What is RNA splicing?

If DNA is a script recording what kind of film that organisms should play, RNAs are movie clips which are filmed according to the script. These clips need some further “editing” to constitute a movie in accordance with the storyline. The team members of “editors” in cells who cooperate with each other are composed of five small nuclear RNAs (snRNAs), and multiple protein factors and protein complexes.

Forty years ago, Dr. Soo-Chen Cheng, a senior of Chemistry Department at National Taiwan University, undertook the project research on echidnotoxin with Dr. Tung-Bin Lo, Academician of Academia Sinica. “Unlike typical chemical experiments, the biochemical research is more related to lives. There are a lot of mysteries awaiting exploration!” She recalled those days when she had been fascinated by biochemical experiments.                                 

Forty years later, Dr. Cheng is still involved in fundamental research, but the object of the study is no longer echidnotoxin. Instead, she researches the mystery of RNA splicing in organisms.

“Editing” Happening in Our Bodies

Walter Murch, one of the most respected film editors, once noted that the best film editing should be as seamless as a blink of an eye. In Nature, a lot of “RNA splicing” is happening in organisms in a blink of an eye or in an even shorter period of time than that.

Before learning more about RNA splicing, we should understand the concept of Central Dogma of molecular biology, which is the process starting from DNA to protein.

There are many types of protein participating in various functions of human bodies, such as metabolism, regulation of muscle contraction, immune response, and so on. The functions of various types of protein are encoded at the original gene segments of DNA. The original gene segments of DNA can be further divided into two sections, which are exon and intron respectively.

“Exons”  remain when RNA splicing happens, and “introns” are cut out. The remaining exons are assembled to generate a piece of mRNA which carries genetic information, and produce corresponding protein according to the genetic information. The protein will later perform its own function in organisms.

All the exons in human bodies, which are the genetic codes that are able to produce protein, actually only constitute 1.5% of the total length of human genome. How can such small number of genes assemble so many types of complicated protein in human bodies? And under some circumstances, the DNA sequence of exons is normal, why does the sequence still produces abnormal protein which causes physiological abnormalities and genetic diseases?

The mystery lies in the fact whether “RNA splicing” functions properly, which means it should remove unwanted introns, and keep the needed exons and assemble them.

The Exquisite Accuracy of “Removing” And “Remaining”

“Editing, after all, is an art largely achieved by subtraction, by a negation of those elements that do not serve the final product. More often than not, that shape is determined as much by what is taken out as what is left in.” Justin Chang, in his book FilmCraft: EDITING, suggested.

The idea is not only applied on film editing but also on RNA splicing in organisms. We can try imagining the process of how RNA splicing produces mRNA with the following graph.

Gene segments of RNA are the clips filmed in accordance with the DNA movie script, and spliceosome is the team of editors in organisms. It keeps the necessary clips for assembly and turns the clips into meaningful storylines. As for the unnecessary cuts, they are removed just like introns.

If there is a mistake in any editing step, which makes missing clips or adds extra clips, the scenario will be confusing for the audience. A movie with discordant scenarios is like the abnormal protein which is produced at the end of the process. The abnormal protein confuses the physiological mechanism in organisms and then results in physiological abnormalities or genetic diseases.

Construct the RNA Splicing Pathway

In order to know about where an abnormality occurs in the process of RNA splicing, first we need to comprehensively understand the pathway of splicing. Dr. Cheng’s team took the budding yeast Saccharomyces cerevisiae as the model system, and studied the molecular mechanism of the RNA splicing reaction using biochemical methods. Her research team found out what kind of “editors”, which are protein factors and protein complexes, are involved in the process.                                   

Are yeasts, such single-celled organisms, able to help us learn more about human bodies? Dr. Cheng explained that RNA splicing is a fundamental biochemical reaction in eukaryotes, which is generally similar to the mechanisms in various other organisms. Although human bodies are far more complicated than yeasts, the number of human genes is actually only four times the number of the genes of yeasts.

In higher organisms, a piece of genetic code can produce more than one type of protein, which is achieved by permutation and combination of “splicing”.

After years of dedication and the information provided by other research teams, the knowledge of the pathway of RNA splicing can be integrated and demonstrated as the following graph. Put it in a simple way, the process of RNA splicing consists of four phases, which are assembly, activation, catalysis, and degradation. In the process, five small nuclear RNAs and many types of protein factors compose the team of editors and take on the mission of splicing.

 

During the process of RNA splicing, some protein factors are responsible for making the exons closer, and the exons are ready to covalently join to each other. Some protein complexes, such as NTC, are responsible for activating the molecular mechanisms and catalyzing splicing reaction. And other protein complexes, such as NTR, are responsible for degradation and making the protein factors able to rejoin in the splicing cycle.

At the beginning, no one knew about the existence and the functions of these protein complexes, NTC and NTR, and these protein factors, Cwc22, Cwc24, Cwc25, Yju2 and so on, until Dr. Cheng’s research team broke down the process of RNA splicing step by step through biochemical experiments, and finally discovered “the team of editors” participating in the reaction.

In natural conditions, mistakes are very likely to occur during the splicing process in organisms. For example, when all the splicing protein factors are bound on gene segments of RNA, which results in a lack of protein factors responsible for splicing new RNA segments, it will cause negative effect on cells. We can imagine the situation as making movies, when the team of editors are all stuck in the same film production project and the efforts are in vain, the editing schedule of new films afterwards will be affected and delayed.

 

RNA Splicing Research Is to Be Continued…

The research requires the research team to repeat the experiment many times for confirmation and meticulously monitor the change of experimental product; moreover, the cause of the change needs to be speculated with imagination. Although it takes much time and effort, Dr. Cheng thinks that the experiment process should be placed on a firm footing for reliable results. The research team sometimes encountered seemingly insurmountable obstacles, but the most exciting part of the research was to solve those problems after having thought about them for a long time!

There are lots of genetic diseases related to RNA splicing. However, we need to have a comprehensive grasp on the mechanism of RNA splicing pathway first if we want to explore new medical possibilities for corresponding genetic diseases. A cheerful news in the end of 2016 was that US Food and Drug Administration (FDA) had approved a medication for Spinal Muscular Atrophy (SMA). The medicine is able to modify and meditate the RNA splicing abnormalities of motor neuron protein. Dr. Cheng mentioned that the foreign research team had unrelentingly studied the molecular mechanism and medical treatment for more than twenty years, and had developed new drugs in cooperation with biotechnological companies and pharmaceutical companies.

Apart from being inspired by the success of the research of Spinal Muscular Atrophy (SMA) medication over the world, another promising light is shining on the future development of RNA splicing research.

In the past, we could only “speculate” the pathway of RNA splicing through biochemical experiments. In recent years, because of the considerable advance of cryo-electron microscopy (cryo-EM), which can analyze the complex structure of splicing complexes and show the intricate structure of spliceosome in different splicing phases. The cryo-electron microscopy directly shows us the process of RNA splicing and proves scientists’ speculation from the previous experiments. The progress makes the researchers very excited since it was considered difficult to analyze the structure of spliceosome due to its complication and dynamic.

“Many founding researchers in this field are all even older than eighty years old. They cannot believe that they are able to witness these structures. They are very excited!” Dr. Cheng said with anticipation in her eyes.

Although now we have more knowledge about RNA splicing, there are a lot more out there to be learned. That’s why we continue to work on in this field.

RNA splicing occurs in our bodies at the moment. A large number of discoveries in the field of RNA splicing have been made, but there are a lot more needed to be explored. The more we learn about spliceosome, the better opportunity we have to deal with the diseases resulting from the defect of RNA splicing.

 

Reference

◎ Article translated from 我們體內竟然住著剪接師!鄭淑珍的 RNA 剪接研究 http://research.sinica.edu.tw/pre-mrna-splicing-cheng-soo-chen/

◎ The website of  Dr. Soo-Chen Cheng http://www.imb.sinica.edu.tw/~mbscc/index_c.html

◎ Chan, S.-P., Kao, D.-I., Tsai, W.-Y. and Cheng, S.-C. (2003) The Prp19p-associated complex in spliceosome activation. Science 302, 279-282.

◎ Tsai, R.-T., Fu, R.-H., Yeh, F.-L., Tseng, C.-K., Lin, Y.-C., Huang, Y.-h. and Cheng, S.-C. (2005) Spliceosome disassembly catalyzed by Prp43 and its associated components Ntr1 and Ntr2. Genes Dev. 19, 2991-3003.

◎ Tseng, C.-K. and Cheng, S.-C. (2008) Both catalytic steps of nuclear pre-mRNA splicing are reversible. Science 320, 1782-1784.

◎ Liu, H.-L. and Cheng, S.-C. (2012) The interaction of Prp2 with a defined region of the intron is required for the first splicing reaction. Mol. Cell. Biol. 32, 5056-5066.

◎ Liang, W.-W. and Cheng, S.-C. (2015) A novel mechanism for Prp5 in prespliceosome formation and proofreading the branch site sequence. Genes Dev. 29, 81-93.

◎ Wu, N.-Y., Chung, C.-S. and Cheng, S.-C. (2017) Roles of Cwc24 in the first catalytic step and fidelity in 5’ splice site selection. Mol. Cell. Biol. 37, e00580-16.

 

Editor: Ting-Hsien Lin

Art editor: Yu-Chen Chang

Translator: Man-Shu Huang