Divide and Conquer: Sequencing two types of RNA in a single cell

Imagine a police officer chasing two suspects down a narrow alleyway. He almost catches up to them, when suddenly, the alleyway splits into two paths going in opposite directions. One of the suspects takes the left path while the accomplice takes the right path. No matter which choice he makes, one convict will go behind bars, and the other will remain at large.

This might seem a far-fetched scenario for scientists, but they in fact face a similar dilemma when genetically sequencing a single cell. Conventionally, much like the one policeman, researchers can sequence either the messenger RNA (mRNA) or micro RNA (microRNA) of a single cell, but never both simultaneously. Therefore, it remains poorly understood how both types of RNA affect and regulate each other within a single cell. Recently, a collaborative study involving the Lu and Fan labs at Yale has led to a method that allows both types of RNA from the same cell to be sequenced. This method could prove useful in a variety of fields, ranging from epigenetics to cancer research.

A look inside the protein factory

RNA is generally known to serve as a template and regulator in protein synthesis, but the true complexity of the specific roles played by various types of RNAs and how they interact with each other is less well-known. Each RNA type is like a worker with a unique job on an assembly line, and each of these workers coordinates with every other component in the overall production pipeline. The three main types are large messenger RNAs (mRNA), the intermediate between DNA and protein, transfer RNAs (tRNA), and ribosomal RNAs (rRNA), both of which are involved in the translation of mRNA into functional proteins. Less commonly known is microRNA, a small RNA composed of only twenty-two nucleotides that inhibits gene expression by attaching itself to specific strands of mRNA, physically blocking translation. MicroRNA, alongside a myriad of other RNA types, is crucial in gene expression regulation. For instance, abnormal activities of some microRNAs have been linked to the development of cancer and the onset of Alzheimer’s disease.

Divide and conquer

As a result, many researchers are interested in better understanding microRNA and other types of small RNA, especially at the single-cell level. For Jun Lu, associate professor of Genetics and co-principal investigator of the study, studying microRNA’s role in gene expression can help explain slight variations in functionally similar cells. “Looking at many genes in a single cell at a time has allowed us to detect variations of gene expression in cells of the same tissue…We are interested in understanding microRNA in single cells and their relationship to this variability,” Lu said.

The only way to exactly determine the effects of microRNA on a cell’s genetic variability would be to also simultaneously sequence its messenger RNA, since the two regulate each other. Although it has long been possible to sequence both large RNA and small RNA in a group of cells, no previous methodology has proven successful at sequencing both large and small RNA types in a single cell. Directly combining current approaches does not work. “Just like how certain cooking ingredients cannot be added together, any technique to sequence large RNA, such as mRNA, cannot be combined with techniques to sequence small RNA like microRNA,” Lu explained.

Therefore, in the first part of their study, Lu and his collaborator Rong Fan, associate professor of Biomedical Engineering, worked together to develop their own methodology that could co-sequence both types of RNA. “If we could somehow split the single cell in half, then we could sequence the microRNA and mRNA separately,” Lu said. In other words, what if the police officer in the beginning of this story had a partner to catch the other suspect? Initially, the team thought about engineering an extremely precise tool to cut the cell in half like a microscopic cake cutter. However, they eventually realized that this approach was nearly impossible to implement in practice, due to physical constraints and the inevitability of damaging the genetic material inside the cell. Instead, they decided to divide the cell using an approach called half-cell genomics. In this method, the membranes of single cells were first broken. The resulting lysate was split into two “half-cell lysates” where one was sequenced for its microRNA while the other was sequenced for its mRNA. To account for the smaller amount of genetic material in a half cell compared to a group of cells, the researchers chose sensitive sequencing techniques.

Perfecting the “cell-splitting” methodology was difficult. For instance, when the researchers realized that each “half-cell” didn’t have equal amounts of microRNA, they had to tinker with freezing and heating processes in the cell lysis protocol to produce lysates that had equal amounts of mRNA and microRNA. With the kinks sorted out, the technique proved to be remarkably successful–even to the surprise of the researchers. Out of twenty trials with single cells, nineteen of them passed the quality check for both microRNA and mRNAs.

Insights from the new method

In addition to verifying their methodology on single cell RNA samples, the researchers also analyzed the resulting sequencing data to determine the degree to which variations in microRNA expression related to mRNA expression in a single cell. As predicted, the variation of abundantly expressed microRNAs was significantly anti-correlated with the mRNAs that those microRNAs targeted, suggesting that differences in cell-specific microRNA expression alone can lead to non-genetic variation between cells. Furthermore, the researchers found evidence suggesting that some large RNAs could also regulate small RNAs like microRNAs, suggesting an in­terdependent relationship rather than con­ventional one-way regulation of microR­NAs inhibiting mRNAs.

Lu and Fan believe that their “half-cell genomics” method can serve as a powerful molecular tool to understand genomics on a cell-by-cell basis. Specifically, this tool could help researchers study the relation­ship between the expression of cancer-ini­tiating microRNAs and differences in in­dividual cells in cancer tissue. “If we can continue improving this methodology, we could look at cancer tissue in high defi­nition…and extensively study one cell’s gene expression patterns in a manner un­like other techniques,” Lu said. Moreover, this methodology opens the door to other methods that can further examine the un­derlying molecular “wiring” between small RNAs and large RNAs. “Since we can now sequence both small and large RNAs in a single cell, we can start to use computa­tional tools to figure out the logic behind how they regulate each other,” Lu said. Ul­timately, however, the researchers hope to have their new method adopted by others, who can apply it to solve a wide variety of specific problems.

Collaboration and continuation

Despite this recent success, both labs acknowledge the limitations of their cur­rent implementation of “half-cell genom­ics” and seek to improve the speed and scalability of this technique. “If you take a look at other single-cell measurements, you will be amazed at how many single cells they can measure in a single run–tens of thousands and even millions. To further advance this technology and broaden its impact, designs of higher throughput is the direction to go.” Chen said. Another fore­seeable direction is to automate and en­hance the microRNA sequencing process using tools like microfluidics.

A large part of this project’s success was attributed to the close collaboration be­tween the Lu and Fan labs, and a shared interest in developing single-cell tech­niques to study microRNA. “It was natural for us. We had expertise on the molecular aspects of small RNA research, while Dr. Fan had expertise in developing single-cell technologies,” Lu said. Zhuo Chen, a grad­uate student in the Fan lab who was part of the project team, spoke to the contrasting strengths of the collaborators. “Engineers are more results-driven and are always try­ing to build things that work. Biologists are more detail-oriented and curious about why things happen,” he said.

The collaboration between the Lu and Fan labs has led to the creation of a new and important method in single-cell gene sequencing and will likely continue to reap benefits given the multiple promising di­rections in sight. As in the case of co-se­quencing, it often takes two collaborators to divide and conquer.