Ribonuclease Dicer Research Articles

Exosomes-mediated synthetic Dicer substrates delivery for intracellular Dicer imaging detection

Ribonuclease Dicer initiates gene-silencing process by cleaving exogenously long RNA duplexes into small interfering RNA (siRNA) or endogenous precursor microRNAs (pre-miRNAs) into mature miRNAs. It holds great promise in cancer diagnosis and therapeutics due to its molecular ruler role. However, the intracellular Dicer detection remains a key challenge and Dicer related gene therapy has never been explored. In this study, we design a fluorescent labeling Dicer substrate and effectively deliver it into cell by exosomes derived from the target parent cells for intracellular Dicer expression level monitor and gene therapy. Using pre-miRNA let-7a as a model, the Dicer substrates with two terminals labeled with fluorescent and quencher group respectively was obtained by T4 RNA mediated ligase reaction from two short RNA sequences. Then, the substrate was packaged into exosomes by electroporation and delivered to target cells for intracellular dicer imaging detection. After packaging substrates into exosomes with little immunogenicity and good innate biocompatibility by electroporation and delivered to target cells, the Dicer mediated substrate cleavage was effectively monitored by the fluorescence recovery, providing a powerful tool for Dicer analysis. Importantly, the cleaved product exhibited significant suppression toward tumor cell growth and regulated cancer cells cycle. This work might open a new avenue for Dicer analysis and Dicer-related clinical application.

The RNA\u2013RNA base pairing potential of human Dicer and Ago2 proteins

The ribonuclease Dicer produces microRNAs (miRNAs) and small interfering RNAs that are handed over to Ago proteins to control gene expression by targeting complementary sequences within transcripts. Interestingly, a growing number of reports have demonstrated that the activity of Dicer may extend beyond the biogenesis of small regulatory RNAs. Among them, a report from our latest studies revealed that human Dicer facilitates base pairing of complementary sequences present in two nucleic acids, thus acting as a nucleic acid annealer. Accordingly, in this manuscript, we address how RNA structure influences the annealing activity of human Dicer. We show that Dicer supports hybridization between a small RNA and a complementary sequence of a longer RNA in vitro, even when both complementary sequences are trapped within secondary structures. Moreover, we show that under applied conditions, human Ago2, a core component of RNA-induced silencing complex, displays very limited annealing activity. Based on the available data from new-generation sequencing experiments regarding the RNA pool bound to Dicer in vivo, we show that multiple Dicer-binding sites within mRNAs also contain miRNA targets. Subsequently, we demonstrate in vitro that Dicer but not Ago2 can anneal miRNA to its target present within mRNA. We hypothesize that not all miRNA duplexes are handed over to Ago proteins. Instead, miRNA-Dicer complexes could target specific sequences within transcripts and either compete or cooperate for binding sites with miRNA-Ago complexes. Thus, not only Ago but also Dicer might be directly involved in the posttranscriptional control of gene expression.

MiR-302: The Multifunctional MicroRNA

MicroRNAs (miRNAs) are short single-stranded noncoding RNAs (20- to 25-nucleotide (nt) long) representing a class of small regulatory RNAs. By inhibiting the translation of target mRNAs, miRNAs regulate gene expression posttranscriptionally and thus play an important role in a wide range of cellular processes. Currently, there are two known types of miRNAs: intergenic and intronic miRNAs. Biogenesis of an intergenic miRNA starts with the synthesis of a primary miRNA transcript (pri-miRNA) catalyzed by types-II or -III RNA polymerase (Pol-II/III). Pri-miRNAs are processed in the nucleus by the ribonuclease Drosha into a miRNA precursor (pre-miRNA) approximately 60-nt in length. After being transported into the cytoplasm, these pre-miRNAs are further processed into mature and functional miRNAs by the cytoplasmic ribonuclease Dicer. Mature miRNAs then associate with a number of proteins to form the RNA-induced silencing complex (RISC) that bind with target mRNAs having total or partial complementary sequences to the miRNAs and initiate the inhibition of subsequent protein translation via RNA interference (RNAi).

The Effects on Ishikawa Endometrial Cancer Cell Lines upon Treatment with Hormonal Agents and Novel Drug Target

Background and Hypothesis: Endometrial cancer is the most common gynecological malignancy in the US and will claim the lives of over 12,000 women in 2019. Women with high-risk histologic tumors, such as high-grade endometrial adenocarcinoma, represent over 50% of these deaths. Novel treatments are needed to treat these aggressive tumors. Decreased expression of the ribonuclease DICER is associated with high-grade and recurrent endometrial adenocarcinoma. To study the role of DICER in endometrial cancer, we used CRISPR/Cas9 to delete DICER in an endometrial cancer cell line. RNA sequencing revealed dysregulation of steroid hormone receptor signaling and high expression of APEX1 [apurinic/apyrimidinic endonuclease 1 (Ref-1/APE1)]. Elevated levels of Ref-1/APE1 have been found in various tumor types, and Ref1/APE1 inhibitors are in clinical trials. We hypothesize that our DICER deleted cell lines will be hormone insensitive yet respond to Ref-1/APE1 inhibitors by decreasing cellular proliferation.
 Project Methods: Ingenuity Pathway Analysis provided an unbiased approach to determine dysregulated genes in steroid hormone signaling in our RNA transcriptomic data. QPCR was used to confirm gene expression in independent samples. Cells were treated with steroid hormones or Ref-1/APE1 inhibitors and proliferation was assessed using MTS.
 Results: DICER deleted cell lines had downregulated expression of steroid hormone receptors. Treatment with steroid hormones did not have a significant effect on proliferation. However, treatment with Ref-1/APE1 inhibitors resulted in a significant decrease in proliferation.
 Conclusion and Potential Impact: Ref-1/APE1 inhibitors affect cellular proliferation of endometrial cancer cell lines. Because survival has actually worsened for women with endometrial cancer, novel therapies such as Ref1/APE1 inhibitors deserve future study.

Abstract 2355: IKKa serves as a molecular switch linking miRNA biogenesis and inflammation in skin tumorigenesis

Abstract Our previous studies have shown that the absence of tumor suppressor IKKα leads to development of spontaneous mouse squamous cell carcinomas (SCCs) in the skin, lungs, and esophagus, which is partially mediated by aberrant epigenetic alterations and inflammation. However, it remains unknown whether there is a crosstalk between the above-mentioned mechanisms. As a part of the epigenetic machinery, miRNAs regulate many genes post-transcriptionally. Here we show that IKKα deletion in the mouse epidermis results in impaired miRNA processing with markedly reduced expression of ribonuclease Dicer, an enzyme required for miRNA maturation. We found that the lack of IKKα causes Dicer gene silencing through DNA methylation. As a result, the maturation of miR-203-3p, which is the most abundant miRNA in keratinocytes, is blocked in Ikkα-null keratinocytes. Next, we identified Myd88, an important component of innate immunity and inflammation, as one of miR-203-3p targets. The level of Myd88 expression is robustly increased in Ikkα-null keratinocytes due to the lack of miR-203-3p. We also observed increased activity of caspase 1 cleavage, an indicator of inflammasome activation, in Ikkα-null keratinocytes. Interestingly, Myd88 deletion or 5-aza-2’-deoxycytidine (DNA methyltransferase inhibitor) treatment rescued the survival of Ikkαf/f;K5Cre mice, which usually die within three weeks after birth. Ikkαf/f;Myd88f/f;K5Cre mice also develop much fewer spontaneous skin tumors. By analyzing a cohort of human head and neck SCCs from TCGA database, we found that Dicer expression is concurrently downregulated among 32% of patients with low IKKα expression. Taken together, our finding illustrated how IKKα loss mediates Dicer gene silencing, which turns off miRNA processing, including miR-203-3p maturation, and eventually triggers Myd88-dependent inflammation. Thus, our study revealed a novel IKKα/Dicer/miR-203-3p/Myd88 signaling loop, in which IKKα serves as a molecular switch linking microRNA biogenesis and inflammation in skin tumorigenesis. Citation Format: Feng Zhu, Jian Zhang, Jami Willette-Brown, Ling Su, Xiaolin Wu, Yinling Hu. IKKa serves as a molecular switch linking miRNA biogenesis and inflammation in skin tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2355.

Functional MoS2 nanosheets for precursor and mature microRNA detection in living cells.

Mature microRNAs (miRNAs) are small-sized RNAs cleaved from precursor microRNAs (pre-miRNAs) by the RNase Dicer. Various miRNAs play key regulatory roles in tumorigenesis and metastasis, and are therefore potential diagnostic and prognostic cancer biomarkers. However, detecting miRNAs and pre-miRNAs accurately and selectively in living cells remains a major challenge, as the mature miRNA sequence is also present in its pre-miRNA and current sequence probes exhibit poor gene delivery efficiency. Herein, we report a strategy for selectively and accurately detecting miRNA-21 and pre-miRNA-21 in living cells using functional MoS2 nanosheets (NSs) loaded with rationally engineered molecular beacons (MBs). The exfoliated MoS2 nanosheets (NSs) with a mean lateral diameter of 50-70nm were functionalized with the aptamer AS1411 and polyethylene glycol (MoS2-PEG-AS) to achieve target-cell-specific delivery and to enhance biocompatibility. The large available surface of the MoS2-PEG-AS was loaded with MB probes. The resulting MoS2-PEG-AS/MBs present cancer-cell-targeting ability, good protection properties, good optical stability, and biocompatibility. We demonstrated that the resulting nanoprobes can selectively image miRNA-21 and pre-miRNA-21 in various cell lines by facilitating enhanced fluorescence in the presence of miRNA-21 and pre-miRNA-21. Thus, these MoS2-PEG-AS/MBs are potentially a tool to discriminate between intracellular miRNA and pre-miRNA at different expression levels. Graphical abstract.

Main constraints for RNAi induced by expressed long dsRNA in mouse cells.

RNAi is the sequence-specific mRNA degradation guided by siRNAs produced from long dsRNA by RNase Dicer. Proteins executing RNAi are present in mammalian cells but rather sustain the microRNA pathway. Aiming for a systematic analysis of mammalian RNAi, we report here that the main bottleneck for RNAi efficiency is the production of functional siRNAs, which integrates Dicer activity, dsRNA structure, and siRNA targeting efficiency. Unexpectedly, increased expression of Dicer cofactors TARBP2 or PACT reduces RNAi but not microRNA function. Elimination of protein kinase R, a key dsRNA sensor in the interferon response, had minimal positive effects on RNAi activity in fibroblasts. Without high Dicer activity, RNAi can still occur when the initial Dicer cleavage of the substrate yields an efficient siRNA. Efficient mammalian RNAi may use substrates with some features of microRNA precursors, merging both pathways even more than previously suggested. Although optimized endogenous Dicer substrates mimicking miRNA features could evolve for endogenous regulations, the same principles would make antiviral RNAi inefficient as viruses would adapt to avoid efficacy.

Sensitively distinguishing intracellular precursor and mature microRNA abundance.

Mature microRNAs (miRNAs) produced from precursor microRNAs (pre-miRNAs) by the RNase Dicer have showed significant potential for cancer diagnosis and prognosis due to their key regulatory roles in various pathological processes. However, discriminatory detection of low-abundance miRNAs and pre-miRNAs remains a key challenge since the mature sequence is also present in the pre-miRNA forms. Herein, we report a novel cascade reaction to sensitively distinguish miRNAs versus pre-miRNAs in living cells based on two pairs of programmable hairpin oligonucleotide probes with a simple sequence design. The programmable hairpin probes can metastably coexist until the introduction of miRNAs or pre-miRNAs, which can trigger a specific hybridization chain reaction (HCR), respectively, leading to the self-assembly of nicked DNA duplex structures and a remarkable specific fluorescence intensity increase. The system can readily and sensitively assess the miRNA or pre-miRNA abundance in a homogeneous solution. The intracellular miRNA and pre-miRNA expression level assessment in different living cells is realized. Thus, we provide a novel investigation tool for discriminatorily and accurately assessing miRNA and pre-miRNA abundance, which could be useful for the biomedical application of miRNAs.

Dicer promotes endothelial recovery and limits lesion formation after vascular injury through miR-126-5p

BackgroundThe recovery of endothelial cells (ECs) after vascular injury is mainly mediated by the proliferation of resident ECs, thereby reducing neointima formation. The RNase Dicer processes microRNAs (miRNAs) and regulates EC function by controlling miRNA-mediated regulation of gene expression. This study aimed to investigate the impact of miRNA biogenesis in ECs on endothelial repair during lesion formation after vascular injury. Methods and resultsTo study the effect of Dicer on ECs during neointima formation, conditional deletion of Dicer was induced in Apoe−/− mice (EC-Dicerflox) by tamoxifen injection. Following wire-induced injury to carotid arteries of EC-Dicerflox mice, the EC recovery was impaired and the neointima formation and lesional macrophage accumulation was increased. Moreover, conditional deletion of Dicer in ECs diminished the expression of miR-126-5p in EC-Dicerflox mice. Notably, reconstitution of miR-126-5p in the injured arteries of EC-Dicerflox mice using miR-126-5p mimic, prevented the impaired endothelial recovery and increased lesion formation observed in EC-Dicerflox mice. ConclusionsDeficiency of endothelial Dicer diminished endothelial recovery and promoted neointima formation probably due to impaired miR-126-5p expression. Treatment with miR-126-5p mimics promotes endothelial recovery and thereby limits neointima formation. Thus, miR-126-5p therapy represents a potential approach to improve endothelial recovery and prevent restenosis following vascular injury.

Structural basis of siRNA recognition by TRBP double‐stranded RNA binding domains

The accurate cleavage of pre‐micro(mi)RNAs by Dicer and mi/siRNA guide strand selection are important steps in forming the RNA‐induced silencing complex (RISC). The role of Dicer binding partner TRBP in these processes remains poorly understood. Here, we solved the solution structure of the two N‐terminal dsRNA binding domains (dsRBDs) of TRBP in complex with a functionally asymmetric siRNA using NMR, EPR, and single‐molecule spectroscopy. We find that siRNA recognition by the dsRBDs is not sequence‐specific but rather depends on the RNA shape. The two dsRBDs can swap their binding sites, giving rise to two equally populated, pseudo‐symmetrical complexes, showing that TRBP is not a primary sensor of siRNA asymmetry. Using our structure to model a Dicer‐TRBP‐siRNA ternary complex, we show that TRBP's dsRBDs and Dicer's RNase III domains bind a canonical 19 base pair siRNA on opposite sides, supporting a mechanism whereby TRBP influences Dicer‐mediated cleavage accuracy by binding the dsRNA region of the pre‐miRNA during Dicer cleavage.

Dicer and microRNAs protect adult dopamine neurons

MicroRNAs (miRs) are important post-transcriptional regulators of gene expression implicated in neuronal development, differentiation, aging and neurodegenerative diseases, including Parkinson’s disease (PD). Several miRs have been linked to PD-associated genes, apoptosis and stress response pathways, suggesting that deregulation of miRs may contribute to the development of the neurodegenerative phenotype. Here, we investigate the cell-autonomous role of miR processing RNAse Dicer in the functional maintenance of adult dopamine (DA) neurons. We demonstrate a reduction of Dicer in the ventral midbrain and altered miR expression profiles in laser-microdissected DA neurons of aged mice. Using a mouse line expressing tamoxifen-inducible CreERT2 recombinase under control of the DA transporter promoter, we show that a tissue-specific conditional ablation of Dicer in DA neurons of adult mice led to decreased levels of striatal DA and its metabolites without a reduction in neuronal body numbers in hemizygous mice (DicerHET) and to progressive loss of DA neurons with severe locomotor deficits in nullizygous mice (DicerCKO). Moreover, we show that pharmacological stimulation of miR biosynthesis promoted survival of cultured DA neurons and reduced their vulnerability to thapsigargin-induced endoplasmic reticulum stress. Our data demonstrate that Dicer is crucial for maintenance of adult DA neurons, whereas a stimulation of miR production can promote neuronal survival, which may have direct implications for PD treatment.

How short RNAs impact the human ribonuclease Dicer activity: putative regulatory feedback-loops and other RNA-mediated mechanisms controlling microRNA processing.

Ribonuclease Dicer plays a pivotal role in RNA interference pathways by processing long double-stranded RNAs and single-stranded hairpin RNA precursors into small interfering RNAs (siRNAs) and microRNAs (miRNAs), respectively. While details of Dicer regulation by a variety of proteins are being elucidated, less is known about non-protein factors, e.g. RNA molecules, that may influence this enzyme's activity. Therefore, we decided to investigate the question of whether the RNA molecules can function not only as Dicer substrates but also as its regulators. Our previous in vitro studies indicated that the activity of human Dicer can be influenced by short RNA molecules that either bind to Dicer or interact with its substrates, or both. Those studies were carried out with commercial Dicer preparations. Nevertheless, such preparations are usually not homogeneous enough to carry out more detailed RNA-binding studies. Therefore, we have established our own system for the production of human Dicer in insect cells. In this manuscript, we characterize the RNA-binding and RNA-cleavage properties of the obtained preparation. We demonstrate that Dicer can efficiently bind single-stranded RNAs that are longer than ~20-nucleotides. Consequently, we revisit possible scenarios of Dicer regulation by single-stranded RNA species ranging from ~10- to ~60-nucleotides, in the context of their binding to this enzyme. Finally, we show that siRNA/miRNA-sized RNAs may affect miRNA production either by binding to Dicer or by participating in regulatory feedback-loops. Altogether, our studies suggest a broad regulatory role of short RNAs in Dicer functioning.

Canonical microRNAs Enable Differentiation, Protect Against DNA Damage, and Promote Cholesterol Biosynthesis in Neural Stem Cells

Neural stem cells (NSCs) have the capacity to differentiate into neurons, astrocytes, and oligodendrocytes, and therefore represent a promising donor tissue source for treating neurodegenerative diseases and repairing injuries of the nervous system. However, it remains unclear how canonical microRNAs (miRNAs), the subset of miRNAs requiring the Drosha-Dgcr8 microprocessor and the type III RNase Dicer for biogenesis, regulate NSCs. In this study, we established and characterized Dgcr8-/- NSCs from conditionally Dgcr8-disrupted mouse embryonic brain. RNA-seq analysis demonstrated that disruption of Dgcr8 in NSCs causes a complete loss of canonical miRNAs and an accumulation of pri-miRNAs. Dgcr8-/- NSCs can be stably propagated in vitro, but progress through the cell cycle at reduced rates. When induced for differentiation, Dgcr8-/- NSCs failed to differentiate into neurons, astrocytes, or oligodendrocytes under permissive conditions. Compared to Dgcr8+/- NSCs, Dgcr8-/- NSCs exhibit significantly increased DNA damage. Comparative RNA-seq analysis and gene set enrichment analysis (GSEA) revealed that Dgcr8-/- NSCs significantly downregulate genes associated with neuronal differentiation, cell cycle progression, DNA replication, protein translation, and DNA damage repair. Furthermore, we discovered that Dgcr8-/- NSCs significantly downregulate genes responsible for cholesterol biosynthesis and demonstrated that Dgcr8-/- NSCs contain lower levels of cholesterol. Together, our data demonstrate that canonical miRNAs play essential roles in enabling lineage specification, protecting DNA against damage, and promoting cholesterol biosynthesis in NSCs.

TWEAK Negatively Regulates Human Dicer.

The ribonuclease Dicer plays a central role in the microRNA pathway by processing microRNA precursors (pre-microRNAs) into microRNAs, a class of 19- to 24-nucleotide non-coding RNAs that regulate expression of ≈60% of the genes in humans. To gain further insights into the function and regulation of Dicer in human cells, we performed a yeast two-hybrid (Y2HB) screen using human Dicer double-stranded RNA-binding domain (dsRBD) as bait. This approach identified tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) as a Dicer-interacting protein candidate. Confocal immunofluorescence microscopy revealed the colocalization of Dicer and TWEAK proteins at the perinuclear region of HeLa cells. The Dicer-TWEAK protein interaction was confirmed by coimmunoprecipitation and found not likely to be mediated by RNA. TWEAK dose-dependently reduced pre-microRNA conversion into mature microRNA in Dicer activity assays using extracts of transfected human HEK 293 cells. TWEAK expression also impaired microRNA-guided RNA silencing of a reporter gene induced by a pre-microRNA. These findings suggest a role for TWEAK—a pro-inflammatory cytokine—in regulating Dicer function and microRNA biogenesis, and its possible involvement in regulating gene expression during inflammatory processes and diseases.

MiR-124 is differentially expressed in derivatives of the sympathoadrenal cell lineage and promotes neurite elongation in chromaffin cells

The neural-crest-derived sympathoadrenal cell lineage gives rise to sympathetic neurons and to endocrine chromaffin cells of the adrenal medulla. Both cell types express a largely overlapping set of genes, including those coding for the molecular machinery related to the synthesis and exocytotic release of catecholamines. During their early development, sympathetic neurons and chromaffin cells rely on a shared transcription factor network that controls the establishment of these common features. Despite many similarities, mature sympathetic neurons and chromaffin cells significantly differ regarding their morphology and function. Most prominently, sympathetic neurons possess axons that are absent in mammalian adrenal chromaffin cells. The molecular mechanism underlying the divergent development of sympathoadrenal cells into neuronal and endocrine cells remains elusive. Mutational inactivation of the ribonuclease dicer hints at the importance of microRNAs in this diversification. We show here that miR-124 is detectable in developing sympathetic neurons but absent in chromaffin cell precursors. We further demonstrate that miR-124 promotes neurite elongation when transfected into cultured chromaffin cells indicating its capability to support the establishment of a neuronal morphology in non-neuronal sympathoadrenal cells. Our results also show that treatment of PC12 cells with the neurotrophin nerve growth factor leads to an upregulation of miR-124 expression and that inhibition of miR-124 reduces nerve-growth-factor-induced neurite outgrowth in PC12 cells. Thus, our data indicate that miR-124 contributes to the establishment of specific neuronal features in developing sympathoadrenal cells.

Revealing a new activity of the human Dicer DUF283 domain in vitro.

The ribonuclease Dicer is a multidomain enzyme that plays a fundamental role in the biogenesis of small regulatory RNAs (srRNAs), which control gene expression by targeting complementary transcripts and inducing their cleavage or repressing their translation. Recent studies of Dicer’s domains have permitted to propose their roles in srRNA biogenesis. For all of Dicer’s domains except one, called DUF283 (domain of unknown function), their involvement in RNA substrate recognition, binding or cleavage has been postulated. For DUF283, the interaction with Dicer’s protein partners has been the only function suggested thus far. In this report, we demonstrate that the isolated DUF283 domain from human Dicer is capable of binding single-stranded nucleic acids in vitro. We also show that DUF283 can act as a nucleic acid annealer that accelerates base-pairing between complementary RNA/DNA molecules in vitro. We further demonstrate an annealing activity of full length human Dicer. The overall results suggest that Dicer, presumably through its DUF283 domain, might facilitate hybridization between short RNAs and their targets. The presented findings reveal the complex nature of Dicer, whose functions may extend beyond the biogenesis of srRNAs.

Endothelial Dicer promotes atherosclerosis and vascular inflammation by miRNA-103-mediated suppression of KLF4.

MicroRNAs regulate the maladaptation of endothelial cells (ECs) to naturally occurring disturbed blood flow at arterial bifurcations resulting in arterial inflammation and atherosclerosis in response to hyperlipidemic stress. Here, we show that reduced endothelial expression of the RNAse Dicer, which generates almost all mature miRNAs, decreases monocyte adhesion, endothelial C–X–C motif chemokine 1 (CXCL1) expression, atherosclerosis and the lesional macrophage content in apolipoprotein E knockout mice (Apoe−/−) after exposure to a high-fat diet. Endothelial Dicer deficiency reduces the expression of unstable miRNAs, such as miR-103, and promotes Krüppel-like factor 4 (KLF4)-dependent gene expression in murine atherosclerotic arteries. MiR-103 mediated suppression of KLF4 increases monocyte adhesion to ECs by enhancing nuclear factor-κB-dependent CXCL1 expression. Inhibiting the interaction between miR-103 and KLF4 reduces atherosclerosis, lesional macrophage accumulation and endothelial CXCL1 expression. Overall, our study suggests that Dicer promotes endothelial maladaptation and atherosclerosis in part by miR-103-mediated suppression of KLF4.

Human ribonuclease Dicer inhibition using RNA aptamers

Human ribonuclease Dicer inhibition using RNA aptamers

Mutation Scanning of D1705 and D1709 in the RNAse IIIb Domain of MicroRNA Processing Enzyme Dicer in Cutaneous Melanoma.

Since the discovery of microRNAs (miRNAs) there have been performed several studies showing perturbations in the expression of miRNAs and the miRNA expression machinery in cutaneous melanoma. Dicer, a pivotal cytosolic enzyme of miRNA maturation has shown to be affected by both somatic and germline mutations in a variety of cancers. Recent studies have shown that recurrent somatic mutations of Dicer frequently affect the metal-ion-binding sites D1709 and D1705 of its RNase IIIb domain, therefore called hot spot mutations. The present study investigates metal-ion-binding sites D1709 and D1705 of the Dicer RNase IIIb domain in cutaneous melanomas and melanoma metastasis by Sanger sequencing. All investigated samples showed wildtype sequence and no single mutation was detected. The miRNA processing enzyme Dicer of melanoma and melanoma metastasis does not appear to be affected by mutation in the metal-ion-binding sites D1709 and D1705 of its RNase IIIb domain.

Nucleotide sequence of miRNA precursor contributes to cleavage site selection by Dicer

The ribonuclease Dicer excises mature miRNAs from a diverse group of precursors (pre-miRNAs), most of which contain various secondary structure motifs in their hairpin stem. In this study, we analyzed Dicer cleavage in hairpin substrates deprived of such motifs. We searched for the factors other than the secondary structure, which may influence the length diversity and heterogeneity of miRNAs. We found that the nucleotide sequence at the Dicer cleavage site influences both of these miRNA characteristics. With regard to cleavage mechanism, we demonstrate that the Dicer RNase IIIA domain that cleaves within the 3′ arm of the pre-miRNA is more sensitive to the nucleotide sequence of its substrate than is the RNase IIIB domain. The RNase IIIA domain avoids releasing miRNAs with G nucleotide and prefers to generate miRNAs with a U nucleotide at the 5′ end. We also propose that the sequence restrictions at the Dicer cleavage site might be the factor that contributes to the generation of miRNA duplexes with 3′ overhangs of atypical lengths. This finding implies that the two RNase III domains forming the single processing center of Dicer may exhibit some degree of flexibility, which allows for the formation of these non-standard 3′ overhangs.