Extraskeletal chondroma of the toe in a child with DICER1 tumor predisposition syndrome: support for a dominant negative mechanism.

DICER1 tumor predisposition syndrome is a pleiotropic disorder that gives rise to various mainly pediatric-onset lesions. We report an extraskeletal chondroma (EC) of the great toe occurring in a child who, unusually, carries a germline "hotspot" missense DICER1 variant rather than the more usual loss-of-function (LOF) variant. No heterozygous LOF allele was identified in the EC. We demonstrate this variant impairs 5p cleavage of precursor-miRNA (pre-miRNA) and competes with wild-type (WT) DICER1 protein for pre-miRNA processing. These results suggest a mechanism through which a germline RNase IIIb variant could impair pre-miRNA processing without complete LOF of the WT DICER1 allele.

Uncovering a mammalian neural-specific poly(A) binding protein with unique properties.

The mRNA 3' poly(A) tail plays a critical role in regulating both mRNA translation and turnover. It is bound by the cytoplasmic poly(A) binding protein (PABPC), an evolutionarily conserved protein that can interact with translation factors and mRNA decay machineries to regulate gene expression. Mammalian PABPC1, the prototypical PABPC, is expressed in most tissues and interacts with eukaryotic translation initiation factor 4G (eIF4G) to stimulate translation in specific contexts. In this study, we uncovered a new mammalian PABPC, which we named neural PABP (neuPABP), as it is predominantly expressed in the brain. neuPABP maintains a unique architecture as compared with other PABPCs, containing only two RNA recognition motifs (RRMs) and maintaining a unique N-terminal domain of unknown function. neuPABP expression is activated in neurons as they mature during synaptogenesis, where neuPABP localizes to the soma and postsynaptic densities. neuPABP interacts with the noncoding RNA BC1, as well as mRNAs coding for ribosomal and mitochondrial proteins. However, in contrast to PABPC1, neuPABP does not associate with actively translating mRNAs in the brain. In keeping with this, we show that neuPABP has evolved such that it does not bind eIF4G and as a result fails to support protein synthesis in vitro. Taken together, these results indicate that mammals have expanded their PABPC repertoire in the brain and propose that neuPABP may support the translational repression of select mRNAs.

The EDC4-XRN1 interaction controls P-body dynamics to link mRNA decapping with decay.

Deadenylation-dependent mRNA decapping and decay is the major cytoplasmic mRNA turnover pathway in eukaryotes. Many mRNA decapping and decay factors are associated with each other via protein-protein interaction motifs. For example, the decapping enzyme DCP2 and the 5'-3' exonuclease XRN1 interact with the enhancer of mRNA-decapping protein 4 (EDC4), a large scaffold that has been reported to stimulate mRNA decapping. mRNA decapping and decay factors are also found in processing bodies (P-bodies), evolutionarily conserved ribonucleoprotein granules that are often enriched with mRNAs targeted for decay, yet paradoxically are not required for mRNA decay to occur. Here, we show that disrupting the EDC4-XRN1 interaction or altering their stoichiometry inhibits mRNA decapping, with microRNA-targeted mRNAs being stabilized in a translationally repressed state. Importantly, we demonstrate that this concomitantly leads to larger P-bodies that are responsible for preventing mRNA decapping. Finally, we demonstrate that P-bodies support cell viability and prevent stress granule formation when XRN1 is limiting. Taken together, these data demonstrate that the interaction between XRN1 and EDC4 regulates P-body dynamics to properly coordinate mRNA decapping with 5'-3' decay in human cells.

DICER1 platform domain missense variants inhibit miRNA biogenesis and lead to tumor susceptibility.

The endoribonuclease DICER1 plays an essential role in the microRNA (miRNA) biogenesis pathway, cleaving precursor miRNA (pre-miRNA) stem-loops to generate mature single-stranded miRNAs. Germline pathogenic variants (GPVs) in DICER1 result in DICER1 tumor predisposition syndrome (DTPS), a mainly childhood-onset tumor susceptibility disorder. Most DTPS-causing GPVs are nonsense or frameshifting, with tumor development requiring a second somatic missense hit that impairs the DICER1 RNase IIIb domain. Interestingly, germline DICER1 missense variants that cluster in the DICER1 Platform domain have been identified in some persons affected by tumors that also associate with DTPS. Here, we demonstrate that four of these Platform domain variants prevent DICER1 from producing mature miRNAs and as a result impair miRNA-mediated gene silencing. Importantly, we show that in contrast to canonical somatic missense variants that alter DICER1 cleavage activity, DICER1 proteins harboring these Platform variants fail to bind to pre-miRNA stem-loops. Taken together, this work sheds light upon a unique subset of GPVs causing DTPS and provides new insights into how alterations in the DICER1 Platform domain can impact miRNA biogenesis.

miRNA biogenesis and inherited disorders: clinico-molecular insights.

MicroRNAs (miRNAs) play vital roles in the regulation of gene expression, a process known as miRNA-induced gene silencing. The human genome codes for many miRNAs, and their biogenesis relies on a handful of genes, including DROSHA, DGCR8, DICER1, and AGO1/2. Germline pathogenic variants (GPVs) in these genes cause at least three distinct genetic syndromes, with clinical manifestations that range from hyperplastic/neoplastic entities to neurodevelopmental disorders (NDDs). Over the past decade, DICER1 GPVs have been shown to lead to tumor predisposition. Moreover, recent findings have provided insight into the clinical consequences arising from GPVs in DGCR8, AGO1, and AGO2. Here we provide a timely update with respect to how GPVs in miRNA biogenesis genes alter miRNA biology and ultimately lead to their clinical manifestations.

P-bodies directly regulate MARF1-mediated mRNA decay in human cells.

Processing bodies (P-bodies) are ribonucleoprotein granules that contain mRNAs, RNA-binding proteins and effectors of mRNA turnover. While P-bodies have been reported to contain translationally repressed mRNAs, a causative role for P-bodies in regulating mRNA decay has yet to be established. Enhancer of decapping protein 4 (EDC4) is a core P-body component that interacts with multiple mRNA decay factors, including the mRNA decapping (DCP2) and decay (XRN1) enzymes. EDC4 also associates with the RNA endonuclease MARF1, an interaction that antagonizes the decay of MARF1-targeted mRNAs. How EDC4 interacts with MARF1 and how it represses MARF1 activity is unclear. In this study, we show that human MARF1 and XRN1 interact with EDC4 using analogous conserved short linear motifs in a mutually exclusive manner. While the EDC4-MARF1 interaction is required for EDC4 to inhibit MARF1 activity, our data indicate that the interaction with EDC4 alone is not sufficient. Importantly, we show that P-body architecture plays a critical role in antagonizing MARF1-mediated mRNA decay. Taken together, our study suggests that P-bodies can directly regulate mRNA turnover by sequestering an mRNA decay enzyme and preventing it from interfacing with and degrading targeted mRNAs.

PABP prevents the untimely decay of select mRNA populations in human cells.

Gene expression is tightly regulated at the levels of both mRNA translation and stability. The poly(A)-binding protein (PABP) is thought to play a role in regulating these processes by binding the mRNA 3' poly(A) tail and interacting with both the translation and mRNA deadenylation machineries. In this study, we directly investigate the impact of PABP on translation and stability of endogenous mRNAs in human cells. Remarkably, our transcriptome-wide analysis only detects marginal mRNA translation changes in PABP-depleted cells. In contrast, rapidly depleting PABP alters mRNA abundance and stability, albeit non-uniformly. Otherwise stable transcripts, including those encoding proteins with constitutive functions, are destabilized in PABP-depleted cells. In contrast, many unstable mRNAs, including those encoding proteins with regulatory functions, decay at similar rates in presence or absence of PABP. Moreover, PABP depletion-induced cell death can partially be suppressed by disrupting the mRNA decapping and 5'-3' decay machinery. Finally, we provide evidence that the LSM1-7 complex promotes decay of "stable" mRNAs in PABP-depleted cells. Taken together, these findings suggest that PABP plays an important role in preventing the untimely decay of select mRNA populations.

A Cell-Free System for Investigating Human MARF1 Endonuclease Activity.

Experiments in cell cultures have been useful for investigating a number of RNA endonucleases. However, endonuclease decay intermediates are often challenging to study in cellulo, as decay intermediates are rapidly degraded by exoribonucleases. Thus, cell-free assays have been critical for assessing endonuclease kinetics. Here, we describe such an in vitro assay to analyze endoribonuclease activity using recombinant proteins and end-radiolabeled RNA oligonucleotides. Specifically, we detail a protocol for assaying the endoribonuclease activity and kinetics of the human MARF1 protein.

A non-canonical role for the EDC4 decapping factor in regulating MARF1-mediated mRNA decay.

EDC4 is a core component of processing (P)-bodies that binds the DCP2 decapping enzyme and stimulates mRNA decay. EDC4 also interacts with mammalian MARF1, a recently identified endoribonuclease that promotes oogenesis and contains a number of RNA binding domains, including two RRMs and multiple LOTUS domains. How EDC4 regulates MARF1 action and the identity of MARF1 target mRNAs is not known. Our transcriptome-wide analysis identifies bona fide MARF1 target mRNAs and indicates that MARF1 predominantly binds their 3' UTRs via its LOTUS domains to promote their decay. We also show that a MARF1 RRM plays an essential role in enhancing its endonuclease activity. Importantly, we establish that EDC4 impairs MARF1 activity by preventing its LOTUS domains from binding target mRNAs. Thus, EDC4 not only serves as an enhancer of mRNA turnover that binds DCP2, but also as a repressor that binds MARF1 to prevent the decay of MARF1 target mRNAs.

DGCR8 microprocessor defect characterizes familial multinodular goiter with schwannomatosis.

BACKGROUNDDICER1 is the only miRNA biogenesis component associated with an inherited tumor syndrome, featuring multinodular goiter (MNG) and rare pediatric-onset lesions. Other susceptibility genes for familial forms of MNG likely exist.METHODSWhole-exome sequencing of a kindred with early-onset MNG and schwannomatosis was followed by investigation of germline pathogenic variants that fully segregated with the disease. Genome-wide analyses were performed on 13 tissue samples from familial and nonfamilial DGCR8-E518K-positive tumors, including MNG, schwannomas, papillary thyroid cancers (PTCs), and Wilms tumors. miRNA profiles of 4 tissue types were compared, and sequencing of miRNA, pre-miRNA, and mRNA was performed in a subset of 9 schwannomas, 4 of which harbor DGCR8-E518K.RESULTSWe identified c.1552G>A;p.E518K in DGCR8, a microprocessor component located in 22q, in the kindred. The variant identified is a somatic hotspot in Wilms tumors and has been identified in 2 PTCs. Copy number loss of chromosome 22q, leading to loss of heterozygosity at the DGCR8 locus, was found in all 13 samples harboring c.1552G>A;p.E518K. miRNA profiling of PTCs, MNG, schwannomas, and Wilms tumors revealed a common profile among E518K hemizygous tumors. In vitro cleavage demonstrated improper processing of pre-miRNA by DGCR8-E518K. MicroRNA and RNA profiling show that this variant disrupts precursor microRNA production, impacting populations of canonical microRNAs and mirtrons.CONCLUSIONWe identified DGCR8 as the cause of an unreported autosomal dominant mendelian tumor susceptibility syndrome: familial multinodular goiter with schwannomatosis.FUNDINGCanadian Institutes of Health Research, Compute Canada, Alex's Lemonade Stand Foundation, the Mia Neri Foundation for Childhood Cancer, Cassa di Sovvenzioni e Risparmio fra il Personale della Banca d'Italia, and the KinderKrebsInitiative Buchholz/Holm-Seppensen.

Communication Is Key: 5'-3' Interactions that Regulate mRNA Translation and Turnover.

Most eukaryotic mRNAs maintain a 5' cap structure and 3' poly(A) tail, cis-acting elements that are often separated by thousands of nucleotides. Nevertheless, multiple paradigms exist where mRNA 5' and 3' termini interact with each other in order to regulate mRNA translation and turnover. mRNAs recruit translation initiation factors to their termini, which in turn physically interact with each other. This physical bridging of the mRNA termini is known as the "closed loop" model, with years of genetic and biochemical evidence supporting the functional synergy between the 5' cap and 3' poly(A) tail to enhance mRNA translation initiation. However, a number of examples exist of "non-canonical" 5'-3' communication for cellular and viral RNAs that lack 5' cap structures and/or poly(A) tails. Moreover, in several contexts, mRNA 5'-3' communication can function to repress translation. Overall, we detail how various mRNA 5'-3' interactions play important roles in posttranscriptional regulation, wherein depending on the protein factors involved can result in translational stimulation or repression.

Of seeds and supplements: structural insights into extended microRNA-target pairing.

Metazoan micro(mi) RNAs guide Argonaute proteins to their targets via perfect pairing to the seed region, located near the 5' end of the miRNA. In this issue of The EMBO Journal, Sheu-Gruttadauria et al report the crystal structure of human Argonaute 2 in complex with both a miRNA and target RNA and show that miRNA 3' supplementary nucleotides can increase target affinity and may contribute more to miRNA-mediated silencing than is currently appreciated.

Mesenchymal Hamartoma of the Liver and DICER1 Syndrome.

Mesenchymal hamartoma of the liver (MHL) is a benign tumor affecting children that is characterized by a primitive myxoid stroma with cystically dilated bile ducts. Alterations involving chromosome 19q13 are a recurrent underlying cause of MHL; these alterations activate the chromosome 19 microRNA cluster (C19MC). Other cases remain unexplained. We describe two children with MHLs that harbored germline DICER1 pathogenic variants. Analysis of tumor tissue from one of the children revealed two DICER1 "hits." Mutations in DICER1 dysregulate microRNAs, mimicking the effect of the activation of C19MC. Our data suggest that MHL is a new phenotype of DICER1 syndrome. (Funded by the Canadian Institutes of Health Research and others.).

Leukodystrophy-associated POLR3A mutations down-regulate the RNA polymerase III transcript and important regulatory RNA BC200.

RNA polymerase III (Pol III) is an essential enzyme responsible for the synthesis of several small noncoding RNAs, a number of which are involved in mRNA translation. Recessive mutations in POLR3A, encoding the largest subunit of Pol III, cause POLR3-related hypomyelinating leukodystrophy (POLR3-HLD), characterized by deficient central nervous system myelination. Identification of the downstream effectors of pathogenic POLR3A mutations has so far been elusive. Here, we used CRISPR-Cas9 to introduce the POLR3A mutation c.2554A→G (p.M852V) into human cell lines and assessed its impact on Pol III biogenesis, nuclear import, DNA occupancy, transcription, and protein levels. Transcriptomic profiling uncovered a subset of transcripts vulnerable to Pol III hypofunction, including a global reduction in tRNA levels. The brain cytoplasmic BC200 RNA (BCYRN1), involved in translation regulation, was consistently affected in all our cellular models, including patient-derived fibroblasts. Genomic BC200 deletion in an oligodendroglial cell line led to major transcriptomic and proteomic changes, having a larger impact than those of POLR3A mutations. Upon differentiation, mRNA levels of the MBP gene, encoding myelin basic protein, were significantly decreased in POLR3A-mutant cells. Our findings provide the first evidence for impaired Pol III transcription in cellular models of POLR3-HLD and identify several candidate effectors, including BC200 RNA, having a potential role in oligodendrocyte biology and involvement in the disease.

Mechanistic Insights into MicroRNA-Mediated Gene Silencing.

MicroRNAs (miRNAs) posttranscriptionally regulate gene expression by repressing protein synthesis and exert a broad influence over development, physiology, adaptation, and disease. Over the past two decades, great strides have been made toward elucidating how miRNAs go about shutting down messenger RNA (mRNA) translation and promoting mRNA decay.

Human MARF1 is an endoribonuclease that interacts with the DCP1:2 decapping complex and degrades target mRNAs.

Meiosis arrest female 1 (MARF1) is a cytoplasmic RNA binding protein that is essential for meiotic progression of mouse oocytes, in part by limiting retrotransposon expression. MARF1 is also expressed in somatic cells and tissues; however, its mechanism of action has yet to be investigated. Human MARF1 contains a NYN-like domain, two RRMs and eight LOTUS domains. Here we provide evidence that MARF1 post-transcriptionally silences targeted mRNAs. MARF1 physically interacts with the DCP1:DCP2 mRNA decapping complex but not with deadenylation machineries. Importantly, we provide a 1.7 Å resolution crystal structure of the human MARF1 NYN domain, which we demonstrate is a bona fide endoribonuclease, the activity of which is essential for the repression of MARF1-targeted mRNAs. Thus, MARF1 post-transcriptionally represses gene expression by serving as both an endoribonuclease and as a platform that recruits the DCP1:DCP2 decapping complex to targeted mRNAs.

Molecular architecture of LSM14 interactions involved in the assembly of mRNA silencing complexes.

The LSM domain-containing protein LSM14/Rap55 plays a role in mRNA decapping, translational repression, and RNA granule (P-body) assembly. How LSM14 interacts with the mRNA silencing machinery, including the eIF4E-binding protein 4E-T and the DEAD-box helicase DDX6, is poorly understood. Here we report the crystal structure of the LSM domain of LSM14 bound to a highly conserved C-terminal fragment of 4E-T. The 4E-T C-terminus forms a bi-partite motif that wraps around the N-terminal LSM domain of LSM14. We also determined the crystal structure of LSM14 bound to the C-terminal RecA-like domain of DDX6. LSM14 binds DDX6 via a unique non-contiguous motif with distinct directionality as compared to other DDX6-interacting proteins. Together with mutational and proteomic studies, the LSM14-DDX6 structure reveals that LSM14 has adopted a divergent mode of binding DDX6 in order to support the formation of mRNA silencing complexes and P-body assembly.

Anaplastic sarcomas of the kidney are characterized by DICER1 mutations.

Anaplastic sarcoma of the kidney is a rare tumor (≤25 reported cases) characterized by the presence of cysts, and solid areas composed of bundles of undifferentiated spindle cells, showing marked cellular anaplasia (usually accompanied by TP53 overexpression). These tumors often feature prominent areas of cartilage or chondroid material. Germline mutations in DICER1, encoding the microRNA (miRNA) processor DICER1, cause an eponymous syndrome. Recent reports suggest that anaplastic sarcoma of the kidney should be included in DICER1 syndrome as germline DICER1 mutations are associated with the occurrence of such tumors. Therefore, we sought to determine the following: (1) what proportion of anaplastic sarcoma of the kidney have DICER1 mutations; (2) whether the identified mutations affect both alleles of DICER1 (ie, are biallelic); (3) whether somatic missense mutations in the DICER1 RNase IIIb domain impact miRNA generation; and (4) whether TP53 alteration always occurs in these tumors. DICER1 mutations were evaluated by Sanger sequencing and next-generation sequencing in nine tumor/normal pairs. Impact of DICER1 mutations on miRNA generation was evaluated via an in vitro DICER1 cleavage assay. TP53 status was assessed by immunohistochemistry and next-generation sequencing. Eight of the nine cases had at least one RNase IIIb DICER1 mutation that impacted the generation of miRNAs. There were six tumors with truncating DICER1 mutations and in four of them, the mutation found in the tumor was also detected in adjacent normal tissue, and therefore was likely to be either mosaic or germline in origin. Analysis of mutation phase revealed that two of three tumors had biallelic DICER1 mutations. Six of nine anaplastic sarcomas of the kidney had aberrant TP53 immunohistochemisty with damaging TP53 mutations identified in three cases. Taken together, these data suggest that the great majority of anaplastic sarcomas of the kidney have DICER1 mutations and confirm that these tumors are part of the DICER1 syndrome.

Structural Dynamics of the GW182 Silencing Domain Including its RNA Recognition motif (RRM) Revealed by Hydrogen-Deuterium Exchange Mass Spectrometry.

The human GW182 protein plays an essential role in micro(mi)RNA-dependent gene silencing. miRNA silencing is mediated, in part, by a GW182 C-terminal region called the silencing domain, which interacts with the poly(A) binding protein and the CCR4-NOT deadenylase complex to repress protein synthesis. Structural studies of this GW182 fragment are challenging due to its predicted intrinsically disordered character, except for its RRM domain. However, detailed insights into the properties of proteins containing disordered regions can be provided by hydrogen-deuterium exchange mass spectrometry (HDX/MS). In this work, we applied HDX/MS to define the structural state of the GW182 silencing domain. HDX/MS analysis revealed that this domain is clearly divided into a natively unstructured part, including the CCR4-NOT interacting motif 1, and a distinct RRM domain. The GW182 RRM has a very dynamic structure, since water molecules can penetrate the whole domain in 2 h. The finding of this high structural dynamics sheds new light on the RRM structure. Though this domain is one of the most frequently occurring canonical protein domains in eukaryotes, these results are - to our knowledge - the first HDX/MS characteristics of an RRM. The HDX/MS studies show also that the α2 helix of the RRM can display EX1 behavior after a freezing-thawing cycle. This means that the RRM structure is sensitive to environmental conditions and can change its conformation, which suggests that the state of the RRM containing proteins should be checked by HDX/MS in regard of the conformational uniformity. Graphical Abstract.

CTCF facilitates DNA double-strand break repair by enhancing homologous recombination repair.

The repair of DNA double-strand breaks (DSBs) is mediated via two major pathways, nonhomologous end joining (NHEJ) and homologous recombination (HR) repair. DSB repair is vital for cell survival, genome stability, and tumor suppression. In contrast to NHEJ, HR relies on extensive homology and templated DNA synthesis to restore the sequence surrounding the break site. We report a new role for the multifunctional protein CCCTC-binding factor (CTCF) in facilitating HR-mediated DSB repair. CTCF is recruited to DSB through its zinc finger domain independently of poly(ADP-ribose) polymers, known as PARylation, catalyzed by poly(ADP-ribose) polymerase 1 (PARP-1). CTCF ensures proper DSB repair kinetics in response to γ-irradiation, and the loss of CTCF compromises HR-mediated repair. Consistent with its role in HR, loss of CTCF results in hypersensitivity to DNA damage, inducing agents and inhibitors of PARP. Mechanistically, CTCF acts downstream of BRCA1 in the HR pathway and associates with BRCA2 in a PARylation-dependent manner, enhancing BRCA2 recruitment to DSB. In contrast, CTCF does not influence the recruitment of the NHEJ protein 53BP1 or LIGIV to DSB. Together, our findings establish for the first time that CTCF is an important regulator of the HR pathway.