bioinformatical analysis of large data sets is also required. The candidate is rigorous, able to conduct independent research, but also enjoys interacting with colleagues of different disciplines and nations. Good presentation skills and experience on project management (importance of scheduling, deliverables, reporting) will be highly valued.

Funding for a total of 3 years is available and the position is open immediately. Your application (in English language) must include a CV, a summary of academic records, your research interests, and at least two letters of recommendation (or contact details of potential referees). For further question, please contact Dr. Bruno Galy (b.galy@dkfz.de).