New method provides panoramic view of protein-RNA interactions in living cells

November 03, 2008

DNA, it has turned out, isn't all it was cracked up to be. In recent years we learned that the molecule of life, the discovery of the 20th century, did not -- could not -- by itself explain the huge differences in complexity between a human and a worm. Forced to look elsewhere, scientists turned to RNA, a direct yet more complex transcript of DNA. But methodological problems have historically plagued the study of RNA regulation in living cells, limiting not only the accuracy of results but also our understanding of RNA's role in human disease.

But now, in research to appear in the November 2 advance online issue of Nature, Robert B. Darnell, head of the Laboratory of Molecular Neuro-oncology at Rockefeller University and a Howard Hughes Medical Institute investigator, and his team have changed all that.

By adapting techniques mastered in the test tube and combining them with high throughput technology, the team has developed a genome-wide platform to study how specialized proteins regulate RNA in living, intact cells. The platform allows researchers to identify, in a single experiment, every sequence within every strand of RNA to which proteins bind. The result is an unbiased and unprecedented look at how differences in RNA can explain how a worm and a human can each have 25,000 genes yet be so different.

"RNA offers a way to make the cell much more complex than what this limited set of genes can offer," says Darnell, who is Robert and Harriet Heilbrunn Professor at Rockefeller. "But how is RNA being regulated in different conditions and diseases, and in different cell types? With this platform, we now have a way to address all these questions."

Traditional methods used molecules to extract protein-RNA complexes from living tissue. But often the molecule only extracted the RNA. Other times, the protein bound too weakly to survive the purification process, which involved stripping the complex of unwanted debris. To address the issue, Darnell and his team used a trick from test-tube biochemistry that molecularly cements these regulatory proteins to RNA at the moment they touch. The technique, when applied to high throughput sequencing, is called high throughput sequencing-cross linking immunoprecipitation, or HITS-CLIP for short.

Since the RNA and RNA-binding protein are fused together, the researchers can really beat up the extract and rigorously purify the protein without fear of losing the RNA. At the end of the day, they are left with the RNA sequence to which the protein was bound. They can then take these sequences to Rockefeller's high throughput sequence facility, and with the help of Research Support Specialist Scott Dewell, overlay them onto the genome and see where they match. What they get is a map of every position on every transcribed RNA where the RNA binding protein is binding.

When DNA is transcribed into RNA, the primary transcript is divided into many blocks called exons, which are separated by empty spaces. In order to convert the transcript into some sort of message, all the spaces need to be removed; but if an exon is dropped, a different version of that protein, which could carry a very different message, is created. "That's RNA splicing," says first author Donny Licatalosi, a postdoctoral associate in the lab. "It is what gives rise to this massive pool of diverse and complex tissues with a relatively small number of genes."

In the past, the group used a sophisticated process of evidence and inference to make predictions of the points of regulation along the transcript. "Now, we have direct biochemical validation that these interactions occur in the brain to regulate splicing," says Licatalosi.

And as it turns out, "The observed map -- and this was amazing -- looked just like our predicted map," says Darnell.

Darnell, Licatalosi and their colleagues Aldo Mele, a research assistant, John Fak, a research assistant, Sung-Wook Chi, a graduate fellow in computational biology and medicine, Xuning Wang, assistant director of biocomputing and Jennifer Darnell, a research associate professor, looked at an RNA-binding protein called Nova2 that is found exclusively in neurons. They found that depending on where Nova2 binds to RNA, they could predict and directly observe whether an exon would be included or excluded in the final transcript, and which protein version it created. "The cell seems to be going through great trouble to regulate these RNAs in different conditions and different cell types," says Darnell. "When RNA developed the ability to make a more stable copy of itself -- DNA -- it didn't write itself off as a relic for the textbooks. It stayed at the core of complex processes in the cell."

Rockefeller University

---

---

	RNA: Protein Interactions: A Practical Approach by Christopher W. J. Smith (Editor) RNA-Protein Interactions: A Practical Approach provides a clear and comprehensive guide to the many experimental procedures used in studying RNA-protein interactions. The approaches covered range from those initially used to detect a novel RNA-protein interaction to various biochemical and genetic approaches, and from in-depth methods for analyzing the structural basis of the interaction to methods for purifying and cloning RNA binding proteins. The volume contains a number of procedures that have not previously been covered in this type of manual, including the production of site-specifically modified RNAs by enzymatic and chemical methods and in vivo screening for novel RNA-protein interactions in yeast and E. coli. This informative book will be a valuable aid to laboratory workers in...
	RNA-Protein Interaction Protocols (Methods in Molecular Biology) by Ren-Jang Lin (Editor) Due to the vital biological importance of RNA and proteins functioning together within a cell, a protocol volume describing experimental procedures to study their interactions should find a home in many laboratories. RNA-Protein Interaction Protocols, Second Edition updates, complements, and expands upon the popular first edition by providing a collection of cutting-edge techniques developed or refined in the past few years along with tried-and-true methods. The expert contributors explore the isolation and characterization of RNA-protein complexes, the analysis and measurement of RNA-protein interaction, and related novel techniques and strategies. Written in the highly successful Methods in Molecular Biology™ series format, the chapters include brief introductions to the material,...
	RNA-Protein Interactions (Frontiers in Molecular Biology) by Kiyoshi Nagai (Editor), Iain W. Mattaj (Editor) The study of RNA-protein interactions is crucial to understanding the mechanisms and control of gene expression and protein synthesis. The realization that RNAs are often far more biologically active than was previously appreciated has stimulated a great deal of new research in this field. Uniquely, in this book, the world's leading researchers have collaborated to produce a comprehensive and current review of RNA-protein interactions for all scientists working in this area. Timely, comprehensive, and authoritative, this new Frontiers title will be invaluable for all researchers in molecular biology, biochemistry and structural biology.
	Adenovirus Methods and Protocols: Volume 2: Ad Proteins and RNA, Lifecycle and Host Interactions, and Phyologenetics (Methods in Molecular Medicine) (v. 2) by William S. M. Wold (Editor), Ann E. Tollefson (Editor) Adenovirus Methods and Protocols, Second Edition, now in two volumes, is an essential resource for adenovirus (Ad) researchers beginning in the field, and an inspirational starting point for researchers looking to branch into new areas of Ad study. In addition to updating and expanding important chapters from the first edition, the authors have added new chapters that address innovative, exciting areas of emphasis in Ad research, including Ad vector construction and use, real-time PCR, use of new animal models, and methods for quantification of Ad virus or virus expression/interactions. The protocols presented are written by trendsetting researchers.
	Adenovirus Methods and Protocols: Volume 2: Ad Proteins and RNA, Lifecycle and Host Interactions, and Phyologenetics (Methods in Molecular Medicine) by William S. M. Wold (Editor), Ann E. Tollefson (Editor) Adenovirus Methods and Protocols, Second Edition, now in two volumes, is an essential resource for adenovirus (Ad) researchers beginning in the field, and an inspirational starting point for researchers looking to branch into new areas of Ad study. In addition to updating and expanding important chapters from the first edition, the authors have added new chapters that address innovative, exciting areas of emphasis in Ad research, including Ad vector construction and use, real-time PCR, use of new animal models, and methods for quantification of Ad virus or virus expression/interactions. The protocols presented are written by trendsetting researchers.
	Global Analysis of Protein-RNA Interactions: Discovery of RNA-Protein Networks and Their Implications in Human Disease by André Gerber (Author) It is becoming increasingly evident that post-transcriptional regulation has profound impact on when, where, and to what level genes are expressed. Hundreds of RNA-binding proteins and non-coding RNAs mediate post-transcriptional gene regulation – but how they are coordinated and what their RNA targets are remains largely elusive. The development of genome-wide analysis tools enables us now to systematically study the extent and logic of post-transcriptional gene regulation on global scale. In this book, the author André Gerber explains some of these new techniques, which led to the discovery of novel principles of gene expression control such as RNA regulons. He also provides an overview of current knowledge about the implications of RNA-binding proteins and non-coding RNAs in human...
	PostTranscriptional Regulation by STAR Proteins: Control of RNA Metabolism in Development and Disease (Advances in Experimental Medicine and Biology) by Talila Volk (Editor), Karen Artzt (Editor) This book aims to bring to the forefront a field that has been developing since the late 1990s called the STAR pathway for Signal Transduction and Activation of RNA. It is a signaling pathway that targets RNA directly; in contrast to the canonical signal—kinase cascade—transcription factor—DNA—RNA. It is proposed to allow quick responses to environment changes such as those necessary in many biological phenomenon such as the nervous system and during development.
	Structural Studies of Protein-Nucleic Acid Interaction: The Sources of Sequence-Specific Binding by Thomas A. Steitz (Author) The study of the interactions of proteins, whether they be enzymes, regulatory proteins or structural proteins, with nucleic acids is one of the most exciting areas of modern molecular biology. In this 1993 text, Professor Steitz reviews the wide-ranging research in structural studies of DNA-binding proteins and their complexes with DNA. The author clearly and concisely describes the uses of techniques in molecular genetics, DNA synthesis, protein crystallography and nuclear magnetic response. The book, by a now Nobel Prize-winning author, will continue to be enjoyed by anyone with an interest in this exciting area of research.
	Protein-Nucleic Acid Interactions: Structural Biology (RSC Biomolecular Sciences) by Phoebe A. Rice (Editor), Carl C. Correll (Editor) This book provides both in-depth background and up-to-date information in this area. The chapters are organized by general themes and principles, written by experts who illustrate topics with current findings. Topics covered include: - the role of ions and hydration in protein-nucleic acid interactions - transcription factors and combinatorial specificity - indirect readout of DNA sequence - single-stranded nucleic acid binding proteins - nucleic acid junctions and proteins, - RNA protein recognition - recognition of DNA damage. It will be a key reference for both advanced students and established scientists wishing to broaden their horizons.
	Rna-Protein Interactions in the Bacteriophage Ms2 (Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science & Technology) by Elin Grahn (Author)