Noncoding RNAs (ncRNAs) play increasingly appreciated gene-regulatory roles. Here, we describe a regulatory network centered on four ncRNAs—a long ncRNA, a circular RNA, and two microRNAs—using gene ...editing in mice to probe the molecular consequences of disrupting key components of this network. The long ncRNA Cyrano uses an extensively paired site to miR-7 to trigger destruction of this microRNA. Cyrano-directed miR-7 degradation is much more effective than previously described examples of target-directed microRNA degradation, which come primarily from studies of artificial and viral RNAs. By reducing miR-7 levels, Cyrano prevents repression of miR-7–targeted mRNAs and enables accumulation of Cdr1as, a circular RNA known to regulate neuronal activity. Without Cyrano, excess miR-7 causes cytoplasmic destruction of Cdr1as in neurons, in part through enhanced slicing of Cdr1as by a second miRNA, miR-671. Thus, several types of ncRNAs can collaborate to establish a sophisticated regulatory network.
Display omitted
•A long noncoding RNA, a circular RNA, and two microRNAs form a regulatory network•The Cyrano long noncoding RNA directs potent, multiple-turnover destruction of miR-7•Unchecked miR-7 prevents accumulation of Cdr1as circular RNA in cytoplasm of neurons•miR-7 prevents this accumulation by enhancing the miR-671–directed slicing of Cdr1as
Three different classes of noncoding RNA converge in a regulatory network whereby a long noncoding RNA represses a microRNA via target-directed microRNA degradation, which in turn enables the accumulation of a circular RNA in the mouse brain.
The biochemical basis of microRNA targeting efficacy McGeary, Sean E; Lin, Kathy S; Shi, Charlie Y ...
Science (American Association for the Advancement of Science),
12/2019, Letnik:
366, Številka:
6472
Journal Article
Recenzirano
Odprti dostop
MicroRNAs (miRNAs) act within Argonaute proteins to guide repression of messenger RNA targets. Although various approaches have provided insight into target recognition, the sparsity of miRNA-target ...affinity measurements has limited understanding and prediction of targeting efficacy. Here, we adapted RNA bind-n-seq to enable measurement of relative binding affinities between Argonaute-miRNA complexes and all sequences ≤12 nucleotides in length. This approach revealed noncanonical target sites specific to each miRNA, miRNA-specific differences in canonical target-site affinities, and a 100-fold impact of dinucleotides flanking each site. These data enabled construction of a biochemical model of miRNA-mediated repression, which was extended to all miRNA sequences using a convolutional neural network. This model substantially improved prediction of cellular repression, thereby providing a biochemical basis for quantitatively integrating miRNAs into gene-regulatory networks.
MicroRNAs (miRNAs) associate with Argonaute (AGO) proteins to direct widespread posttranscriptional gene repression. Although association with AGO typically protects miRNAs from nucleases, extensive ...pairing to some unusual target RNAs can trigger miRNA degradation. We found that this target-directed miRNA degradation (TDMD) required the ZSWIM8 Cullin-RING E3 ubiquitin ligase. This and other findings support a mechanistic model of TDMD in which target-directed proteolysis of AGO by the ubiquitin-proteasome pathway exposes the miRNA for degradation. Moreover, loss-of-function studies indicated that the ZSWIM8 Cullin-RING ligase accelerates degradation of numerous miRNAs in cells of mammals, flies, and nematodes, thereby specifying the half-lives of most short-lived miRNAs. These results elucidate the mechanism of TDMD and expand its inferred role in shaping miRNA levels in bilaterian animals.
MicroRNAs (miRNAs) pair to sites in mRNAs to direct the degradation of these RNA transcripts. Conversely, certain RNA transcripts can direct the degradation of particular miRNAs. This target-directed ...miRNA degradation (TDMD) requires the ZSWIM8 E3 ubiquitin ligase. Here, we report the function of ZSWIM8 in the mouse embryo.
embryos were smaller than their littermates and died near the time of birth. This highly penetrant perinatal lethality was apparently caused by a lung sacculation defect attributed to failed maturation of alveolar epithelial cells. Some mutant individuals also had heart ventricular septal defects. These developmental abnormalities were accompanied by aberrant accumulation of more than 50 miRNAs observed across 12 tissues, which often led to enhanced repression of their mRNA targets. These ZSWIM8-sensitive miRNAs were preferentially produced from genomic miRNA clusters, and in some cases, ZSWIM8 caused a switch in the dominant strand or isoform that accumulated from a miRNA hairpin-observations suggesting that TDMD provides a mechanism to uncouple coproduced miRNAs from each other. Overall, our findings indicate that the regulatory influence of ZSWIM8, and presumably TDMD, in mammalian biology is widespread and consequential, and posit the existence of many yet-unidentified transcripts that trigger miRNA degradation.
MicroRNAs (miRNAs) are small, ~21 nucleotide RNAs that guide Argonaute (AGO) proteins to complementary target RNAs using base-pairing interactions. In canonical miRNA-mediated repression, AGO ...recruits repressive trans-acting factors to bound targets to promote their deadenylation, decapping, and subsequent decay. Although knowledge surrounding miRNA biogenesis and effector functions is extensive, the understanding of how miRNAs decay is still relatively nascent. Recent global measurements of miRNA stabilities in cells of mice and flies concluded that most miRNAs are very stable, with half-lives ranging from many hours to days. However, a minority of miRNAs were also observed to be labile, with half-lives of a few hours. Prior to our work, it was not known how this rapid turnover is specified and actuated, although a number of miRNA-degradation processes and associated effectors had been proposed. One particular pathway discovered in 2010, named “target-directed miRNA degradation” (TDMD), was shown to cause miRNA degradation in the presence of unusually highly complementary target RNAs, and held particular explanatory appeal. However, neither the mechanism nor the biological scope of this pathway, beyond a handful of examples, were definitively known. We searched for factors involved in TDMD, and unexpectedly found that the ZSWIM8 Cullin-RING ligase was required for all known instances of TDMD that we tested. We then showed that, contrary to the prior model, TDMD proceeds by a mechanism where in the presence of a highly complementary target RNA, ZSWIM8 polyubiquitinates AGO and marks it for destruction, thereby exposing the normally protected miRNA to degradation. By perturbing ZSWIM8, we were able to confidently identify dozens of miRNAs likely undergoing targeted degradation in cells of flies, mice, and worms. Our data showed that TDMD was able to quantitatively explain the half-lives of most rapidly turned over miRNAs in cells of mice and flies, implying that this regulatory mode is pervasively used in bilaterian animals. To interrogate the biological functions mediated by TDMD, we knocked out ZSWIM8 in mice and found that mutants died soon after birth with small body size, cyanotic appearance, and defects in heart and lung development. These aberrations were accompanied by accumulation of ~50 miRNAs across various tissues – in many cases resulting in significant repression of their targets. This work is consistent with the possibility that TDMD mediates important biological functions in animals, and further work will be needed to clarify the precise molecular and cellular bases of the phenotypes.
MicroRNAs (miRNAs) are small, ~21 nucleotide RNAs that guide Argonaute (AGO) proteins to complementary target RNAs using base-pairing interactions. In canonical miRNA-mediated repression, AGO ...recruits repressive trans-acting factors to bound targets to promote their deadenylation, decapping, and subsequent decay. Although knowledge surrounding miRNA biogenesis and effector functions is extensive, the understanding of how miRNAs decay is still relatively nascent. Recent global measurements of miRNA stabilities in cells of mice and flies concluded that most miRNAs are very stable, with half-lives ranging from many hours to days. However, a minority of miRNAs were also observed to be labile, with half-lives of a few hours. Prior to our work, it was not known how this rapid turnover is specified and actuated, although a number of miRNA-degradation processes and associated effectors had been proposed. One particular pathway discovered in 2010, named “target-directed miRNA degradation” (TDMD), was shown to cause miRNA degradation in the presence of unusually highly complementary target RNAs, and held particular explanatory appeal. However, neither the mechanism nor the biological scope of this pathway, beyond a handful of examples, were definitively known.;
;
We searched for factors involved in TDMD, and unexpectedly found that the ZSWIM8 Cullin-RING ligase was required for all known instances of TDMD that we tested. We then showed that, contrary to the prior model, TDMD proceeds by a mechanism where in the presence of a highly complementary target RNA, ZSWIM8 polyubiquitinates AGO and marks it for destruction, thereby exposing the normally protected miRNA to degradation. By perturbing ZSWIM8, we were able to confidently identify dozens of miRNAs likely undergoing targeted degradation in cells of flies, mice, and worms. Our data showed that TDMD was able to quantitatively explain the half-lives of most rapidly turned over miRNAs in cells of mice and flies, implying that this regulatory mode is pervasively used in bilaterian animals.;
;
To interrogate the biological functions mediated by TDMD, we knocked out ZSWIM8 in mice and found that mutants died soon after birth with small body size, cyanotic appearance, and defects in heart and lung development. These aberrations were accompanied by accumulation of ~50 miRNAs across various tissues – in many cases resulting in significant repression of their targets. This work is consistent with the possibility that TDMD mediates important biological functions in animals, and further work will be needed to clarify the precise molecular and cellular bases of the phenotypes.
Ph.D.
Intestinal intraepithelial lymphocytes (IELs) are distributed along the length of the intestine and are considered the frontline of immune surveillance. The precise molecular mechanisms, especially ...epigenetic regulation, of their development and function are poorly understood. The trimethylation of histone 3 at lysine 27 (H3K27Me3) is a kind of histone modifications and associated with gene repression. Kdm6b is an epigenetic enzyme responsible for the demethylation of H3K27Me3 and thus promotes gene expression. Here we identified Kdm6b as an important intracellular regulator of small intestinal IELs. Mice genetically deficient for Kdm6b showed greatly reduced numbers of TCRαβ
CD8αα
IELs. In the absence of Kdm6b, TCRαβ
CD8αα
IELs exhibited increased apoptosis, disturbed maturation and a compromised capability to lyse target cells. Both IL-15 and Kdm6b-mediated demethylation of histone 3 at lysine 27 are responsible for the maturation of TCRαβ
CD8αα
IELs through upregulating the expression of Gzmb and Fasl. In addition, Kdm6b also regulates the expression of the gut-homing molecule CCR9 by controlling H3K27Me3 level at its promoter. However, Kdm6b is dispensable for the reactivity of thymic precursors of TCRαβ
CD8αα
IELs (IELPs) to IL-15 and TGF-β. In conclusion, we showed that Kdm6b plays critical roles in the maturation and cytotoxic function of small intestinal TCRαβ
CD8αα
IELs.
Seven of 120 consecutive patients with inducible sustained ventricular tachycardia (from September 1, 1988 to January 1, 1991) had bundle branch reentrant tachycardia and underwent percutaneous ...radiofrequency ablation of the right bundle branch. The seven patients had been unsuccessfully treated with a mean of 3 ± 1 drugs. Four patients presented with syncope and three with aborted sudden death. The baseline electrocardiogram revealed a left bundle branch block pattern in three patients and an intraventricular conduction defect in four.
The baseline HV interval was prolonged in each case (79 ± 2 ms). With use of programmed ventricular extrastimuli, sustained bundle branch reentrant tachycardia was inducible in all patients at a mean cycle length of 283 ± 17 ms (range 230 to 350). Bundle branch reentrant tachycardia characteristics included atrioventricular dissociation, a His deflection that preceded each QRS complex and spontaneous His to His variation that preceded changes in ventricular tachycardia cycle length.
A quadripolar catheter was positioned across the tricuspid valve with the distal electrode tip of the catheter near the right bundle branch. One to three applications of continuous unmodulated radiofrequency current at 300 kHz between the distal electrode and a large posterior skin patch resulted in complete right bundle branch block in all patients, after which none had inducible bundle branch reentrant tachycardia on restudy. On restudy, three of the seven patients had ventricular tachycardia of myocardial origin (not bundle branch reentry). One patient required no therapy; drug or defibrillator therapy was used in the others. After a mean follow-up interval of 12 ± 3 months (range 6 to 29) complete right bundle branch block persisted, there were no spontaneous episodes of ventricular tachycardia and no patient required a permanent pacemaker.
Radiofrequency catheter ablation of the right bundle branch is easily performed and is a safe and effective treatment for bundle branch reentrant tachycardia. It is probably the procedure of choice for these highly symptomatic patients.
PDF
