In modern science, ‘big data’ is a highly trendy issue,-biology is no exception. This is no surprise given that the study of life and living organisms is one of themost complicated subjects in the natural world and the data for biological research exponentially increases with each new advance in research methodology or analysis techniques. Large-scale experiments that produce immense amounts of data, and data analysis that finds the answers one needs – like finding pearls in the mud – have become extremely important in biology. Therefore collaboration is not merely encouraged, it is indispensable to extract and analyze big data. In their pursuit of analyzing the subtleties of poly(A) tails, Hyeshik Chang (industrial engineering major) and Jaechul Lim (veterinary science major) of the Center for RNA Research (Director V. Narry Kim) at the Institute for Basic Science, have shown a prime example of how their different background and skills are combined to achieve great results.

In March of 2014, a paper written by Korean scientists was published in the Molecular Cell, the sister journal of Cell. This paper featured what may be deemed the fundamentals of molecular biology and genetics, and yet it has attracted overwhelming attention. Thanks to their research, what has not been much investigated compared to its significance, due to the lack of research methodology was now at the center of interest in the field. Some people even deem their research to serve as a catalyst for mRNA research that has recently slowed down. A new technique called ‘TAIL-seq Analysis’ is the theme of the paper that jointly published by Hyeshik Chang and Jaechul Lim of the Center for RNA Research at the Institute for Basic Science (IBS).

Poly(A) Tail Research: The Road to CollaborationThis research did not start out as collaboration. It all started with Dr. Jaechul Lim of the Center for RNA Research at IBS, who was working on a miRNA study. His focus was on the mechanism for the generation and destruction of the mRNA along with how those mechanisms were controlled. As his research progressed, he began to focus on the connection between RNA’s destruction mechanism and how it related to the poly(A) tail. In addition to its connection to mRNA degradation, the tails also serves to enhance the gene expression by increase the production of proteins. This was not new information but, rather part of basic RNA theory which is familiar to every undergraduate biology student - however this was just the beginning.

“The poly(A) tail is known to be one of the major elements in mRNA. The tail prevents RNA degradation from decay enzymes and subsequently leads to increased lifespan of RNA.It also regulates the rate of translations which produces proteins from the genetic information through the interactions with ribosome,”explained Dr. Lim. “Despite the important role, the poly(A) tail research was sluggish. The recently-developed high-throughput sequencing has been just for templated parts of RNA, thus a more specialized method was required for analyzing the poly(A) tail and for more thorough study.”

For most studies dealing with mRNA, consecutive appearance of single type of base is extremely rare and analyzing highly repeating sequence is not required. For analyzing the length and sequence of poly(A) tails however, more than several dozens of such repetitions could be distinguished from shorter or longer ones for each RNA tail.

Lim explains his research methodology. “It wasn’t like what we expected in the beginning.The existing method was started by dissecting them RNA strand randomly in regard to dissected region, but we excluded that part to keep the tail intact. Instead, applying few more modifications,we could finish constructing our first sample to analyze. However, the RNA sequencing did not produce any good results as to the extremely high amount of short noncoding RNAs as compared to mRNA, the result showed a severe contamination. In our search to eliminate the contaminants and purify the mRNA fragments including tails, we applied not only web-based research, but a variety of methods which were detailed in a number of old and unpopular research papers. In our search for a viable method, we selected the most simple and efficient way to complete the experiment,” he said. The first experiment, however, did not show any long poly(A) tails. The reason for this was the repeated appearance of adenine in long stretch, which misled image processors in the sequencer.

As we know, genetic information is expressed in the arrangement of ‘bases’ in DNA or RNA, and ‘base sequencing’ is the process of checking the order of base arrangement and determining what kind of information on biological phenomena is contained within. The main problem with the modern sequencers is that they are optimized to catalog ‘complex’ base arrangements which are dynamically changing,not the highly homogenous compositions of the poly(A) tail.Dr. Hyeshik Chang illustrates the difficulty in analyzing the data. “I think that the measurement of the length of poly(A) tails is similar to the job of counting the number of character ‘A’s in a text containing hundreds of ‘A’ characters. Due to the repetition of the same letter, it is hard to count ‘A’s exactly. When we analyzed the poly(A) tail, using the standard software provided with a sequencer,the results were quite disappointing. Even for short tails, sequencer reported the tail as much longer stretches. Of course, quantitative analysis was impossible. We needed something new.”

Chang and Lim were were driven and creative in their ways of searching for a new quantitative analysis. The Center for RNA Research was equipped with a relatively recent sequencer at that time, but the results were still unsatisfactory.“It didn’t matter how recent the equipment was,it was not able to count the number of the same base patterns,” said Chang.

A Combination of Talents Creates OpportunityDr. Chang recounts how the TAIL-seq technique evolved. It was an unexpected harvest. “While attempting to perform many iterations of trial and-error in sequencing, we found that the signals coming from the long poly(A) tails are distinct from shorter tails. The sequencer incorrectly reported all of the bases were adenines. To get around this problem, we thought that using a more fundamental signal would lead to better analysis of the poly(A) tails.”

The group realized that the defect in the machine could be used to their advantage. The challenging issue was, how? Their assumption was that the analysis results varied depending on the length of tail, but it was not easy to find a specific way to test it. They decided to synthesize artificial tails to sequence and observe what signal the tail had produced.

Hyeshik Chang: “We thought about making various lengths of poly(A) tails for the test. If we figured out the quantitative relationship between the length of tail and specific pattern of signal in the analysis, we were fairly certain that we could measure the exact length of the tail. As you know, the sequencer works by labeling DNA bases with fluorescent dyes and converting them into signals, which can then be translated into base information. We focused on the duration of maintaining high level of signal rather than the actual base content. From there, we could design a working algorithm.”

In this approach, there was a natural division of labor. Lim synthesized various lengths of poly(A) tails and prepared samples to produce data for analysis while Dr. Chang modified and polished the algorithm for the sequencer using the collected data. As we know now, this approach was a great success.

Jaechul Lim: “The whole process took six months. Considering the frustration we felt in the beginning, our goal was realized quite quickly.”

Hyeshik Chang: “Working in the same physical space was very helpful. As soon as we encountered a problem, we could discuss it and find a solution. Especially because we watched things unfold in real time, we each understood the process more specifically which made it easier to find the optimal solution, regardless of any individual’s limitations. The difference of our skills or backgrounds was not a barrier to our research.”
While Lim majored in veterinary medicine going on to study RNA, Chang majored in industrial engineering, later specializing in bioinformatics and was ultimately attracted to biology. Dr.Chang stressed that seamless incorporation of methods developed in information science into classical biology is more critical than ever.

Hyeshik Chang: “The international trend in the field of biology, especially in molecular biology, is ‘high-throughput analysis.’ In the study of RNA, as the volume of information increases exponentially, computational analysis becomes more important. World leading research groups are training students so that they are versed both in both biological experiments and data analysis.”

Jaechul Lim: “The Center for RNA Research is also hiring many researchers who majored in other fields. Internally, the Center for RNA Research is hosting workshops once a month to actively exchange our thoughts.”What type of effect can occur as a result of the collaboration between researchers from different educational backgrounds? Chang and Lim’s collaboration was a great example of interdisciplinary research. Since the publication of their findings in the journal Molecular Cell, they have been contacted by six overseas laboratories also seeking advice on the study of poly(A) tails.Considering the great success their research has achieved, there may be a long process of trial and error in the search for a new technology.

Hyeshik Chang: “In some ways, this study is no different from our routine research work. Our job was to train the machine so that it could analyze the signal. The hard part was that we needed to validate and confirm in every perspective related to the measurement: accuracy, biases,reproducibility, efficiency, technical easiness and biological relevance, for example. Until we were confident that our new algorithm was enough for actual biological studies, we continued to change and repeat the experiment. We had to synthesize various poly(A) tails and alter each of them for testing. It was painstaking work.”

Jaechul Lim: “When we first designed the experiment, there were many problems. If ribosomal RNA were not removed enough, the analysis would not represent the profile of tails with accuracy. Over-fragmentation of the RNA resulted in the loss of wanted tail region. We applied every experimental method that we could find, which even includes old methods developed over thirty years ago, and also discussed with many scientists in internet communities for unpublished knowledge regarding the subject.What we eventually realized was that we needed to rethink why such approach was taken, even if it is just a minor part of old experiment methods.”

A Look to The FutureWhat is the next step for Chang and Lim? Both of them say that this performance is just the beginning. This means that subsequent studies are needed to get a more biologically meaningful result. In fact, upon the publication of their findings, they already began their subsequent study.

Jaechul Lim: “The starting point of my research was disease. As I continue to study RNA, I hope to extend the topic to protein and disease. I think that researches connecting the basic research with clinical trials are relatively weak, and hope to make this aspect more solid.My wish is to understand the causes of disease or clinical phenomena through basic study. I would like to see the expression of my research in the clinical field.”

Hyeshik Chang: “I think that I am a born engineer. Since I was young, I like making and disassembling things. My best hobby has been computer programming, something which I have done for almost thirty years. In the long run, I hope to further develop programming technologies of ‘life’, the living things. Actually, that is why I was attracted to protein design. As you know, the protein is also thought to be a kind of life-machine. Early on, I was disappointed when I realized that it was not handy to modify and design the structure of protein significantly. Single protein molecule cannot have a complex function as a genetic circuit. I thought that the programming of life needed to be done for more general purposes, and RNA could be one of the general-purpose parts that can rewire biological molecules as I intend. This is why I selected RNA as my research topic in my PhD course. In the future, I hope to find a way of designing new life.”

Bases, such as RNA and DNA maintain their stability through complementary bonding. Paired bases perfectly fit with each other. Literally, this bonding supplements each other, making each more complete. Thanks to this process, creatures can make protein based on genetic material capable of all kinds of chemical reactions for their survival. The blueprint for these reaction scan be seen as long-term storage through its transmission to descendants.

This team’s synergistic collaboration mirrors their area of research, genetic material. In other words, just as bases stabilize each other through complementary bonding, these scientists are able to supplement their needs with each other’s strengths through a kind of ‘social complementary bonding,’ making the team strong through connection and interaction.Someday, they will part ways to achieve their own goals, but until that day comes, we await their next collaborative project at IBS with great anticipation.