Unpopular Opinion: MicroRNAs Are Spicy
An Explanation of the Underappreciated Role of Cilia
Background
Have you ever wondered how certain genes are regulated at certain points in time? Or how early development takes place? Or what the purpose of those tiny hair-like structures throughout your body is? Well, this paper will hopefully answer some of those questions.
The normal function of a microRNA (miRNA) is to bind to the 3’ untranslated region (UTR) of an mRNA and either cause degradation of the mRNA or inhibit translation. The seed sequence of the miRNA, the portion that always binds to the mRNA, is 100% complementary to the 3’UTR of the mRNA. If the remaining sequence of the miRNA is 100% complementary to the mRNA, then the mRNA is degraded. On the other hand, if the remainder of the miRNA is only somewhat complementary to the remainder of the mRNA, then translation is inhibited. In either case, the result is preventing the action of the protein encoded by the mRNA.
These miRNAs play an important role in regulating certain genes and molecules necessary for development. miR-430a is a specific miRNA that targets sqt during development of zebrafish and controls its expression. Sqt is a nodal-related molecule that aids in developing cilia of proper length, and when miR-430a is present, it essentially prevents sqt from doing its job. Nodal molecules are signaling molecules that are expressed exclusively on the left side of the embryo during development. They also have an important role in heart development.
Cilia are structures composed mainly of microtubules and are present throughout the body. You might be more familiar with the motile cilia in your airways, for example, that move to push any debris out of your lungs. Motile cilia are also important for establishing proper patterning in the embryos of many organisms, including humans and zebrafish. There are more similarities between zebrafish and humans than you would probably expect, which is one reason why they make great model organisms. Like humans, zebrafish are not completely symmetrical. This asymmetry requires an asymmetric distribution of certain genes and signaling molecules in order for the organism to develop with that asymmetry. Humans and zebrafish have an organ called a left-right organizer (in zebrafish, it’s called Kupffer’s Vesicle — KV) that has cilia. Sqt is the specific gene that is important in the development of the KV, which is why examining the function of miR-430a is crucial. The expression of sqt is needed to develop a proper KV and functioning cilia, both of which are needed for a properly patterned organism.
Results
The two main experiments performed in this paper are in situ hybridization (ISH) and immunohistochemistry (IHC). In situ hybridization detects expression of a certain gene at a given point in development. An RNA probe, generated to be complementary to the gene of interest, is introduced into a zebrafish embryo and will bind to the complementary DNA present throughout that embryo. It allows researchers to visualize the location of the expression of a certain gene. Immunohistochemistry uses antibodies to detect certain proteins in a given tissue. An antibody against the protein of interest will be introduced into the embryo and when binding occurs, fluorescence will result.
To examine the effects of miR-430a on cilia function, an IHC was performed to visualize the cilia. When miR-430a was injected into the embryo, the IHC revealed shortened cilia when compared to the control. However, when Posqt, the sqt that is in Japanese flounder, was co-injected with miR-430a, the cilia appear to be normal size. The sequence of Posqt is conserved just enough to restore the function of sqt in zebrafish, but the 3' UTR, the region that binds to miRNAs, contains just enough difference in sequence that miR-430a cannot inhibit Posqt. Because the cilia are restored to their normal length, this is called a rescue experiment. By inhibiting the function of sqt with miR-430a, a significant loss of function occurs in the cilia that are needed early on in development. (Figure 8 — scroll down to view the image)
Functional motile cilia are needed early on in development because they are the driving force behind asymmetric patterning that is especially important for the proper development of the heart. The effect miR-430a has on left-right patterning was observed in an ISH. The gene of interest was cmlc2 because it is a cardiac marker. Ultimately, the authors are determining the effect of miR-430a on the heart’s development. Again, when comparing the embryo injected with miR-430a to the embryo co-injected with miR-430a and Posqt, left-right patterning was rescued in the latter experiment, revealing the necessary role of sqt in development. (Figure 6 — you will have to scroll down again)
Conclusion
Genes and signaling molecules play an important role throughout life, especially during development. The expression of both must be closely monitored so that there is just the right amount of expression at just the right time. This is why miRNAs are so important, however, scenarios where too much miRNA is present can also occur. Too much miR-430a can have an extremely negative effect on the viability and development of zebrafish. More specifically, abnormally high miR-430a levels can inhibit the proper function of cilia. Some humans experience ciliopathies, a condition in which they lack functioning cilia and as a result, display unpleasant symptoms and phenotypes. By studying the many ways cilia can be regulated, hopefully we will be able to better treat these individuals.
