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: 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.

The new (dis)order in RNA biology

The current paradigm of RNA-binding proteins is that they contain regions, or domains, that fold tightly into an ordered interaction platform that mediate RNA binding. In this review, we describe how this paradigm has been challenged by studies showing that other, hitherto neglected regions in RNA-binding proteins, which in spite of being intrinsically disordered, can play key functional roles in protein-RNA interactions. Proteins harbouring such disordered regions are involved in virtually every step of RNA regulation and, in some instances, have been implicated in disease. Based on exciting recent discoveries that indicate their unexpectedly pervasive role in RNA binding, we propose that the systematic study of disordered regions within RNA-binding proteins will shed light on poorly understood aspects of RNA biology and their implications in health and disease.