This piece was written LIVE during the 5th annual RNA Symposium: Processing RNA. Follow us on Twitter or the tag #umichrna!

Live blogger: Logan Walker

Editor: Alyse Krausz

Over the last two days, we have heard talks all about how RNA is a key building block in myriad biochemical processes, both natural and artificial. But, with all of this RNA floating around, we are left with a simple question: what happens to the RNA molecules once our cells are “done” with them? The answer turns out to be a constellation of proteins that work together to detect incorrect sequences, turnover old RNA molecules, perform post-translational modifications, and remove invasive sequences, such as viral RNA molecules. In the case of RNA turnover, much of this processing is performed by the RNA exosome complex, making it an important target of study for diseases where it is dysregulated, such as multiple myeloma, pulmonary fibrosis, and many subtypes of cancer.

RNA Exosome Structure

In pursuit of a better understanding of the exosome structure, Dr. Christopher Lima’s lab has recently published a protein structural model (Figure 1) of this complex. In this study, they created a 3D model of the 3 top and 6 bottom proteins that make up a barrel-shaped complex.  The exosome consumes power in the form of ATP and uses it first to unwind, then disassemble RNA molecules (Figure 1, top). As the RNA is unwound, it is passed through a pore in the center of the complex. To capture this 3D model of the molecular structure, his students used a technique called cryo-electron microscopy (CryoEM), wherein the protein is frozen in vitreous (flash-frozen) ice and then viewed through an electron microscope (Figure 1, bottom). These individual electron micrographs can then be used to predict the 3D structure of the protein complex by computationally modeling how the 2D micrograph would appear.

Diagram

Description automatically generated
Figure 1: Overview panel from Weick, et al. Cell 2018

After solving the protein complex’s structure, there are many experiments that can be performed to further interrogate and improve the 3D model. In particular, they were interested in how the complex actually performs the RNA degradation functions which have been well studied. To this end, they engineered “RNA-origami”, which forced the protein complex to be “stuck” in place, by combining an RNA and DNA molecule. Using this chimera, they repeated the cryoEM experiment.

The RNA Exosome is Picky

After completing their studies on the structure of the exosome complex, Dr. Lima’s team looked to better understand how the activity of the exosome can be regulated from a structural view. They observed that the helicase, the molecular machine that unwinds RNA before processing in the exosome, can often decide what types of RNAs it will “accept” for processing. In the RNA world, any number of RNA classes are known to exist such as microRNAs (miRNA), messenger RNA (mRNA), transfer RNAs (tRNA), and ribosomal RNAs (rRNA). They found this target selectivity by looking in many different organisms (including human and yeast cells) at the types of RNAs that the exosome will react with. Notably, they found that the helicase is always in physical contact with a scaffold protein, called ZCCHC8 in humans. This protein combines with another called RBM7 to form an oligomer called NEXT. The NEXT oligomer was hypothesized to be responsible for stabilizing the exosome complex and required for its activity. The ZCCHC8 protein was also found to be binding with itself between complexes, resulting in a dimer structure being observed in the exosome.

To test this model, their lab generated a cell line where the NEXT complex is mutated in different ways. When they tested that mutated complex, they found that even minor changes to the NEXT binding domains result in an exosome that reacts much slower than in the wild-type cells. It is notable, however, that this mutant does not fully suppress the exosome activity fully. From this, they conclude that the exosome can function as a way to sequester RNA.

With this focus on the NEXT complex, they looked for other ways that it could be implicated in disease. It had been previously found that ZCCHC8 can be fused with a second protein called ROS1 in the N-terminal region of the protein. The ROS1 protein being fused to another through chromosome remodeling (translocation) has been found in many cancers, and the chemical activity caused by the translocation is important for cancer development (oncogenesis). As a result, the Lima lab chose to interrogate the activity of ZCCHC8-ROS1. They found that this fusion protein is more active than just ROS1 by itself, indicating that the dimerization is also contributing to the oncogenicity of the ROS1 translocations.

In closing, Dr. Lima presented a complex model, where the naively-simple RNA regulation models that are taught in textbooks are actually constructed out of many different protein complexes, each with their own unique regulation machinery like he showed today. There is an entire network of interactions to discover!


Photo: HHMI

Christopher Lima received his BA from Ohio State University and his Ph.D. from Northwestern University under the supervision of Dr. Alfonso Mondragon studying the E. coli topoisomerase I protein. After a postdoc in Dr. Wayne Hendrickson’s lab at Columbia University, he joined the faculty at the Weill Medical College of Cornell University. Shortly after in 2003, he moved to the Sloan-Kettering Institute, where he remains as the chair of the structural biology program.

During his time at Sloan-Kettering, he was named a Howard Hughes Medical Institute Investigator and has recently been instated as a member of the National Academy of Sciences.

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out /  Change )

Google photo

You are commenting using your Google account. Log Out /  Change )

Twitter picture

You are commenting using your Twitter account. Log Out /  Change )

Facebook photo

You are commenting using your Facebook account. Log Out /  Change )

Connecting to %s