All life forms are able to survive because they can replicate themselves faithfully and pass on their genes to their progeny. We live in a world where our genetic blueprint is double-stranded DNA built from nucleotides A, T, C, and G. Our DNA sequences are used as a template to form an intermediate molecule called RNA. This messenger RNA is used as a template to form functional molecules called proteins. But this RNA (called pre-mRNA) needs to be processed (into mRNA) before it can be read into a protein. A long time ago it was believed that only other proteins possessed the ability to process pre-mRNA. But one fine day, researchers discovered that RNA could carry out (aka catalyze) its own processing! RNA molecules possessing catalytic ability were termed ‘ribozymes’ (akin to how protein catalysts are called ‘enzymes’) (1).
In particular, scientists discovered RNA sequences called "introns" that can self-catalyze their removal from an mRNA. This is called self-splicing. These self-splicing introns are found in very ancient cells. As cells became more “modern”, their introns could no longer self-splice but depend on large molecular machines called the "spliceosome" to help them splice. Spliceosomes are complexes made up of catalytic RNAs (ribozymes) and stabilizing protein subunits (2). Since the spliceosome is made up of RNA and protein, it's called a ribonucleoprotein. Below, is a video that shows this process in action!
[If you have trouble viewing the video, go here]
Had modern day introns fully lost their dependence on RNA to carry out splicing? To answer that question, scientists needed to learn more about the structure of the spliceosome. Would RNA play a large role? One component of the spliceosome is the U1 small nuclear ribonucleoprotein. I know that’s a mouthful, so we’ll henceforth refer to it as the U1 snRNP. The spliceosome consists of 5 snRNP complexes which come together during every new splicing reaction. An early step in this process involves the interaction of U1 snRNP with the start of an intron in the pre-mRNA. This interaction stimulates the remaining four snRNAs of the spliceosome to assemble together with U1 (3).
The spliceosome is a big and complex machine. So, it was hard to solve its structure. After many attempts, researchers finally obtained a highly-detailed crystal structure of U1 snRNP which clarified its role in the splicing process. For instance, at first it was thought that a protein component in U1 snRNP called U1-C was needed to recognize introns on its own. However, the crystal structure (3) showed that U1 RNA was the only part of the U1 snRNP that recognized and bound the intron. The protein (U1-C) just helped make the binding be more efficient and stronger, but the RNA played the key role (Figure 1).
Since errors in splicing can lead to dysfunctional proteins, it makes sense that broken components of the spliceosome could lead to diseases. Because when introns aren't removed from mRNA, the extra protein sequence can cause the protein to misfold. Many, if not all neurodegenerative diseases are characterized by misfolded proteins, so scientists were curious if there could be a connection to splicing.
The spliceosome is a big and complex machine. So, it was hard to solve its structure. After many attempts, researchers finally obtained a highly-detailed crystal structure of U1 snRNP which clarified its role in the splicing process. For instance, at first it was thought that a protein component in U1 snRNP called U1-C was needed to recognize introns on its own. However, the crystal structure (3) showed that U1 RNA was the only part of the U1 snRNP that recognized and bound the intron. The protein (U1-C) just helped make the binding be more efficient and stronger, but the RNA played the key role (Figure 1).
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| Figure 1: In this image taken at 3.3 angstrom resolution, we can observe the different portions of the U1 snRNP co-crystallized with one end of the intron called the "5-prime splice site" (labelled 5'-SS in gold). Notice that the RNA portion of the snRNP is colored in grey, while the proteins that associate with the RNA are colored in red, orange, yellow, blue, green, purple, and brown! Among all these parts, the protein U1-C (shown in a deep red color) is visible near the 5'SS, but it is actually the grey U1 snRNA that most intimately pairs with the intron's 5'-SS. Based off of this crystal structure, scientists were finally convinced that the RNA (and not the protein) portion was important for recognizing the intron. |
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| Figure 2: When our DNA is transcribed into pre-mRNA, that pre-mRNA consists of exons and introns. Exons are parts of the pre-mRNA that code for protein, while introns are not. If introns are not removed from pre-mRNA, then the resulting protein sequence will be incorrect since it would have been formed from the RNA coming from exons and introns. Such an incorrect protein sequence may have a higher tendency to misfold and be nonfunctional in the cell. |
The first clue of the connection was that U1 snRNP was found to be mislocalized within cells of patients with Lou Gehrig’s disease, a neurodegenerative disease that leads to death of neurons and atrophy of muscles (4). A second clue followed: In Alzheimer’s disease, U1 snRNP was found to be tangled within amyloid protein aggregates, a hallmark of Alzheimer’s (5). To further study this phenomenon, authors incubated homogenized brain-tissue from Alzheimer’s patients and saw that with increasing concentrations of patient brain tissue U1 snRNP became more and more tangled with amyloid aggregates (Figure 2).
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| Figure 3: This image shows the different conditions used to test how a protein portion of U1 snRNP (called U1-70K) becomes aggregated into insoluble lumps in Alzheimer's Disease. 'AD' refers to brain lysates taken from Alzheimer's Disease (AD) patients, while 'Control' samples were from normal human post-mortem brains. In part 'A' of the image, researchers created six different mixtures containing healthy Control and AD brain tissue, in which they progressively increased the amount of patient-derived AD brain tissue. They then quantified the amount of "tangled", aka insoluble amount of U1 snRNP. In part 'C' of the image, they show two curves. In red, we see the amount of insoluble U1 snRNP that increases in direct proportion to the concentration of brain tissue present from AD patients. In blue, we see a curve representing what is happening when brain tissue from healthy controls is mixed with brain tissue from AD. In this case, there is an even more rapid increase in the amount of U1 snRNP that becomes tangled into insoluble aggregates. Hence, this experiment made the authors realize that it really were the amyloid aggregates from Alzheimer's patients that were inducing U1 snRNP components to get entangled into insoluble aggregates. |
Mislocalization of U1 snRNP, its aggregation, or its degradation can lead to defects in splicing. This can in turn lead to altered expression of how mRNA messages are read into proteins, which can then pave the way for more misfolded proteins, hyper- or hypo-activated genes, and different forms of cancers. These implications of U1 snRNP function in different biological contexts require that it be studied further. Perhaps one day, new insights into the varied roles of U1 snRNP will help find cures to diseases that affect so many people world wide. Overall, the example of U1 snRNP shows us the important roles RNA is capable of playing in catalyzing reactions that occur in the cells of our body, in addition to just being a messenger molecule. By studying the structure and mechanisms of action of different ribonucleoproteins and catalytic RNAs further, we can understand how to target them or use them therapeutically to treat diseases.
References
1. Cech, T. R., Zaug, A. J., and Grabowski, P. J. (1981) In vitro splicing of the ribosomal RNA precursor of tetrahymena: Involvement of a guanosine nucleotide in the excision of the intervening sequence. Cell. 27, 487–496
2. Elliot, D. Molecular Biology of RNA. 2016. Oxford Press.
3. Kondo, Y., Oubridge, C., van Roon, A. M. M., and Nagai, K. (2015) Crystal structure of human U1 snRNP, a small nuclear ribonucleoprotein particle, reveals the mechanism of 5’ splice site recognition. Elife. 4, 1–19
4. Yu, Y., Chi, B., Xia, W., Gangopadhyay, J., Yamazaki, T., Winkelbauer-Hurt, M. E., Yin, S., Eliasse, Y., Adams, E., Shaw, C. E., and Reed, R. (2015) U1 snRNP is mislocalized in ALS patient fibroblasts bearing NLS mutations in FUS and is required for motor neuron outgrowth in zebrafish. Nucleic Acids Res. 43, 3208–3218
5. Diner, I., Hales, C. M., Bishof, I., Rabenold, L., Duong, D. M., Yi, H., Laur, O., Gearing, M., Troncoso, J., Thambisetty, M., Lah, J. J., Levey, A. I., and Seyfried, N. T. (2014) Aggregation properties of the small nuclear ribonucleoprotein U1-70K in Alzheimer disease. J. Biol. Chem. 289, 35296–35313
About the author
Gargi is a Biochemistry major and Mathematics minor at Mount Holyoke College graduating in May 2018. Outside of science, she likes to run, volunteer, and cook.
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