Proteins are like molecular dancers, with the cell acting as their dance floor. Proteins pair up with various partners to perform elaborate dances. Depending on who they partner with, they can carry out different functions in the cell, just like someone might prefer to do the waltz with one partner, but the salsa with another. Identifying key dance partners in health and disease can help us advance treatments for diseases like Huntington’s.

Pairing up for the molecular waltz

Scientists often focus on a protein’s dance partners when investigating a protein’s function. Knowing which proteins are pairing up sheds light on how that protein works and what it does inside the cell. Cells are like an extraordinarily crowded dance floor, with billions of interacting proteins, constantly interacting with and swapping partners in an elaborate rhythm.

Identifying protein interactions is critical to our understanding of disease. Diseases can alter who a protein likes to interact with, which can affect the functions that it carries out in the cell. If our once tango-loving protein refuses to dance with a certain partner, they may no longer like to do the tango. That could be an issue if that’s their signature dance.

Huntingtin on the dance floor

In the case of Huntington’s disease (HD), researchers are working to map the dance partners of the protein Huntingtin. A spelling mistake in the Huntingtin protein causes HD. Knowing the interaction partners of Huntingtin with and without the spelling mistake can help uncover cellular processes altered by HD. Scientists can use this knowledge of protein interactions to improve our understanding of disease mechanisms and, eventually, develop potential treatments to test in clinical trials.

In a recent publication, a team led by Dr. Cheryl Arrowsmith at the University of Toronto devised an experiment to see all the different dance partners that Huntingtin has, not just other proteins. This work showed that huntingtin also binds to a molecule called RNA.

RNA: A new Huntingtin dance partner

RNA, a cousin of DNA, is best known for its role in producing proteins. While DNA primarily serves as a genetic blueprint for building proteins, RNA has a much broader range of activities. The most studied type of RNA is messenger RNA, also called mRNA, which codes for protein.

“While most proteins don’t partner with RNA, the new work from Dr. Arrowsmith’s group suggests that Huntingtin appears to be one of the few that do. This raised the possibility that the spelling mistake in Huntingtin that causes HD could disrupt these interactions. ”

However, up to 90% of RNA molecules don’t code for proteins and are not mRNA. Instead, they interact with proteins, coordinating important cellular processes. In this way, these so-called “non-coding” RNAs act like protein dancers themselves or are at least protein choreographers, helping proteins dance together. While most proteins don’t partner with RNA, the new work from Dr. Arrowsmith’s group suggests that Huntingtin appears to be one of the few that do. This raised the possibility that the spelling mistake in Huntingtin that causes HD could disrupt these interactions.

Scientists had a couple of reasons to suspect that Huntingtin might partner with RNA. For example, when they used powerful microscopes to closely examine the exact shape and physical structure of Huntingtin, they noticed a spot on the protein that could, in theory, fit an RNA molecule right into it.

In addition, previous experiments by Dr. Arrowsmith’s lab showed that RNA molecules strongly partnered with Huntingtin in certain experiments where they were trying to study Huntingtin’s protein interactions. In fact, so much RNA partnered with Huntingtin that they needed to perform additional steps to get rid of the RNA (because they were interested in proteins at the time). However, this led them to wonder, what ARE those RNA molecules that partner with Huntingtin and could they play a role in HD?

The dance between Huntingtin and RNA

Before diving into more complex techniques, the researchers conducted a simpler experiment. They mixed RNA into a kind of “science jello” and subjected it to an electrical current. Because RNA molecules have a negative charge, they migrate toward positive charges positioned on the opposite side of the jello. Scientists compared the speed that the RNA moved through the jello with, and without, Huntingtin mixed in.

They found that, in the presence of Huntingtin, RNA moved through the jello more slowly, suggesting that RNA was in fact partnering with Huntingtin. Importantly, this slowing effect was not observed when DNA was mixed with Huntingtin, suggesting that Huntingtin has a specificity for RNA. This is important because DNA and RNA are chemically quite similar, but functionally very different. This experiment showed that Huntingtin is specifically interested in dancing with RNA, not DNA.

Encouraged by this result, the scientists decided to dig deeper by analyzing all the RNA that partnered with Huntingtin in living cells grown in a dish. As expected, they found that Huntingtin parternerd with many different RNA molecules. They next narrowed their investigation by focusing on some of those RNA partners to learn more.

A NEAT dance sequence

While reviewing Huntingtin’s RNA partners, the researchers noticed some interesting patterns. Many of these RNA molecules were involved in activities that are critical for cell survival. Because these activities are so crucial, they are not the kinds of RNA you would want Huntingtin snubbing on the dance floor!

Huntingtin also prefers to partner with a specific type of RNA containing lots of guanine, a building block of RNA. To confirm this, the scientists made an artificial “lab-made” RNA strand with lots of guanines and, sure enough, Huntingtin sidled right up to it.

The researchers decided to shine the spotlight on one RNA that consistently partnered with Huntingtin and contained lots of guanine: NEAT1. NEAT1 is an RNA that plays a key role in forming something called paraspeckles - tiny structures inside the nucleus of cells that control RNA production. You can think of paraspeckles like the VIP lounge on the molecular dance floor. NEAT1 is the RNA choreographer specifically for the VIP lounge, dancing with partners there and regulating the dances of others. Huntingtin will join the VIP area, but has no problem dancing in other areas of the cell. The researchers found that when Huntingtin joins the VIP lounge paraspeckles, it likes to partner with NEAT1.

Next, the team wanted to know if levels of NEAT1 were changed by the spelling mistake in Huntingtin that causes HD. Although they found levels of NEAT1 were lower in brain cells grown in a dish and mouse brain tissue containing the Huntingtin spelling mistake, the results from human brain tissue were less clear. During the early stages of HD, NEAT1 levels were lower, but NEAT1 levels were higher in later stages of the disease. The scientists suggested this could be caused by the loss of brain cells as the disease progresses. Regardless, these results suggest that NEAT1 levels are changed in Huntington’s disease.

“These findings are significant because they show that Huntingtin interacts with NEAT1, an RNA crucial for paraspeckle formation, and this interaction is disrupted in HD - potentially causing serious problems in the brain. ”

The VIP lounge

To see if there is a direct connection between NEAT1 and Huntingtin, the scientists tested whether changes in Huntingtin levels could affect NEAT1 levels or the paraspeckles that NEAT1 organizes. Afterall, if you know your favorite dance partner will be a no-show, you might not show up either! The researchers found that when Huntingtin levels were lowered, NEAT1 levels rapidly decreased afterward, suggesting that Huntingtin stabilizes NEAT1. So NEAT1 really only wants to be around if Huntingtin is around too. Because NEAT1 is crucial to forming paraspeckles, reducing Huntingtin also led to smaller and fewer paraspeckles in the nucleus. So without Huntingtin, NEAT1 doesn’t even bother organizing the VIP lounge. That’s a serious commitment to your dance partner!

Next, the researchers asked if the spelling error that causes HD effects NEAT1’s role in organizing paraspeckles. They found that brain cells grown in a dish that have HD-causing Huntingtin had fewer paraspeckles, and those that remained were smaller. This suggests that both the loss of Huntingtin and the presence of HD-causing Huntingtin disrupt paraspeckle formation, possibly by destabilizing NEAT1.

These findings are significant because they show that Huntingtin interacts with NEAT1, an RNA crucial for paraspeckle formation, and this interaction is disrupted in HD - potentially causing serious problems in the brain. However, there are still some important unanswered questions. For one, most of these experiments were done in cells grown in a dish, so we don’t know if the same interactions occur in the human brain. Additionally, the consequences of reduced NEAT1 and paraspeckle formation in the brain remain unclear. Previous studies in mice suggest that NEAT1 is not essential for brain development or brain cell survival. Still, the consequences of disrupting NEAT1 or paraspeckles in humans, or during human disease, are unknown.

Zooming out on the dance floor

While most of this work focuses on NEAT1 and paraspeckles, let’s not lose sight of the big picture: Huntingtin interacts with RNA! NEAT1 was just one of up to 571 RNAs the researchers found that may interact with Huntingtin, many of which were involved in important activities like producing energy. Future studies are needed to examine how Huntingtin might affect these other RNAs just as this study analysed NEAT1. For example, if Huntingtin is important for NEAT1 stability, could Huntingtin stabilize other important RNAs?

Let’s think therapeutics - where does this research get us? First of all, this study will certainly motivate additional research into Huntingtin’s RNA connection. And, if an interaction between any specific RNA and Huntingtin were found to be harmful or beneficial, then small molecules that influence that partnership could be designed. In the elaborate choreography of cellular processes, finding the right molecular dance partners is like playing the perfect song – it can set the stage for a harmonious performance or prevent a misstep that disrupts the entire routine.

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