Final Report Summary - MIRTURN (Mechanisms of microRNA biogenesis and turnover)
Although microRNAs (miRNAs) are well-known for their roles in posttranscriptionally regulating gene expression, it has been much less appreciated that miRNAs may be subject to extensive regulation themselves. We uncovered roles of active miRNA degradation pathways in such regulation. Following our initial discovery of the ribonuclease XRN2 as a 'miRNase' in the nematode C. elegans (Chatterjee & Großhans, 2009), in the current project, we identified the novel PAXT-1 (PArtner of XRN-Two) protein as a stable subunit of XRN2 complexes that promotes XRN2 function (Miki et al., 2014b). Targeted construction of a conditional mutant allele of xrn-2 (Miki et al., 2014a) permitted testing of its substrate range, which, strikingly, revealed specificity for a subset of miRNAs. The targets of miRNAs may drive such specificity, as we uncovered an ability of mRNAs to modulate miRNA stability. Thus, highly complementary targets in C. elegans can stabilize their cognate miRNAs (Chatterjee et al., 2011), whereas they can induce miRNA degradation in rodent hippocampal neurons (de la Mata et al., 2015). Unexpectedly, this latter activity appears particularly pronounced in primary neurons as opposed to other cell types and established cell lines, suggesting the possibility of a particularly important function of this process in the brain. Degradation of the miRNA and its target are independent, antagonistic processes, the balance of which can be tilted through appropriate target design. Hence, our knowledge can be applied for efficient depletion of even abundant miRNAs.
As miRNAs frequently repress many targets, but mostly only modestly, it has become a dogma in the field that their key function may be to regulate pathways through coordinate 'tuning' of multiple targets, and/or achieve robustness of gene expression patterns by reducing noise. We tested this notion experimentally for the highly conserved let-7 miRNA, which has many previously identified targets, and which is essential for C. elegans survival. Using genome editing of the endogenous target, we could demonstrate that it is in fact regulation of one target alone, LIN-41/TRIM71, that is necessary and sufficient for this function of let-7 (Ecsedi et al. 2015). Hence, at least a subset of miRNAs may achieve functions in a more 'focused' manner. Conversely, discovery of a large set of regulated genes cannot be equated with physiological functions for each of these. Genome-editing approaches as pioneered here will provide a means to test and validate or refute such functions.
Finally, through gene expression profiling performed on developing C. elegans at high temporal resolution, we discovered an intriguing phenomenon of extensive oscillatory gene expression (Hendriks et al., 2014). Thus, >2,700 transcripts (representing nearly one fifth of active genes) change at least 2.1-fold but frequently > 10-fold, and with a period of ~8 hrs. These oscillations are distinct from circadian rhythms and may serve a developmental function, which we are investigating. Irrespective of the physiological function, a major impact of our finding on C. elegans research in general is that it reveals that gene expression profiling experiments are highly sensitive to temporal deviations in wild-type vs. mutant development, thus requiring exquisite control.
As miRNAs frequently repress many targets, but mostly only modestly, it has become a dogma in the field that their key function may be to regulate pathways through coordinate 'tuning' of multiple targets, and/or achieve robustness of gene expression patterns by reducing noise. We tested this notion experimentally for the highly conserved let-7 miRNA, which has many previously identified targets, and which is essential for C. elegans survival. Using genome editing of the endogenous target, we could demonstrate that it is in fact regulation of one target alone, LIN-41/TRIM71, that is necessary and sufficient for this function of let-7 (Ecsedi et al. 2015). Hence, at least a subset of miRNAs may achieve functions in a more 'focused' manner. Conversely, discovery of a large set of regulated genes cannot be equated with physiological functions for each of these. Genome-editing approaches as pioneered here will provide a means to test and validate or refute such functions.
Finally, through gene expression profiling performed on developing C. elegans at high temporal resolution, we discovered an intriguing phenomenon of extensive oscillatory gene expression (Hendriks et al., 2014). Thus, >2,700 transcripts (representing nearly one fifth of active genes) change at least 2.1-fold but frequently > 10-fold, and with a period of ~8 hrs. These oscillations are distinct from circadian rhythms and may serve a developmental function, which we are investigating. Irrespective of the physiological function, a major impact of our finding on C. elegans research in general is that it reveals that gene expression profiling experiments are highly sensitive to temporal deviations in wild-type vs. mutant development, thus requiring exquisite control.