A paper published June 1st in Nature Genetics began by asking how a defect in micro RNA regulation might affect the heart. The result is the surprising finding that deregulation of a single micro RNA (or miRNA), miR208-a, in heart cells severely affects the ability of heart muscles to contract.
The authors, Jian Ding et al., deleted the gene Trbp specifically in mouse cardiomyocytes, the muscle cells of the heart. Trbp encodes an important protein for the production and regulation of miRNAs, which in turn are thought to regulate the expression levels of target genes. Because miRNAs can each have many target genes, and genes can each have many miRNAs that target them, it is thought that, generally, the effect of losing any one miRNA will be very small.
Trbp is too important to be deleted from the whole mouse. This would lead to a very early death. However, when Trbp was deleted only in cardiomyocytes, mice were able to live up to 8 months (though most died before 4 months). Immediately after birth, there were no obvious defects in the hearts of mice with the Trbp deletion, but by 3 weeks after birth there were signs of trouble. The heart chambers of these mice were dilated and the heart couldn’t contract normally. Eventually, this led to cardiomyopathy and heart failure.
To find out how deletion of a gene for general miRNA regulation could have such a specific effect on heart muscle function, the authors looked for genes that were aberrantly expressed in the deletion mice. They found that the levels of genes for slow and fast twitch muscle fibers were not expressed at their normal ratios due to increased expression of a gene called Sox6. They also noticed that several miRNAs were not processed normally in Trbp deletion mice. One miRNA in particular, miR-208a, was found to be responsible for misregulation of Sox6. Adding normally-processed miR-208a back into the Trbp deletion mice completely reversed the heart defects.
We asked the senior author of the study, Da-Zhi Wang from Harvard Medical School, to tell us a little more about the background of this fascinating discovery.
What motivated you to choose Trbp for this study? Why did you specifically choose to knock this gene out in cardiomyocytes?
We have been interested in microRNA biogenesis [the creation of miRNAs from longer RNA precursors] and the underlying functional mechanisms for the past ten years. It has been generally thought that most (if not all) microRNAs are produced and processed in a similar manner, where they are transcribed by RNA polymerase II and processed by the microprocessor (Drosha/DGCR8 complex) in the nucleus. In the cytoplasm, they are further processed by Dicer and Dicer co-factors. Trbp is interesting because, unlike Dicer, Drosha and Ago2, which were primarily characterized as regulators of miRNA pathway, Trbp was originally identified as an RNA binding protein binding to HIV RNA, suggesting it may play a distinct role in RNA biology. Interestingly, several previous studies, primarily conducted in vitro, suggested that Trbp could function as a Dicer binding partner to modulate miRNA biogenesis. However, few studies have directly linked the biochemical properties of this RNA binding protein to any physiological function.
Our lab studies molecular mechanism of the cardiac system. We have previously knocked out Dicer in mice and we found that cardiac-specific deletion of Dicer resulted in the blocking of miRNA biogenesis. As a consequence, we found that cardiac function was impaired in Dicer mutant mice (Chen et al., PNAS 2008). In order to have our hands on Trbp, we thought that we would first generate cardiac-specific Trbp knockout mice, which will enable us to examine its biological function as well as to define the molecular mechanism in a defined biological system.
Were you surprised to see such a significant cardiac defect in these mice?
Not really. As we predicted that Trbp is functionally important in the heart, we actually expected to see the cardiac defects. On the other hand, we were aware of the presence of a Trbp homologous gene, Pact, which potentially could have resulted in functional redundancy.
What was your initial reaction when you learned that only 60 miRNA transcripts were affected by Trbp deletion? Was this expected? Why or why not?
We were indeed surprised to see that only 60 miRNA transcripts were affected in Trbp-KO hearts. As indicated earlier, prior studies have suggested that Trbp functions as an obligate co-factor of Dicer to regulate miRNA biogenesis globally. Our study, on the other hand, clearly demonstrated that the effect of Trbp on miRNA expression is context-dependent. We would like to emphasize that most previous Trbp-related studies were conducted biochemically in vitro, whereas we took a mouse genetic approach to delete the Trbp gene in vivo. These apparently inconsistent observations further underscore the importance of genetic studies in vivo.
What do you see as the most significant aspect of this study?
Re-expression of a single miRNA (miR-208a) or knockdown of a single mRNA target (Sox6) could fully rescue the loss of Trbp phenotype. This may make us rethink the functional mechanisms of miRNAs and their targets.
How do you envision others using this study in the future?
Although our study clearly demonstrated that the function of Trbp is mediated by miR-208a and Sox6 in the heart, it remains to be determined of how Trbp specifically regulates the biogenesis/processing of miR-208a. In other words, how is the specificity of Trbp conferred/determined?
Another important finding of our study is that the Trbp-miRNA-mediated fast- and slow-twitch myofiber gene program is correlated with cardiac function. The fast- and slow-twitch gene regulation has been extensively studied in skeletal muscle (and linked to skeletal muscle function), but not in heart. It will be important to investigate whether human cardiomyopathy patients are linked to dysregulation or mutation of the fast- and slow-twitch myofiber program.