Scientists have explored long non-coding chains in a variety of ways

RNA2016-2-3 sequencing China

Until today, a concise and reasonable classification system for lncRNAs is still out of reach. In 2013, a group of researchers decided to delve deeper into a human embryonic stem cell line called H1 and received some surprises. H1 is one of the most famous stem cell lines, but the team still successfully discovered over 2000 previously unexplored RNA fragments. More importantly, 146 of them are unique to human embryonic stem cells, providing enticing clues for studying pluripotency - the ability to transform into any cell type in the body.

However, these transcripts have not received much attention because they contain repetitive coding fragments that are often filtered out by sequence analyzers. This is a huge blind spot. Other laboratories have found preliminary evidence of the existence of RNA rich in repetitive coding and highly important in human stem cells. When researchers mainly from Stanford University in California analyzed the results, they realized that they had just found this type of RNA. Team member Vittorio Sebastiano explained that in a list of 146 RNA sequences, three enriched ones - named HPAT2, HPAT3, and HPAT5- seem necessary for establishing pluripotent cells capable of developing into human embryos.

These RNA fragments are examples of long non coding RNAs (lncRNAs) (sequences that are at least 200 bases long and do not encode proteins). LncRNAs exist in many different types of tissues and are often found at specific locations within cells. However, most lncRNAs lack well-defined functions and were only considered transcriptional noise until recently. With the influx of more data, this viewpoint is beginning to change. The data indicates that the genomic regions transcribed by lncRNAs are more highly conserved in evolution than previously thought, which means they possess some functions. However, until today, a concise and reasonable classification system for lncRNAs is still out of reach.

Function priority

Due to the lengthy list of lncRNAs, a crucial step is to determine which ones are prioritized for research. John Rinn, who discovered lncRNAs during his graduate studies about 15 years ago, proposed starting with lncRNAs from genomic regions already associated with diseases. Currently, Rinn manages a laboratory dedicated to studying molecules within the Broad Institute established by MIT and Harvard University.

Another idea is to study where lncRNAs are located - for example, to find lncRNAs near the transcription start point, which means they are involved in regulating nearby genes. Currently, researchers can relatively easily track the location of molecules within cells. Rinn and other researchers from the Broad Institute and the University of Pennsylvania used a technique called single-molecule fluorescence in situ hybridization to successfully identify the locations of 61 lncRNA molecules located within skin, lung, and cervical tumor cells. Nowadays, scientists can also use CRISPR-Cas9 and other gene editing techniques to interfere with the partial DNA sequence of lncRNA transcription or the promoter that commands its transcription, in order to test the function of lncRNA. Some technologies enable laboratories to quickly screen out a large number of lncRNAs. When CRISPR-Cas9 is used to study the function of protein coding genes, the logic is the same: introducing single base deletions or substitutions into DNA and observing the effect of altered transcription.

Howard Chang, a cancer biologist at Stanford University School of Medicine, explained that the only issue is that compared to proteins, lncRNAs are less likely to lose their ability due to minor changes, while sequence changes are usually more intense. This is why RNA researchers create CRISPR-Cas9 themselves. They expanded the CRISPR toolbox to include methods for blocking or initiating specific lncRNA transcription. However, Rinn and his team have developed another approach: a tool called CRISPR Display. Rinn likened it to a drone capable of transporting items - in this case specific lncRNAs - anywhere within a cell. If a task in gene activation is suspected, it can be tested by transporting lncRNA to a different genomic region and observing gene activation at a new site.

RNA interaction group

A completely different approach is to find the object that is interacting with lncRNA. People still believe that although lncRNAs are important, they ultimately cannot perform their functions without auxiliary factors. ”Jeannie Lee, co-founder of RaNA therapy company and molecular biologist at Massachusetts General Hospital, introduced that these auxiliary factors are almost always proteins.

Lee and others have started using an lncRNA called Xist to reveal its interactions. Research has found that Xist is necessary to inactivate one of the two X chromosomes in female mammalian cells, thereby preventing females from having many X chromosome gene products that are twice as abundant as males. Proteins that bind to Xist silence gene expression through various mechanisms. However, last year scientists finally identified the identities of these 'partners'. Nowadays, research has found that these proteins not only attract other molecules that silence transcription, but also repel some molecules that initiate transcription.

There are many techniques available for exploring the protein "partner" group of a certain lncRNA: broadly speaking, researchers use substances such as formaldehyde or ultraviolet light to link RNA and protein together, and then use mass spectrometry to analyze who binds to whom, and propose the concept of "interactome". Usually, these analyses involve multiple steps, requiring scientists to make many strategic choices. How should RNA and protein be linked together? How to distinguish between real signals and false signals of interaction? The problem that envelops all such studies is that RNA typically behaves differently in vitro than in cells.

That's why scientists dedicated to studying Xist interactions focus on various techniques that can identify RNA that binds to proteins in living cells. The recent improvement in sensitivity of mass spectrometry analysis is helping researchers. Last year, Chang's team combined experiments using formaldehyde as a linker with the latest mass spectrometry analysis method and found that Xist can bind to 81 proteins in vitro.

Secrets in Structure

The third method to explore the function of lncRNAs is to study their structure. Although this method cannot directly predict the function of lncRNA like predicting protein function, a better understanding of RNA arching and folding may provide some information. This is a completely open field that requires a lot of work, "said Karissa Sanbonmatsu, a structural biologist at Los Alamos National Laboratory in New Mexico. The methods for establishing the secondary structure of a certain lncRNA include chemical detection, such as the method known as SHAPE. It involves attaching acetyl groups to RNA and modifying its "backbone" only in flexible regions. The modified location will block the enzyme that reads RNA to create complementary DNA sequences, resulting in the generation of short DNA fragments instead of long strands. These fragments can then be sequenced on the gel or sorted by size.

In 2012, the Sanbonmatsu team first described the secondary structure of a human lncRNA: a steroid receptor RNA activator that had been thought to be associated with estrogen receptors for over a decade. However, this structural approach has to address the issue of RNA exhibiting different behaviors in vitro and in cells. Like in combination research, the latest technology is conducted in vitro. In 2012, a team including Chang described a version of SHAPE that could function in living cells and have since improved it to simultaneously depict the structures of thousands of RNAs. Like other methods, structural research requires a significant investment of time, so careful choices must be made when focusing on lncRNAs that are most likely to have functionality. Sanbonmatsu introduced that fortunately, researchers are performing better and better in this classification. She suggested that when assessing the possibility of functional significance, scientists should start with lncRNAs with known phenotypes, and then explore them through chemical methods to obtain secondary structures and examine to what extent they can be preserved in other species.

Reference: Finding function in mystery transcripts. doi: 10.1038/529423a.