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

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."


Contact: Thania Benios
Rockefeller University

Related biology news :

1. Iowa State researcher develops new treatment method for canine eye diseases
2. Fingerprinting method tracks mercury emissions from coal
3. Deep biosphere research points to new methods for recovering petroleum
4. New lab manual focuses on essential methods for purifying and characterizing proteins
5. UC Riverside biochemists devise method for bypassing aluminum toxicity effects in plants
6. Commercial aquatic plants offer cost-effective method for treating wastewater
7. Radioactivity: Discover the lowest amounts with new methods
8. New method identifies meth hot spots
9. Scientists develop new method to investigate origin of life
10. Biophysical method may help to recover hearing
11. LSUHSC research reports new method to protect brain cells from diseases like Alzheimers
Post Your Comments:
(Date:11/18/2015)... 18, 2015  As new scientific discoveries deepen our ... other healthcare providers face challenges in better using that ... In addition, as more children continue to survive pediatric ... and old age. John M. Maris, M.D ... of Philadelphia (CHOP) . --> John ...
(Date:11/17/2015)... 2015 Pressure BioSciences, Inc. (OTCQB: PBIO) ("PBI" ... sale of broadly enabling, pressure cycling technology ("PCT")-based sample ... announced it has received gross proceeds of $745,000 from ... (the "Offering"), increasing the total amount raised to date ... closings are expected in the near future. ...
(Date:11/11/2015)... MINNETONKA, Minn. , Nov. 11, 2015   MedNet ... the entire spectrum of clinical research, is pleased to announce ... Partnerships in Clinical Trials (PCT) event, to be held ... will be able to view live demonstrations of ... platform, and learn how iMedNet has been able ...
Breaking Biology News(10 mins):
(Date:11/24/2015)... ... November 24, 2015 , ... This fall, global software solutions leader ... five states to develop and pitch their BIG ideas to improve health and wellness ... competing for votes to win the title of SAP's Teen Innovator, an all-expenses paid ...
(Date:11/24/2015)... Nov. 24, 2015  Tikcro Technologies Ltd. (OTCQB: TIKRF) today announced that ... 2015 at 11:00 a.m. Israel time, at the ... Yigal Allon Street, 36 th Floor, Tel Aviv, Israel ... Paneth and Izhak Tamir to the Board of Directors; ... external directors; , approval of an amendment to certain terms of ...
(Date:11/24/2015)... ... November 24, 2015 , ... InSphero AG, the leading supplier of ... has promoted Melanie Aregger to serve as Chief Operating Officer. , Having ... management team and was promoted to Head of InSphero Diagnostics in 2014. ...
(Date:11/24/2015)... , Nov. 24, 2015 HemoShear ... on discovering drugs for metabolic disorders, announced today ... to its Board of Directors (BOD). Mr. Watkins ... of Human Genome Sciences (HGS), and also served ... Jim Powers , Chairman and CEO ...
Breaking Biology Technology: