InteractiveFly: GeneBrief

Dorado: Biological Overview | References

Target-directed microRNA (miRNA) degradation (TDMD), which is mediated by the protein ZSWIM8 (mammalian homolog of Dorado), plays a widespread role in shaping miRNA abundances across bilateria. Some endogenous small interfering RNAs (siRNAs) of Drosophila cells have target sites resembling those that trigger TDMD, raising the question as to whether they too might undergo such regulation by Dorado, the Drosophila ZSWIM8 homolog. This study finds that some of these siRNAs are indeed sensitive to Dora when loaded into Ago1, the Argonaute paralog that preferentially associates with miRNAs. Despite this sensitivity when loaded into Ago1, these siRNAs are not detectably regulated by target-directed degradation because most molecules are loaded into Ago2, the Argonaute paralog that preferentially associates with siRNAs, and with siRNAs and miRNAs loaded into Ago2 were found to be insensitive to Dora. One explanation for the protection of these small RNAs loaded into Ago2 is that these small RNAs are 2'-O-methylated at their 3' termini. However, 2'-O-methylation does not protect these RNAs from Dora-mediated target-directed degradation, which indicates that their protection is instead conferred by features of the Ago2 protein itself. Together, these observations clarify the requirements for regulation by target-directed degradation and expand understanding of the role of 2'-O-methylation in small-RNA biology (Kingston, 2021).

Analyses of siRNAs in both wild-type and dora Drosophila S2 cells demonstrated that this class of small RNAs undergoes little regulation by conventional target-directed degradation (TDD). This finding does not support the suggestion that siRNAs loaded into Ago1 instead of Ago2 might be 'purified,' or removed from the cell, by TDD-a model put forth to help explain the high steady-state enrichment of siRNAs within Ago2. Although TDD of some Ago1-loaded siRNAs is seen, most siRNAs loaded in Ago1 escape such regulation. Furthermore, even for the TDD-sensitive siRNAs, up-regulation upon loss of Dora was undetectable when examining total-sRNA samples, because, for each siRNA, the Ago1-loaded fraction was minimal when compared to the Ago2-loaded fraction, and thus any increase in the Ago1-loaded fraction negligibly affected total siRNA levels. Thus, the known TDD pathway, which requires Dora, does not appear to be a major driving force in shaping the siRNA content of Drosophila S2 cells (Kingston, 2021).

Although methylation of small RNAs is proposed to protect these RNAs from TDD, this study observed both that loss of methylation does not make Ago2-loaded species susceptible to the known TDD pathway and that gained methylation does not protect Ago1-loaded species from this pathway. These observations indicate that features of Ago proteins, rather than modifications of small RNAs, dictate the ability of Ago-RNA complexes to be regulated by TDD. This importance of the Ago protein concurs with the new model for TDD, in which Ago proteins must interact with and be ubiquitinated by the ZSWIM8/Dora for TDD to occur (Han. 2020; Shi; 2020). Whereas Ago1 can engage with Dora in a TDD-competent manner, low sequence similarity between Ago2 and Ago1 supports the idea that Ago2 might lack the features necessary for Dora recognition and polyubiquitination. With respect to the sites of polyubiquitination, studies of human AGO2 implicate K493 and at least one other lysine within a cluster of 17 surface lysines as required for maximal ZSWIM8-mediated regulation, of which K493 and 12 of the other candidates sites are conserved in Drosophila Ago1, whereas K493 and all but two of the other candidates sites are not conserved in Drosophila Ago2. By analogy, it is speculated that if piRNAs are also protected from TDD, then this protection would also be conferred by the inability of PIWI proteins to interact with and be ubiquitinated by the ZSWIM8/Dora (Kingston, 2021).

Across many species, 2'-O-methylation occurs on guide RNAs that have extensive pairing to their targets, such as plant miRNAs, but not on guide RNAs that lack extensive pairing to most of their targets, such as metazoan miRNAs. Loss of this methylation leads to increased tailing and trimming that, at least for plant miRNAs, Tetrahymena piRNAs, nematode 26G siRNAs, and some Drosophila siRNAs, is associated with small-RNA destabilization. The realization that the identity of the Ago protein rather than the methylation status of the small RNA dictates susceptibility to TDD reopens the mystery as to why the tendency to be methylated correlates with the degree of complementarity of typical sites for a given class of small RNA (Kingston, 2021).

As a new solution to this mystery, it is suggested that these classes of small regulatory RNAs with highly complementary sites reside in Ago/PIWI proteins that have intrinsically weaker interactions with the 3' termini of their guide RNAs. This weaker intrinsic binding to guide-RNA 3' termini is expected for these proteins because it would favor formation of extensive target pairing, as release of the 3' terminus appears to be required to accommodate pairing to the central region of the guide RNA. In contrast, stronger intrinsic binding to guide-RNA 3' termini is expected for proteins that associate with metazoan miRNAs, as release of the 3' terminus is not required to accommodate target recognition typical of these small RNAs, i.e., seed pairing or seed pairing plus conventional 3'-supplementary pairing. The weaker intrinsic binding proposed for Ago/PIWI proteins with guide RNAs that recognize highly complementarity sites would presumably leave the 3' termini of their guide RNAs constitutively vulnerable to tailing and trimming even when they are not paired to a target, thereby explaining the benefit of terminal 2'-O-methylation. This Hen1-mediated methylation, found in plants and animals, presumably emerged early in eukaryotic evolution and thus would have been available for incorporation into the nascent metazoan miRNA pathway. Indeed, some methylation has been reported on most Nematostella miRNAs. Perhaps, however, as exemplified by the bilaterian lineage, as the miRNA-associated Ago proteins adapted to recognize less extensively paired sites, they acquired greater affinity to their guide-RNA 3' termini, which reduced vulnerability to trimming and tailing thereby obviating a benefit for their methylation (Kingston, 2021).

Several observations support aspects of this model. First, mutations within human Ago2 that reduce binding to miRNA 3' termini promote tailing and trimming even in the absence of an extensively paired target, which confirms the assumption that weaker binding to small-RNA 3' termini imparts constitutive vulnerability to tailing and trimming. Second, the 3' termini of piRNAs and metazoan siRNAs are 2'-O-methylated after these guide RNAs are loaded into Ago/PIWI, implying that the methylation machinery has at least intermittent access to the guide-RNA 3' termini, as would be expected if these proteins have relatively weak binding to the 3' termini of their guide RNAs. Third, loss of Hen1 led to increased tailing and trimming of Ago2-associated siRNAs that were Dora-insensitive when associated with Ago1, supporting the conjecture that Ago2-associated RNAs are vulnerable to tailing and trimming even in the absence of highly complementary sites able to trigger TDD (Kingston, 2021).

The notion that small RNAs with 3' termini not stably protected within Ago are susceptible to increased tailing and trimming might also help explain the observation that methylated miR-7, when loaded in Ago1, undergoes increased trimming relative to unmethylated miR-7. Perhaps the terminal methyl group is not well-accommodated by Ago1, which is typically loaded with unmethylated miRNAs, leading to increased exposure of the 3' terminus of the methylated miR-7. Indeed, conformations of human AGO2 represented by the crystal structures would not accommodate a terminal methyl group, implying that terminal methyl modifications might similarly clash with the ground-state structure of Drosophila Ago1. Although methylation is thought to protect small RNAs from trimming in addition to tailing, the observation that trimming of methylated miR-7 is Nibbler sensitive suggests that, at least in the context of Drosophila Ago1, methylated species can still be trimmed (Kingston, 2021).

In summary, the observation that some classes of small regulatory RNAs are methylated and some are not can be at least partly explained without invoking a TDD phenomenon: Because piRNAs, siRNAs, and plant miRNAs must efficiently pair to targets throughout their length, their corresponding Ago/PIWI proteins might have reduced affinity to guide-RNA 3' termini, and this reduced affinity would render these guide RNAs more susceptible to tailing and trimming even when they are not paired to target-unless they are 2'-O-methylated (Kingston, 2021).

Endogenous transcripts direct microRNA degradation in Drosophila, and this targeted degradation is required for proper embryonic development

MicroRNAs (miRNAs) typically direct degradation of their mRNA targets. However, some targets have unusual miRNA-binding sites that direct degradation of cognate miRNAs. Although this target-directed miRNA degradation (TDMD) is thought to shape the levels of numerous miRNAs, relatively few sites that endogenously direct degradation have been identified. This study identified six sites, five in mRNAs and one in a noncoding RNA named Marge, which serve this purpose in Drosophila cells or embryos. These six sites direct miRNA degradation without collateral target degradation, helping explain the effectiveness of this miRNA-degradation pathway. Mutations that disrupt this pathway are lethal, with many flies dying as embryos. Concomitant derepression of miR-3 and its paralog miR-309 appears responsible for some of this lethality, whereas the loss of Marge-directed degradation of miR-310 miRNAs causes defects in embryonic cuticle development. Thus, TDMD is implicated in the viability of an animal and is required for its proper development (Kingston, 2022).

MicroRNAs (miRNAs) are ~22-nt RNAs that associate with an Argonaute (Ago) effector protein to form a complex that represses gene expression. Within this miRNA-Ago complex, the miRNA recognizes sites in mRNAs-typically through pairing between its seed region (miRNA nucleotides 2-8) and a complementary site within the mRNA 3' UTR. Meanwhile, Ago recruits factors that repress the targeted mRNA, primarily by accelerating its deadenylation. In flies and mice, loss of an individual miRNA (or of several members of the same miRNA family) typically leads to developmental abnormalities or other defects, which are often severe, affecting viability, fertility, or other critical functions (Kingston, 2022).

In some special cases, a target site within either an mRNA or a noncoding RNA (ncRNA) can trigger degradation of the miRNA, inverting the typical regulatory logic. This target-directed miRNA degradation (TDMD) typically requires pairing to not only the miRNA seed region but also extensive pairing to the miRNA 3' region. This additional pairing is thought to induce conformational changes that recruit the ZSWIM8 Cullin-RING E3 ubiquitin-ligase complex, leading to poly-ubiquitination and proteasomal degradation of Ago and subsequent degradation of the miRNA. In mammalian cells, each of the four Ago paralogs are vulnerable to this degradation, whereas in Drosophila cells, Ago1, the paralog primarily loaded with miRNAs, is vulnerable, whereas Ago2, the paralog primarily loaded with endogenous small-interfering RNAs (siRNAs), is resistant (Kingston, 2022).

Triggers of TDMD with important biological functions were first discovered in herpesviruses, which express transcripts that direct degradation of host miRNAs that would otherwise impede their replication. More recently, sites within four mammalian transcripts were found to direct degradation of miR-29b, miR-7, miR-30b/c, and miR-221/222, which showed that TDMD triggered by endogenous transcripts helps to shape normal miRNA levels of vertebrate animals. Indeed, a site within the NREP mRNA, which directs degradation of miR-29b, is required for normal mouse behavior, and an orthologous site plays an analogous role in zebrafish (Kingston, 2022).

The four established examples of endogenous TDMD are thought to represent only a small fraction of the TDMD naturally occurring in animals. Supporting this idea, levels of 30 additional miRNAs increase after perturbing ZSWIM8 in mammalian cell lines, implying that the endogenous TDMD pathway also shapes the levels of these 30 miRNAs (Shi, 2020). Indeed, TDMD quantitatively explains the short half-lives of most short-lived miRNAs (Shi, 2020). Likewise, levels of 10 miRNAs increase after loss of the ZSWIM8 ortholog in Drosophila S2 cells, and levels of another 10 increase upon loss of the ZSWIM8 ortholog in Caenorhabditis elegans adults, implicating these miRNAs as TDMD substrates in each of these invertebrate species (Shi, 2020). Each of these putative TDMD substrates presumably pairs with at least one endogenously expressed, highly complementary target capable of triggering TDMD. However, no trigger has been reported for any of the recently inferred TDMD substrates (Kingston, 2022).

The TDMD pathway might be essential in some animals. Although null mutations in ebax-1, the ZSWIM8 ortholog of C. elegans, are viable, point substitutions within Dora, the ortholog of D. melanogaster, appear lethal (Wang, 2013; Yamamoto, 2014), implying that in flies, this ubiquitin-ligase receptor has an essential function, which might be either its role in TDMD or its recognition of other substrates. Indeed, recognition of other substrates by Dora orthologs is proposed to promote proper axon guidance in C. elegans and repress myogenesis in mammalian cell culture (Wang, 2013; Okumura, 2021). This study analyzed dora mutants and newly identified TDMD substrates and triggers. It was found that TDMD is required for proper development of an animal and implicated in its viability (Kingston, 2022).

This study identified six Drosophila transcripts that each direct degradation of one or more cognate miRNA, thereby demonstrating that Target-directed microRNA degradation (TDMD) operates to shape endogenous levels of miRNAs in an invertebrate animal and presumably has been doing so since the last common ancestor of flies and mammals. Identification of Marge as a TDMD trigger added to the growing list of lncRNAs with known biological functions, and identification of the other five TDMD triggers added to the growing list of mRNAs with known noncoding functions. Together, the six transcripts more than doubled the set of known endogenous TDMD triggers (Kingston, 2022).

Despite this success, the approach of testing one or two of the top computational predictions did not identify triggers for all of the Dora-sensitive miRNAs. Testing more of the top predictions would presumably identify more triggers. However, it is suspected that some sites that direct miRNA degradation were not among the top predictions. For example, sites that fell in coding sequences or 5' UTRs would have been missed by the pipeline used in this study, and sites with functional pairing architectures that differed from known examples, such as the seed-only recognition mode operating for the miR-35 family in C. elegans, would have scored poorly. Going forward, molecular or biochemical detection of transcripts associated with Dora might be the most productive approach for finding additional sites that direct miRNA degradation-especially now that functional sites have been identified in flies, which provide positive internal standards for calibrating experimental approaches in this classic model organism (Kingston, 2022).

Sites were probably not missed because they fell in lowly expressed, poorly annotated transcripts, since only sites that fell in highly expressed transcripts appeared to function to direct detectable degradation. This expression requirement leaves open the possibility that some candidates that failed to validate might nonetheless direct miRNA degradation in contexts where they are expressed more highly. This requirement also emphasized the importance of validating TDMD triggers in the context of their endogenous expression rather than through ectopic expression, since over-expression might impart activity to transcripts that do not normally reach levels sufficient to direct miRNA degradation (Kingston, 2022).

In normal cells and embryos that had the TDMD pathway, sites that directed miRNA degradation failed to also direct degradation of the trigger mRNA/lncRNA. However, in the absence of TDMD, some of these sites appeared to direct trigger degradation. Indeed, even in settings in which widespread repression of predicted targets of the miRNA was not detected, trigger destabilization seemed to occur-perhaps a consequence of extensive 3' pairing associated with sites that direct miRNA degradation, which can dramatically increase the magnitude of miRNA-mediated mRNA repression. Taken together, these results suggested that the TDMD pathway dominates over the pathway that normally degrades miRNA targets. Perhaps this apparent dominance resulted from the kinetics of the two pathways, with Ago degradation proceeding more rapidly than target degradation, or perhaps it resulted from mutual exclusivity of the two pathways, with efficient binding of Dora to Ago blocking association of the target-degradation machinery (Kingston, 2022).

Loss of Dora was lethal, with many dora-defective individuals dying as embryos. The simplest explanation for this embryonic lethality is that TDMD is required for proper embryonic development and viability of flies. Supporting this proposal, 11 embryonic miRNAs, including those of the miR-310 and miR-3 families, were derepressed in dora mutants. Moreover, targeted derepression of the miR-310 family by perturbation of marge revealed that clearance of this family by the TDMD pathway (and not merely degradation of an unrelated Dora E3-ligase substrate) is important for embryonic cuticle development. Thus, TDMD is required for proper development of an animal. Furthermore, genetic reduction of miR-3 family members partially rescued overall lethality, providing an additional connection between miRNA derepression and the dora phenotype (Kingston, 2022).

The 11 miRNAs derepressed upon the loss of Dora in the embryo did not overlap with the 10 miRNAs derepressed upon loss of Dora in S2 cells. Differences in the cohorts of Dora-sensitive miRNAs might have been expected when considering that S2 cells derive from a macrophage-like cell lineage that constitutes only a very small fraction of the cells in mid-to-late embryos. Nonetheless, the lack of any overlap between the embryonic and S2 samples indicated that TDMD substrates are strikingly cell-type specific in Drosophila (Kingston, 2022).

The 11 embryonic miRNAs derepressed upon the loss of Dora included the two embryonically expressed members of the miR-3 family and all six members of the miR-310 family. Both the miR-3 and the miR-310 families normally peak in expression during early embryogenesis; the two miR-3 family members (miR-3 and miR-309), together with other members of the mir-3 cluster, undergo a strong, transient pulse in production at the maternal-to-zygotic transition, and the miR-310 family is maternally deposited. Dora sensitivity of these miRNAs occurs at the time when their levels rapidly decline, which supports the idea that TDMD is enlisted to rapidly clear miRNAs during developmental transitions. Likewise, members of the miR-35 family, which are critical for early embryogenesis in C. elegans and then rapidly cleared during late embryogenesis, are sensitive to loss of Ebax-1, suggesting that this function is conserved across species. TDMD might also be particularly useful for customizing levels of miRNAs whose production is entangled with that of proteins or other miRNAs, due to transcription as part of either an mRNA intron or a larger miRNA cluster, as occurs with miR-3 and miR-309 (Kingston, 2022).

Analyses of marge embryos showed that target-directed degradation of the miR-310 family is important for proper formation of the embryonic cuticle. It is suggested that dysregulation of cuticle patterning in mutant embryos is driven by increased repression of sha, the top predicted target for the miR-310 family. How upregulation of the miR-310 family caused dysregulation of cuticle composition is more difficult to explain. Many mRNAs that encode structural components of the cuticle were upregulated, presumably as a secondary effect of increased repression of direct targets of the miR-310 family. Moreover, upregulation of cuticle structural components might seem counter-intuitive when considering the decreased cuticle integrity observed for marge mutants; whether this upregulation causes the decreased integrity or whether it is instead a consequence of such decreased integrity is unknown (Kingston, 2022).

The embryonic lethality of dora mutants complicated the study of additional roles of TDMD in Drosophila. This lethality might be bypassed through use of conditional dora disruption or depletion. Another approach for bypassing this lethality is to identify a transcript that triggers TDMD and then disrupt its complementary to the affected miRNA, as exemplified by disruption of the miR-310 site in Marge. This approach has the added benefit of disentangling the consequences of degrading multiple Dora substrates, including substrates other than Ago1, and thereby demonstrating TDMD. Despite success in identifying roles of Marge-directed degradation of miR-310 family members, a full account of the biological roles of targeted degradation of the miR-310 family awaits identification of one or more additional trigger that apparently collaborates with Marge to direct robust degradation of this family. Triggers for miR-3 and about half of the other miRNAs known to be Dora-sensitive also remain unidentified, further limiting the ability to use this approach to uncover additional biological roles for the pathway. How many roles for TDMD in Drosophila development and physiology might ultimately be uncovered? When considering that the search for Dora-sensitive miRNAs appears to have been far from saturating-with 21 known Dora-sensitive miRNAs found in only two contexts, 10 in S2 cells, and 11 in embryos, with no overlap-it appears that this study only scratched the surface (Kingston, 2022).

Screening of Drosophila microRNA-degradation sequences reveals Argonaute1 mRNA's role in regulating miR-999

MicroRNAs (miRNA) load onto AGO proteins to target mRNAs for translational repression or degradation. However, miRNA degradation can be triggered when extensively base-paired with target RNAs, which induces confirmational change of AGO and recruitment of ZSWIM8 ubiquitin ligase to mark AGO for proteasomal degradation. This target RNA-directed miRNA degradation (TDMD) mechanism appears to be evolutionarily conserved, but recent studies have focused on mammalian systems. This study performed AGO1-CLASH (cross-linking and sequencing of hybrids) in Drosophila S2 cells, with Dora (ortholog of vertebrate ZSWIM8) knockout mediated by CRISPR-Cas9 to identify five TDMD triggers (sequences that can induce miRNA degradation). Interestingly, one trigger in the 3' UTR of AGO1 mRNA induces miR-999 degradation. CRISPR-Cas9 knockout of the AGO1 trigger in S2 cells and in Drosophila specifically elevates miR-999, with concurrent repression of the miR-999 targets. AGO1 trigger knockout flies respond poorly to hydrogen peroxide-induced stress, demonstrating the physiological importance of this TDMD event (Sheng, 2023).

The ZSWIM8 ubiquitin ligase mediates target-directed microRNA degradation

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. This study 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 (Shi, 2020).

The analyses were extended beyond mammals, identifying 10 miRNAs sensitive to knockout of the ZSWIM8 ortholog (CG34401) in Drosophila S2 cells, each of which increased without a corresponding increase in the passenger strand. These 10 included the Drosophila miR-7 ortholog, which suggested that targeted degradation of miR-7 might have been conserved since the bilaterian ancestor of flies and mammals. For each of these inferred TDMD substrates, the fraction of tailed and trimmed isoforms increased upon CG34401 knockout; these findings suggested that, as in mammals, endogenous TDMD in flies is triggered by targets that pair in a manner that exposes the miRNA 3' termini to cytoplasmic tailing and trimming activities (Shi, 2020).

The conserved Pelado/ZSWIM8 protein regulates actin dynamics by promoting linear actin filament polymerization

Actin filament polymerization can be branched or linear, which depends on the associated regulatory proteins. Competition for actin monomers occurs between proteins that induce branched or linear actin polymerization. Cell specialization requires the regulation of actin filaments to allow the formation of cell type-specific structures, like cuticular hairs in Drosophila, formed by linear actin filaments. This study reports the functional analysis of CG34401/pelado, a gene encoding a SWIM domain-containing protein, conserved throughout the animal kingdom, called ZSWIM8 in mammals. Mutant <i>pelado</i> epithelial cells display actin hair elongation defects. This phenotype is reversed by increasing actin monomer levels or by either pushing linear actin polymerization or reducing branched actin polymerization. Similarly, in hemocytes, Pelado is essential to induce filopodia, a linear actin-based structure. It was further shown that this function of Pelado/ZSWIM8 is conserved in human cells, where Pelado inhibits branched actin polymerization in a cell migration context. In summary, these data indicate that the function of Pelado/ZSWIM8 in regulating actin cytoskeletal dynamics is conserved, favoring linear actin polymerization at the expense of branched filaments (Molina-Pelayo, 2022).

A P-insertion screen identifying novel X-linked essential genes in Drosophila

The recent determination and annotation of the entire euchromatic sequence of the Drosophila melanogaster genome predicted the existence of about 13600 different genes. In parallel, the Berkeley Drosophila Genome Project (BDGP) has undertaken systematic P-insertion screens, to isolate new lethals and misexpressing lines. To date, however, the genes of the X chromosome have been under-represented in the screens performed. In order both to characterize several X-linked genes of prime interest and contribute to the collection of lethal P-insertions available to the community, a P-insertion mutagenesis was performed of the X chromosome. Using the PlacW and PGawB P-elements as mutagens, two complementary sets of enhancer-trap lines, l(1)(T)PL and l(1)(T)PG, respectively, were generated that both contain a reporter gene whose developmental expression can be monitored when driven by nearby enhancer sequences. This study the characterization of 260 new insertions, mapping to 133 different genes or predicted CGs. Of these, 83 correspond to genes for which no lethal mutation had yet been reported. For 64 of those, it was confirmed that lethality was solely due to the P-element insertion. The primary molecular data, reporter gene expression patterns (observed in embryos, third instar larvae and adult ovaries) and proposed CG assignment for each strain can be accessed and updated on a website (Bourbon, 2022) .

A ubiquitin ligase mediates target-directed microRNA decay independently of tailing and trimming

MicroRNAs (miRNAs) act in concert with Argonaute (AGO) proteins to repress target messenger RNAs. After AGO loading, miRNAs generally exhibit slow turnover. An important exception occurs when miRNAs encounter highly complementary targets, which can trigger a process called target-directed miRNA degradation (TDMD). During TDMD, miRNAs undergo tailing and trimming, suggesting that this is an important step in the decay mechanism. A cullin-RING ubiquitin ligase (CRL) was identified, containing the substrate adaptor ZSWIM8, that mediates TDMD. The ZSWIM8 CRL interacts with AGO proteins, promotes TDMD in a tailing and trimming-independent manner, and regulates miRNA expression in multiple cell types. These findings suggest a model in which the ZSWIM8 ubiquitin ligase mediates TDMD by directing proteasomal decay of miRNA-containing complexes engaged with highly complementary targets (Han, 2020).

The ZSWIM8 ubiquitin ligase regulates neurodevelopment by guarding the protein quality of intrinsically disordered Dab1

Protein quality control (PQC) is essential for maintaining protein homeostasis and guarding the accuracy of neurodevelopment. Previous work found that a conserved EBAX-type CRL regulates the protein quality of SAX-3/ROBO guidance receptors in Caenorhabditis elegans. This study reporta that ZSWIM8, the mammalian homolog of EBAX-1, is essential for developmental stability of mammalian brains. Conditional deletion of Zswim8 in the embryonic nervous system causes global cellular stress, partial perinatal lethality and defective migration of neural progenitor cells. CRISPR-mediated knockout of ZSWIM8 impairs spine formation and synaptogenesis in hippocampal neurons. Mechanistic studies reveal that ZSWIM8 controls protein quality of Disabled 1 (Dab1), a key signal molecule for brain development, thus protecting the signaling strength of Dab1. As a ubiquitin ligase enriched with intrinsically disordered regions (IDRs), ZSWIM8 specifically recognizes IDRs of Dab1 through a 'disorder targets misorder' mechanism and eliminates misfolded Dab1 that cannot be properly phosphorylated. Adult survivors of ZSWIM8 CKO show permanent hippocampal abnormality and display severely impaired learning and memory behaviors. Altogether, these results demonstrate that ZSWIM8-mediated PQC is critical for the stability of mammalian brain development (Wang, G, 2023).

A statistical approach for identifying primary substrates of ZSWIM8-mediated microRNA degradation in small-RNA sequencing data

One strategy for identifying targets of a regulatory factor is to perturb the factor and use high-throughput RNA sequencing to examine the consequences. However, distinguishing direct targets from secondary effects and experimental noise can be challenging when confounding signal is present in the background at varying levels. This study presents a statistical modeling strategy to identify microRNAs that are primary substrates of target-directed miRNA degradation (TDMD) mediated by ZSWIM8. This method uses a bi-beta-uniform mixture (BBUM) model to separate primary from background signal components, leveraging the expectation that primary signal is restricted to upregulation and not downregulation upon loss of ZSWIM8. The BBUM model strategy retained the apparent sensitivity and specificity of the previous ad hoc approach but was more robust against outliers, achieved a more consistent stringency, and could be performed using a single cutoff of false discovery rate (FDR). The BBUM model, a robust statistical modeling strategy, was developed to account for background secondary signal in differential expression data. It performed well for identifying primary substrates of TDMD and should be useful for other applications in which the primary regulatory targets are only upregulated or only downregulated. The BBUM model, FDR-correction algorithm, and significance-testing methods are available as an R package (Wang, P. 2023)

ZSWIM8 is a myogenic protein that partly prevents C2C12 differentiation

Cell adhesion molecule-related/downregulated by oncogenes (Cdon) is a cell-surface receptor that mediates cell-cell interactions and positively regulates myogenesis. The cytoplasmic region of Cdon interacts with other proteins to form a Cdon/JLP/Bnip-2/CDC42 complex that activates p38 mitogen-activated protein kinase (MAPK) and induces myogenesis. However, Cdon complex may include other proteins during myogenesis. This study found that Cullin 2-interacting protein zinc finger SWIM type containing 8 (ZSWIM8) ubiquitin ligase is induced during C2C12 differentiation and is included in the Cdon complex. Zswim8 was knocked-down in C2C12 cells to determine the effect of ZSWIM8 on differentiation. However, neither ZSWIM8-dependent ubiquitination nor the degradation of Bnip2, Cdon, or JLP was detected. In contrast, ZSWIM8 knockdown accelerated C2C12 differentiation. These results suggest that ZSWIM8 is a Cdon complex-included myogenic protein that prevents C2C12 differentiation without affecting the stability of Bnip2, Cdon, and JLP (Okumura, 2021).

Widespread microRNA degradation elements in target mRNAs can assist the encoded proteins

Binding of microRNAs (miRNAs) to mRNAs normally results in post-transcriptional repression of gene expression. However, extensive base-pairing between miRNAs and target RNAs can trigger miRNA degradation, a phenomenon called target RNA-directed miRNA degradation (TDMD). This study systematically analyzed Argonaute-CLASH (cross-linking, ligation, and sequencing of miRNA-target RNA hybrids) data and identified numerous candidate TDMD triggers, focusing on their ability to induce nontemplated nucleotide addition at the miRNA 3' end. When exogenously expressed in various cell lines, eight triggers (sequences that can induce miRNA degradation) induced degradation of corresponding miRNAs. Both the TDMD base-pairing and surrounding sequences are essential for TDMD. CRISPR knockout of endogenous trigger or ZSWIM8, a ubiquitin ligase essential for TDMD, reduced miRNA degradation. Furthermore, degradation of miR-221 and miR-222 by a trigger in BCL2L11, which encodes a proapoptotic protein, enhances apoptosis. Therefore, this study uncovered widespread TDMD triggers in target RNAs and demonstrated an example that could functionally cooperate with the encoded protein (Li, 2021).

The ZSWIM8 ubiquitin ligase mediates target-directed microRNA degradation

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. This target-directed miRNA degradation (TDMD) was found to 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 (Shi, 2020).

Search PubMed for articles about Drosophila Dorado

Bourbon, H. M., Gonzy-Treboul, G., Peronnet, F., Alin, M. F., Ardourel, C., Benassayag, C., Cribbs, D., Deutsch, J., Ferrer, P., Haenlin, M., Lepesant, J. A., Noselli, S. and Vincent, A. (2002). A P-insertion screen identifying novel X-linked essential genes in Drosophila. Mech Dev 110(1-2): 71-83. PubMed ID: 11744370

Han, J., LaVigne, C. A., Jones, B. T., Zhang, H., Gillett, F. and Mendell, J. T. (2020). A ubiquitin ligase mediates target-directed microRNA decay independently of tailing and trimming. Science 370(6523). PubMed ID: 33184234

Kingston, E. R. and Bartel, D. P. (2021). Ago2 protects Drosophila siRNAs and microRNAs from target-directed degradation, even in the absence of 2'-O-methylation. RNA. PubMed ID: 33853897

Kingston, E. R., Blodgett, L. W. and Bartel, D. P. (2022). Endogenous transcripts direct microRNA degradation in Drosophila, and this targeted degradation is required for proper embryonic development. Mol Cell. PubMed ID: 36150386

Li, L., Sheng, P., Li, T., Fields, C. J., Hiers, N. M., Wang, Y., Li, J., Guardia, C. M., Licht, J. D. and Xie, M. (2021). Widespread microRNA degradation elements in target mRNAs can assist the encoded proteins. Genes Dev 35(23-24): 1595-1609. PubMed ID: 34819352

Molina-Pelayo, C., Olguin, P., Mlodzik, M. and Glavic, A. (2022). The conserved Pelado/ZSWIM8 protein regulates actin dynamics by promoting linear actin filament polymerization. Life Sci Alliance 5(12). PubMed ID: 35940847

Okumura, F., Oki, N., Fujiki, Y., Ikuta, R., Osaki, K., Hamada, S., Nakatsukasa, K., Hisamoto, N., Hara, T. and Kamura, T. (2021). ZSWIM8 is a myogenic protein that partly prevents C2C12 differentiation. Sci Rep 11(1): 20880. PubMed ID: 34686700

Sheng, P., Li, L., Li, T., Wang, Y., Hiers, N. M., Mejia, J. S., Sanchez, J. S., Zhou, L. and Xie, M. (2023). Screening of Drosophila microRNA-degradation sequences reveals Argonaute1 mRNA's role in regulating miR-999. Nat Commun 14(1): 2108. PubMed ID: 37055443

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date revised: 20 June 2023

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