Biochemical characterization of Bombyx mori Dicer-2 that dices double-stranded RNAs into 20-nt small RNA.

We detected enzymatic activity that generates 20-nucleotide (nt) RNA from double-stranded RNAs (dsRNAs) in crude extracts prepared from various silkworm (Bombyx mori) organs. The result using knocked-down cultured cells indicated that this dicing activity originated from B. mori Dicer-2 (BmDcr2). Biochemical analyses revealed that BmDcr2 preferentially cleaves 5'-phosphorylated dsRNAs at the 20-nt site-counted from the 5'-phosphorylated end-and required ATP and magnesium ions for the dicing reaction. This is the first report of the biochemical characterization of Dicer-2 in lepidopteran insects. This enzymatic property of BmDcr2 in vitro is consistent with the in vivo small interfering RNA profile in virus-infected silkworm cells.

The Evolution and Characterization of the RNA Interference Pathways in Lophotrochozoa.

In animals, three main RNA interference mechanisms have been described so far, which respectively maturate three types of small noncoding RNAs (sncRNAs): miRNAs, piRNAs, and endo-siRNAs. The diversification of these mechanisms is deeply linked with the evolution of the Argonaute gene superfamily since each type of sncRNA is typically loaded by a specific Argonaute homolog. Moreover, other protein families play pivotal roles in the maturation of sncRNAs, like the DICER ribonuclease family, whose DICER1 and DICER2 paralogs maturate respectively miRNAs and endo-siRNAs. Within Metazoa, the distribution of these families has been only studied in major groups, and there are very few data for clades like Lophotrochozoa. Thus, we here inferred the evolutionary history of the animal Argonaute and DICER families including 43 lophotrochozoan species. Phylogenetic analyses along with newly sequenced sncRNA libraries suggested that in all Trochozoa, the proteins related to the endo-siRNA pathway have been lost, a part of them in some phyla (i.e. Nemertea, Bryozoa, Entoprocta), while all of them in all the others. On the contrary, early diverging phyla, Platyhelminthes and Syndermata, showed a complete endo-siRNA pathway. On the other hand, miRNAs were revealed the most conserved and ubiquitous mechanism of the metazoan RNA interference machinery, confirming their pivotal role in animal cell regulation.

[The clinicopathological features of adult thyroid tumors with DICER1 mutation].

A total of 37 cases of thyroid tumors with pathological features suggestive of DICER1 gene mutation were selected to detect the DICER1 gene and BRAF gene using Sanger sequencing. A total of 10 patients (27.0%) exhibited DICER1 gene mutation all of whom were female with an age of [M(Q1, Q3)] 38.0 (30.5, 47.5) years. All patients had wild-type BRAFV600E gene. The ultrasound examination showed high-low echogenic well-demarcated intra-thyroidal nodules with abundant peripheral and internal blood flow signals in the DICER1 mutated thyroid tumor. The tumor was confined within the thyroid gland, with a diameter of (3.68±1.31) cm. The pathological features are as follows: the majority of tumors are encapsulated, which mainly composed of large follicles rich in colloid and some are small and micro follicles. The nucleus is round and deeply stained or slightly light stained, small to medium-sized, with occasional nuclear grooves and a lack of nuclear pseudoinclusion bodies within the nucleus. Immunohistochemical staining shows that Ki67 proliferation index of approximately 2%-10%. All cases were followed up for 11 to 18 months, and there was no recurrences or distant metastase. This study confirmed that the DICER1 gene mutation is mutually exclusive with the BRAFV600E gene mutation. The thyroid tumor with DICER1 mutation are in big size and are more common in young females with a good prognosis. Cases with the wild-type DICER1 gene may exhibit similar morphological features, and molecular testing is recommended. If somatic DICER1 mutation is confirmed, patients should undergo germline mutation testing to rule out DICER1 syndrome in order to define whether genetic counseling is necessary.

Caenorhabditis elegans Dicer acts with the RIG-I-like helicase DRH-1 and RDE-4 to cleave dsRNA.

Invertebrates use the endoribonuclease Dicer to cleave viral dsRNA during antiviral defense, while vertebrates use RIG-I-like Receptors (RLRs), which bind viral dsRNA to trigger an interferon response. While some invertebrate Dicers act alone during antiviral defense, Caenorhabditis elegans Dicer acts in a complex with a dsRNA binding protein called RDE-4, and an RLR ortholog called DRH-1. We used biochemical and structural techniques to provide mechanistic insight into how these proteins function together. We found RDE-4 is important for ATP-independent and ATP-dependent cleavage reactions, while helicase domains of both DCR-1 and DRH-1 contribute to ATP-dependent cleavage. DRH-1 plays the dominant role in ATP hydrolysis, and like mammalian RLRs, has an N-terminal domain that functions in autoinhibition. A cryo-EM structure indicates DRH-1 interacts with DCR-1's helicase domain, suggesting this interaction relieves autoinhibition. Our study unravels the mechanistic basis of the collaboration between two helicases from typically distinct innate immune defense pathways.

Inducible deletion of microRNA activity in kidney mesenchymal cells exacerbates renal fibrosis.

MicroRNAs (miRNAs) are sequence-specific inhibitors of post-transcriptional gene expression. However, the physiological functions of these non-coding RNAs in renal interstitial mesenchymal cells remain unclear. To conclusively evaluate the role of miRNAs, we generated conditional knockout (cKO) mice with platelet-derived growth factor receptor-ß (PDGFR-ß)-specific inactivation of the key miRNA pathway gene Dicer. The cKO mice were subjected to unilateral ureteral ligation, and renal interstitial fibrosis was quantitatively evaluated using real-time polymerase chain reaction and immunofluorescence staining. Compared with control mice, cKO mice had exacerbated interstitial fibrosis exhibited by immunofluorescence staining and mRNA expression of PDGFR-ß. A microarray analysis showed decreased expressions of miR-9-5p, miR-344g-3p, and miR-7074-3p in cKO mice compared with those in control mice, suggesting an association with the increased expression of PDGFR-ß. An analysis of the signaling pathways showed that the major transcriptional changes in cKO mice were related to smooth muscle cell differentiation, regulation of DNA metabolic processes and the actin cytoskeleton, positive regulation of fibroblast proliferation and Ras protein signal transduction, and focal adhesion-PI3K/Akt/mTOR signaling pathways. Depletion of Dicer in mesenchymal cells may downregulate the signaling pathway related to miR-9-5p, miR-344g-3p, and miR-7074-3p, which can lead to the progression of chronic kidney disease. These findings highlight the possibility for future diagnostic or therapeutic developments for renal fibrosis using miR-9-5p, miR-344g-3p, and miR-7074-3p.

Caspase-mediated processing of TRBP regulates apoptosis during viral infection.

RNA silencing is a post-transcriptional gene-silencing mechanism mediated by microRNAs (miRNAs). However, the regulatory mechanism of RNA silencing during viral infection is unclear. TAR RNA-binding protein (TRBP) is an enhancer of RNA silencing that induces miRNA maturation by interacting with the ribonuclease Dicer. TRBP interacts with a virus sensor protein, laboratory of genetics and physiology 2 (LGP2), in the early stage of viral infection of human cells. Next, it induces apoptosis by inhibiting the maturation of miRNAs, thereby upregulating the expression of apoptosis regulatory genes. In this study, we show that TRBP undergoes a functional conversion in the late stage of viral infection. Viral infection resulted in the activation of caspases that proteolytically processed TRBP into two fragments. The N-terminal fragment did not interact with Dicer but interacted with type I interferon (IFN) signaling modulators, such as protein kinase R (PKR) and LGP2, and induced ER stress. The end results were irreversible apoptosis and suppression of IFN signaling. Our results demonstrate that the processing of TRBP enhances apoptosis, reducing IFN signaling during viral infection.

Imaging Features of Primary Intracranial Sarcoma with DICER1 Mutation: A Multicenter Case Series.

Primary intracranial sarcoma, DICER1-mutant, is a rare, recently described entity in the fifth edition of the WHO Classification of CNS Tumors. Given the entity's rarity and recent description, imaging data on primary intracranial sarcoma, DICER1-mutant, remains scarce. In this multicenter case series, we present detailed multimodality imaging features of primary intracranial sarcoma, DICER1-mutant, with emphasis on the appearance of the entity on MR imaging. In total, 8 patients were included. In all 8 patients, the lesion demonstrated blood products on T1WI. In 7 patients, susceptibility-weighted imaging was obtained and demonstrated blood products. Primary intracranial sarcoma, DICER1-mutant, is a CNS neoplasm that primarily affects pediatric and young adult patients. In the present case series, we explore potential imaging findings that are helpful in suggesting this diagnosis. In younger patients, the presence of a cortical lesion with intralesional blood products on SWI and T1-weighted MR imaging, with or without extra-axial blood products, should prompt the inclusion of this entity in the differential diagnosis.

Deep mutational scanning of the RNase III-like domain in Trypanosoma brucei RNA editing protein KREPB4.

Kinetoplastid pathogens including Trypanosoma brucei, T. cruzi, and Leishmania species, are early diverged, eukaryotic, unicellular parasites. Functional understanding of many proteins from these pathogens has been hampered by limited sequence homology to proteins from other model organisms. Here we describe the development of a high-throughput deep mutational scanning approach in T. brucei that facilitates rapid and unbiased assessment of the impacts of many possible amino acid substitutions within a protein on cell fitness, as measured by relative cell growth. The approach leverages several molecular technologies: cells with conditional expression of a wild-type gene of interest and constitutive expression of a library of mutant variants, degron-controlled stabilization of I-SceI meganuclease to mediate highly efficient transfection of a mutant allele library, and a high-throughput sequencing readout for cell growth upon conditional knockdown of wild-type gene expression and exclusive expression of mutant variants. Using this method, we queried the effects of amino acid substitutions in the apparently non-catalytic RNase III-like domain of KREPB4 (B4), which is an essential component of the RNA Editing Catalytic Complexes (RECCs) that carry out mitochondrial RNA editing in T. brucei. We measured the impacts of thousands of B4 variants on bloodstream form cell growth and validated the most deleterious variants containing single amino acid substitutions. Crucially, there was no correlation between phenotypes and amino acid conservation, demonstrating the greater power of this method over traditional sequence homology searching to identify functional residues. The bloodstream form cell growth phenotypes were combined with structural modeling, RECC protein proximity data, and analysis of selected substitutions in procyclic form T. brucei. These analyses revealed that the B4 RNaseIII-like domain is essential for maintenance of RECC integrity and RECC protein abundances and is also involved in changes in RECCs that occur between bloodstream and procyclic form life cycle stages.

Multiple and hereditary renal tumors: a review for radiologists.

80% of renal carcinomas (RC) are diagnosed incidentally by imaging. 2-4% of "sporadic" multifocality and 5-8% of hereditary syndromes are accepted, probably with underestimation. Multifocality, young age, familiar history, syndromic data, and certain histologies lead to suspicion of hereditary syndrome. Each tumor must be studied individually, with a multidisciplinary evaluation of the patient. Nephron-sparing therapeutic strategies and a radioprotective diagnostic approach are recommended. Relevant data for the radiologist in major RC hereditary syndromes are presented: von-Hippel-Lindau, Chromosome-3 translocation, BRCA-associated protein-1 mutation, RC associated with succinate dehydrogenase deficiency, PTEN, hereditary papillary RC, Papillary thyroid cancer- Papillary RC, Hereditary leiomyomatosis and RC, Birt-Hogg-Dubé, Tuberous sclerosis complex, Lynch, Xp11.2 translocation/TFE3 fusion, Sickle cell trait, DICER1 mutation, Hereditary hyperparathyroidism and jaw tumor, as well as the main syndromes of Wilms tumor predisposition. The concept of "non-hereditary" familial RC and other malignant and benign entities that can present as multiple renal lesions are discussed.

FGFR1 fusions as a novel molecular driver in rhabdomyosarcoma.

The wide application of RNA sequencing in clinical practice has allowed the discovery of novel fusion genes, which have contributed to a refined molecular classification of rhabdomyosarcoma (RMS). Most fusions in RMS result in aberrant transcription factors, such as PAX3/7::FOXO1 in alveolar RMS (ARMS) and fusions involving VGLL2 or NCOA2 in infantile spindle cell RMS. However, recurrent fusions driving oncogenic kinase activation have not been reported in RMS. Triggered by an index case of an unclassified RMS (overlapping features between ARMS and sclerosing RMS) with a novel FGFR1::ANK1 fusion, we reviewed our molecular files for cases harboring FGFR1-related fusions. One additional case with an FGFR1::TACC1 fusion was identified in a tumor resembling embryonal RMS (ERMS) with anaplasia, but with no pathogenic variants in TP53 or DICER1 on germline testing. Both cases occurred in males, aged 7 and 24, and in the pelvis. The 2nd case also harbored additional alterations, including somatic TP53 and TET2 mutations. Two additional RMS cases (one unclassified, one ERMS) with FGFR1 overexpression but lacking FGFR1 fusions were identified by RNA sequencing. These two cases and the FGFR1::TACC1-positive case clustered together with the ERMS group by RNAseq. This is the first report of RMS harboring recurrent FGFR1 fusions. However, it remains unclear if FGFR1 fusions define a novel subset of RMS or alternatively, whether this alteration can sporadically drive the pathogenesis of known RMS subtypes, such as ERMS. Additional larger series with integrated genomic and epigenetic datasets are needed for better subclassification, as the resulting oncogenic kinase activation underscores the potential for targeted therapy.

Clinicopathological analysis of 22 Müllerian adenosarcomas and the sequencing of DICER1 mutation.

BACKGROUND: Müllerian adenosarcoma, a rare malignancy, presents diagnostic and therapeutic challenges. In this study, we conducted an analysis of the clinicopathological characteristics of 22 adenosarcomas, with a particular focus on screening for DICER1 hot mutations. METHODS: The cohort consisted of patients with adenosarcoma who were registered at the West China Second Hospital between the years 2020 and June 2022. Sanger sequencing was employed to screen for somatic Hotspot mutations in the RNase IIIb domain of DICER1 in the 22 adenosarcomas. RESULTS: Only one patient exhibited a DICER1 mutation that was not a DICER1 Hotspot mutation. Among the 22 patients, all underwent total hysterectomy with bilateral salpingo-oophorectomy, and 14 out of these 22 patients received adjuvant treatment. CONCLUSION: In summary, our study of 22 Müllerian adenosarcomas focused on the clinicopathological features and the presence of DICER1 Hotspot mutations. Although our findings did not reveal any DICER1 mutations in the studied samples, this negative result provides valuable information for the field by narrowing down the genetic landscape of adenosarcomas and highlighting the need for further research into alternative molecular pathways driving this malignancy.

Estrogen Receptor Expression in DICER1-related Lesions is Associated With the Presence of Cystic Components.

DICER1 tumor predisposition syndrome results from pathogenic variants in DICER1 and is associated with a variety of benign and malignant lesions, typically involving kidney, lung, and female reproductive system. Over 70% of sarcomas in DICER1 tumor predisposition syndrome occur in females. Notably, pediatric cystic nephroma (pCN), a classic DICER1 tumor predisposition syndrome lesion, shows estrogen receptor (ER) expression in stromal cells. There are also renal, hepatic, and pancreatic lesions unassociated with DICER1 tumor predisposition syndrome that have an adult female predominance and are characterized/defined by ER-positive stromal cells. Except for pCN, the expression of ER in DICER1-associated lesions remains uninvestigated. In the present study, ER expression was assessed by immunohistochemistry in 89 cases of DICER1-related lesions and 44 lesions lacking DICER1 pathogenic variants. Expression was seen in stromal cells in pCN and pleuropulmonary blastoma (PPB) types I and Ir, whereas anaplastic sarcoma of kidney and PPB types II and III were typically negative, as were other solid tumors of non-Müllerian origin. ER expression was unrelated to the sex or age of the patient. Expression of ER showed an inverse relationship to preferentially expressed antigen in melanoma (PRAME) expression; as lesions progressed from cystic to solid (pCN/anaplastic sarcoma of kidney, and PPB types I to III), ER expression was lost and (PRAME) expression increased. Thus, in DICER1 tumor predisposition syndrome, there is no evidence that non-Müllerian tumors are hormonally driven and antiestrogen therapy is not predicted to be beneficial. Lesions not associated with DICER1 pathogenic variants also showed ER-positive stromal cells, including cystic pulmonary airway malformations, cystic renal dysplasia, and simple renal cysts in adult kidneys. ER expression in stromal cells is not a feature of DICER1 perturbation but rather is related to the presence of cystic components.

Recurrent primary intracranial sarcoma, DICER1-mutant in a pediatric patient with DICER1 syndrome: the importance of molecular testing.

Pediatric intracranial sarcomas are rare, aggressive tumors with a poor prognosis in general. Here we report the case of a child who was initially diagnosed with a primary intracranial sarcoma, DICER1-mutant; subsequent genetic analyses confirmed a pathogenic germline DICER1 mutation. She received multimodal standard treatments consisting of surgery, radiotherapy and chemotherapy. The tumor recurred 2.5 years later within the surgical cavity. Following the gross tumor resection of this new lesion, the same multimodal standard approach was used. From a molecular perspective, evidence of hyperactivation of the MAPK-kinase pathway with a pathogenic KRAS mutation at both diagnosis and recurrence was present. The patient is currently in remission, 18 months post-end of treatment.

The functional small RNA interactome reveals targets for the vancomycin-responsive sRNA RsaOI in vancomycin-tolerant Staphylococcus aureus.

Small RNAs have been found to control a broad range of bacterial phenotypes including tolerance to antibiotics. Vancomycin tolerance in multidrug resistance Staphylococcus aureus is correlated with dysregulation of small RNAs although their contribution to antibiotic tolerance is poorly understood. RNA-RNA interactome profiling techniques are expanding our understanding of sRNA-mRNA interactions in bacteria; however, determining the function of these interactions for hundreds of sRNA-mRNA pairs is a major challenge. At steady-state, protein and mRNA abundances are often highly correlated and lower than expected protein abundance may indicate translational repression of an mRNA. To identify sRNA-mRNA interactions that regulate mRNA translation, we examined the correlation between gene transcript abundance, ribosome occupancy, and protein levels. We used the machine learning technique self-organizing maps (SOMs) to cluster genes with similar transcription and translation patterns and identified a cluster of mRNAs that appeared to be post-transcriptionally repressed. By integrating our clustering with sRNA-mRNA interactome data generated in vancomycin-tolerant S. aureus by RNase III-CLASH, we identified sRNAs that may be mediating translational repression. We have confirmed sRNA-dependant post-transcriptional repression of several mRNAs in this cluster. Two of these interactions are mediated by RsaOI, a sRNA that is highly upregulated by vancomycin. We demonstrate the regulation of HPr and the cell-wall autolysin Atl. These findings suggest that RsaOI coordinates carbon metabolism and cell wall turnover during vancomycin treatment. IMPORTANCE: The emergence of multidrug-resistant Staphylococcus aureus (MRSA) is a major public health concern. Current treatment is dependent on the efficacy of last-line antibiotics like vancomycin. The most common cause of vancomycin treatment failure is strains with intermediate resistance or tolerance that arise through the acqusition of a diverse repertoire of point mutations. These strains have been shown to altered small RNA (sRNA) expression in response to antibiotic treatment. Here, we have used a technique termed RNase III-CLASH to capture sRNA interactions with their target mRNAs. To understand the function of these interactions, we have looked at RNA and protein abundance for mRNAs targeted by sRNAs. Messenger RNA and protein levels are generally well correlated and we use deviations from this correlation to infer post-transcriptional regulation and the function of individual sRNA-mRNA interactions. Using this approach we identify mRNA targets of the vancomycin-induced sRNA, RsaOI, that are repressed at the translational level. We find that RsaOI represses the cell wall autolysis Atl and carbon transporter HPr suggestion a link between vancomycin treatment and suppression of cell wall turnover and carbon metabolism.

Non-canonical RNA substrates of Drosha lack many of the conserved features found in primary microRNA stem-loops.

The RNase III enzyme Drosha has a central role in microRNA (miRNA) biogenesis, where it is required to release the stem-loop intermediate from primary (pri)-miRNA transcripts. However, it can also cleave stem-loops embedded within messenger (m)RNAs. This destabilizes the mRNA causing target gene repression and appears to occur primarily in stem cells. While pri-miRNA stem-loops have been extensively studied, such non-canonical substrates of Drosha have yet to be characterized in detail. In this study, we employed high-throughput sequencing to capture all polyA-tailed RNAs that are cleaved by Drosha in mouse embryonic stem cells (ESCs) and compared the features of non-canonical versus miRNA stem-loop substrates. mRNA substrates are less efficiently processed than miRNA stem-loops. Sequence and structural analyses revealed that these mRNA substrates are also less stable and more likely to fold into alternative structures than miRNA stem-loops. Moreover, they lack the sequence and structural motifs found in miRNA stem-loops that are required for precise cleavage. Notably, we discovered a non-canonical Drosha substrate that is cleaved in an inverse manner, which is a process that is normally inhibited by features in miRNA stem-loops. Our study thus provides valuable insights into the recognition of non-canonical targets by Drosha.

Structural atlas of human primary microRNAs generated by SHAPE-MaP.

MicroRNA (miRNA) maturation is critically dependent on structural features of primary transcripts (pri-miRNAs). However, the scarcity of determined pri-miRNA structures has limited our understanding of miRNA maturation. Here, we employed selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP), a high-throughput RNA structure probing method, to unravel the secondary structures of 476 high-confidence human pri-miRNAs. Our SHAPE-based structures diverge substantially from those inferred solely from computation, particularly in the apical loop and basal segments, underlining the need for experimental data in RNA structure prediction. By comparing the structures with high-throughput processing data, we determined the optimal structural features of pri-miRNAs. The sequence determinants are influenced substantially by their structural contexts. Moreover, we identified an element termed the bulged GWG motif (bGWG) with a 3' bulge in the lower stem, which promotes processing. Our structure-function mapping better annotates the determinants of pri-miRNA processing and offers practical implications for designing small hairpin RNAs and predicting the impacts of miRNA mutations.

let-7 miRNAs repress HIC2 to regulate BCL11A transcription and hemoglobin switching.

ABSTRACT: The switch from fetal hemoglobin (γ-globin, HBG) to adult hemoglobin (ß-globin, HBB) gene transcription in erythroid cells serves as a paradigm for a complex and clinically relevant developmental gene regulatory program. We previously identified HIC2 as a regulator of the switch by inhibiting the transcription of BCL11A, a key repressor of HBG production. HIC2 is highly expressed in fetal cells, but the mechanism of its regulation is unclear. Here we report that HIC2 developmental expression is controlled by microRNAs (miRNAs), as loss of global miRNA biogenesis through DICER1 depletion leads to upregulation of HIC2 and HBG messenger RNA. We identified the adult-expressed let-7 miRNA family as a direct posttranscriptional regulator of HIC2. Ectopic expression of let-7 in fetal cells lowered HIC2 levels, whereas inhibition of let-7 in adult erythroblasts increased HIC2 production, culminating in decommissioning of a BCL11A erythroid enhancer and reduced BCL11A transcription. HIC2 depletion in let-7-inhibited cells restored BCL11A-mediated repression of HBG. Together, these data establish that fetal hemoglobin silencing in adult erythroid cells is under the control of a miRNA-mediated inhibitory pathway (let-7 ⊣ HIC2 ⊣ BCL11A ⊣ HBG).