Then and Now: Studying Essential Proteins of the Nuclear Pore Complex

By Paul Elizalde

Researchers can study the role of specific proteins by mutating or getting rid of the protein and seeing what happens to the cell. However, some proteins are essential for cellular growth and survival, so how do you study a protein when removing it kills the cell?

Dr. Vasilisa Aksenova encountered just this problem while studying nuclear pore complex (NPC) proteins, known as nucleoporins, which are needed to move molecules, such as RNA, between the nucleus and cytoplasm.

“RNA export is a highly regulated point for all human gene expression,” Dr. Aksenova explained. “Understanding it has broad implications for understanding disease mechanisms and designing targeted therapeutics.” Her research teases out the role of individual nucleoporins and simultaneously supports a way forward in studying essential proteins.

Dr. Aksenova received her doctorate in cell biology from the Russian Academy of Science. When she wanted to focus on the cell biology of the nucleus, Dr. Mary Dasso’s lab at the NICHD was her first choice. She joined as a visiting postdoctoral fellow interested in nucleoporin function in mitosis. Continuing her work in the same lab, she says that her interest has broadened beyond mitosis. Her recent publication in Nature Communications focuses on the role of the basket nucleoporin, in mRNA export.¹

Based on biology’s “central dogma,” there are three levels that you can target to affect the protein: (1) the DNA, (2) the RNA, or (3) the protein itself. Since the proteins that Dr. Aksenova studies are essential, they cannot be deleted at the gene level due to deleterious effects on the nuclear pore complex. Instead, she and her colleagues deplete the proteins conditionally.

Historically, RNA interference (RNAi) has been a popular method to deplete a target protein. RNAi uses double-stranded RNA to target complementary mRNA for degradation. The phenomenon was first described as “co-suppression” in plants. Researchers found that overexpression of an enzyme necessary for deep violet pigment synthesis blocked biosynthesis resulting in white petunias.² Later studies clarified the mechanism in Caenorhabditis elegans making it a useful method for gene knockdown.³

However, RNAi is an imperfect system. It can take time before the effects of RNAi are observed, and knockdown is not always complete. Dr. Aksenova noted that RNAi knockdown needs multiple rounds of cell division over several days to degrade protein. This makes it hard to study nucleoporins since the complexes break down with the nuclear envelope during cell division in mammalian cell lines.

With the development of the Auxin- Induced Degron (AID) system in 2009 and of CRISPR-Cas three years later,^4,5 Dr. Aksenova and her team had the tools necessary to study the basket nucleoporins when they began the project five years ago.

The AID system is much faster and more efficient than RNAi in knocking down protein levels. With the addition of auxin, protein levels are depleted on the scale of hours rather than days. This allows Dr. Aksenova to observe shorter time points and decipher an order of events among nucleoporin interactions. The CRISPR-Cas system makes it possible for the team to insert necessary AID components into cell lines.

Even though Dr. Aksenova acknowledged the usefulness of RNAi, she commented that “AID and CRISPR changed [their] lives” to the extent that she “forgot about RNAi after using AID.” They were able to knockdown systematically each component of the three subunits of the basket nucleoporin—NUP153, NUP50, and TPR—to determine their distinct roles.

Interestingly, they found that changes in mRNA export upon depletion of TPR were more like changes observed after depletion of mRNA export receptor NXF1, or the GANP subunit of the TREX-2 mRNA export complex, than to loss of the other basket nucleoporins (NUP153 or NUP50).

The application of the AID system and subsequent findings published by Dr. Aksenova and her collaborators could impact how we study different diseases and their mechanisms moving forward. The method pioneered by the team could serve as a template for the systematic study of individual subunits of protein complexes across a wide span of biomedical research.

References

1. Aksenova V, Smith A, Lee H, Baht P, Esnault C, Chen S, Iben J, Kaufhold R, Yau KC, Echeverria C, Fontoura B, Arnaoutov A, Dasso M. (2020). “Nucleoporin TPR is an integral component of the TREX-2 mRNA export pathway.” Nat Commun . 11(1):4577. doi: 10.1038/s41467-020-18266-2.
2. Napoli C, Lemieux C, Jorgensen R. (1990). “Introduction of a Chimeric Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes in trans.” Plant Cell 2(4):279-289. doi: 10.1105/tpc.2.4.279.
3. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. (1998). “Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.” Nature 391(6669):806-811. doi: 10.1038/35888.
4. Nishimura K, Fukagawa T, Takisawa H, Kakimoto T, Kanemaki M. (2009). “An auxin-based degron system for the rapid depletion of proteins in nonplant cells.” Nat Methods . 6(12):917-922. doi: 10.1038/nmeth.1401.
5. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. (2012). “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Science 337(6096):816-821. doi: 10.1126/science.1225829.