Capture and proteomic analysis of single RNP species

It is challenging to determine the composition of a given ribonucleoprotein. We recently approached this problem by adapting the original RNA interactome capture protocol (Castello et al., Cell 2012), to the use of specific antisense LNA probes to capture specific RNA species. We use this method to elucidate the composition of luciferase containing reporters and ribosomal RNA in vitro and in vivo. We were able to recapitulate well-established protein-RNA interactions and to discover new ones.

Specific RNP capture with antisense LNA/DNA mixmers. Rogell B, Fischer B, Rettel M, Krijgsveld J, Castello A, Hentze MW. RNA. 2017 Aug;23(8):1290-1302. doi: 10.1261/rna.060798.117. Epub 2017 May 5.

RNA2017 – Prague

We are back from the RNA2107 in Prague. Very exciting talks!

One highlight, Adrian Krainer talked about SPINRAZA, a new therapy against spinal muscular atrophy (SMA) based on antisense oligos. SMA produces the loss of motor neurons that ends up in muscle wasting and early dead (the life expectation of a new born baby with type 1 SMA is ten months). The recently developed treatment has shown excellent results in phase III clinical trials. Adrian guided us through the conception and development of the drug and included emotive videos of how SPINRAZA improves the life of children affected with SMA. An inspiring talk that demonstrates that basic science is fundamental for advancing applied research.

A Molecular View of HIV Therapy

After HIV enters a T-cell, three enzymes play essential roles in the life cycle of the virus. Reverse transcriptase copies the viral RNA genome and makes a DNA copy. Integrase inserts this viral DNA into the cell’s DNA. In the last steps of the viral life cycle, HIV protease cuts HIV proteins into their functional parts.

This animation was created based on atomic structures from the Protein Data Bank: Reverse Transcriptase: 3hvt, 3dlk, 3v6d, 3v4i, 3klg, 3v81 Integrase: 3os1, 3os0, 3oya Protease: 3pj6, 1kj4, 1hxb, 2az9, 2azc HIV Polyprotein, Capsid Protein, Matrix Protein: 1l6n, 2m8l, 1tam

Story: David S. Goodsell

Animation and Video Editing: Maria Voigt

Narration: Brian Hudson

Music: Gosta Berling

Discovering protein smartphones


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:

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: