• Canadian RNA research contributions
• RNA Canada ARN Organization
• RNA bioinformatics
• RNA experimentation
• RNA regulation
• RNA therapeutics

• Humans
• Biomedical Research
• Canada
• COVID-19/epidemiology
• RNA/genetics
• SARS-CoV-2/genetics

Dicer is a ribonuclease III enzyme in biosynthesis of miRNAs, regulators of gene expression involved in macrophage differentiation. We found a specific truncation of Dicer in monocytic cells resulting from apparently constitutive cleavage by a serine protease. Inhibition of this proteolytic truncation, which occurred during macrophage differentiation in presence of TLR ligands or prostaglandin E₂, up-regulates full-length Dicer and promotes miR biosynthesis. Regulation of transcription of pri-miRNA is one mode to regulate biosynthesis of mature miRNA. Inhibition of constitutive proteolysis of Dicer, as described here, provides a second layer of regulation, at the level of miRNA processing. Our data provide insights to Dicer and miRNAs in macrophage polarization/differentiation, a key process in the innate immune response.

Dicer is a ribonuclease III enzyme in biosynthesis of micro-RNAs (miRNAs). Here we describe a regulation of Dicer expression in monocytic cells, based on proteolysis. In undifferentiated Mono Mac 6 (MM6) cells, full-length Dicer was undetectable; only an ∼50-kDa fragment appeared in Western blots. However, when MM6 cells were treated with zymosan or LPS during differentiation with TGF-β and 1,25diOHvitD3, full-length Dicer became abundant together with varying amounts of ∼170- and ∼50-kDa Dicer fragments. Mass spectrometry identified the Dicer fragments and showed cleavage about 450 residues upstream from the C terminus. Also, PGE₂ (prostaglandin E2) added to differentiating MM6 cells up-regulated full-length Dicer, through EP2/EP4 and cAMP. The TLR stimuli strongly induced miR-146a-5p, while PGE₂ increased miR-99a-5p and miR-125a-5p, both implicated in down-regulation of TNFα. The Ser protease inhibitor AEBSF (4-[2-aminoethyl] benzene sulfonyl fluoride) up-regulated full-length Dicer, both in MM6 cells and in primary human blood monocytes, indicating a specific proteolytic degradation. However, AEBSF alone did not lead to a general increase in miR expression, indicating that additional mechanisms are required to increase miRNA biosynthesis. Finally, differentiation of monocytes to macrophages with M-CSF or GM-CSF strongly up-regulated full-length Dicer. Our results suggest that differentiation regimens, both in the MM6 cell line and of peripheral blood monocytes, inhibit an apparently constitutive Dicer proteolysis, allowing for increased formation of miRNAs.

• PGE2
• macrophage
• miRNA

• Animals
• Embryonic Development/genetics
• Female
• Oogenesis/genetics
• RNA, Small Untranslated/genetics
• RNA, Small Untranslated/metabolism
• Ribonuclease III/metabolism

• Dicer
• Dicer-like helicase
• MDA5
• RIG-I
• antiviral immunity
• helicase

• Dicer
• Helicase
• PAZ
• dsRNA
• miRNA
• siRNA

The human dicer1 gene has been predicted to produce several mRNA variants that encode truncated Dicer1 proteins of varying lengths. One of these Dicer1 variants, Dicer1e, was recently found to be differentially expressed in breast cancer cells. Because the expression and function of the Dicer1e protein variant has not been well characterized and the underlying molecular mechanisms for the development of oral squamous cell carcinomas (OSCCs) are poorly understood, the present study sought to characterize the biological role of Dicer1e and determine its relationship, if any, to OSCC pathogenesis.

Western blot analyses were used to examine Dicer1e expression levels in a panel of oral cancer cells/tissues and during epithelial-mesenchymal transition (EMT), followed by 5′/3′-RACE analyses to obtain the full-length Dicer1e transcript. Biochemical fractionation and indirect immunofluorescent studies were performed to determine the cellular localization of Dicer1e and the effects of Dicer1e silencing on cancer cell proliferation, clonogenicity, and drug sensitivity were also assessed.

Dicer1e protein levels were found to be overexpressed in OSCC cell lines of epithelial phenotype and in OSCC tissues with its levels downregulated during EMT. Moreover, the Dicer1e protein was observed to predominantly localize in the nucleus. 5′/3′-RACE analyses confirmed the presence of the Dicer1e transcript and silencing of Dicer1e impaired both cancer cell proliferation and clonogenicity by inducing either apoptosis and/or G2/M cell cycle arrest. Lastly, Dicer1e knockdown enhanced the chemosensitivity of oral cancer cells to cisplatin.

The expression levels of Dicer1e influence the pathogenesis of oral cancer cells and alter their response to chemosensitivity, thus supporting the importance of Dicer1e as a therapeutic target for OSCCs.

The online version of this article (doi:10.1186/1476-4598-13-190) contains supplementary material, which is available to authorized users.

Growing evidence suggests that breast cancer cell plasticity arises due to a partial reactivation of epithelial-mesenchymal transition (EMT) programs in order to give cells pluripotency, leading to a stemness-like phenotype. A complete EMT would be a dead end program that would render cells unable to fully metastasize to distant organs. Evoking the EMT-mesenchymal-to-epithelial transition (MET) cascade promotes successful colonization of distal target tissues. It is unlikely that direct reprogramming or trans-differentiation without passing through a pluripotent stage would be the preferred mechanism during tumor progression. This review focuses on key EMT transcriptional regulators, EMT-transcription factors involved in EMT (TFs) and the miRNA pathway, which are deregulated in breast cancer, and discusses their implications in cancer cell plasticity. Cross-regulation between EMT-TFs and miRNAs, where miRNAs act as co-repressors or co-activators, appears to be a pivotal mechanism for breast cancer cells to acquire a stem cell-like state, which is implicated both in breast metastases and tumor recurrence. As a master regulator of miRNA biogenesis, the ribonuclease type III endonuclease Dicer plays a central role in EMT-TFs/miRNAs regulating networks. All these EMT-MET key regulators represent valuable new prognostic and predictive markers for breast cancer as well as promising new targets for drug-resistant breast cancers.

• Embryonic transcription factors
• Epithelial to mesenchymal transition
• Breast cancer
• MicroRNAs
• Dicer
• Feedback loop

• crispr
• lncrna
• mirna
• pirna
• sirna

• CRISPR-Cas Systems
• Epigenetic Repression
• RNA Interference
• RNA, Untranslated/classification
• RNA, Untranslated/genetics
• Ribonucleoproteins/genetics

microRNAs function in diverse developmental and physiological processes by regulating target gene expression at the post-transcriptional level. ALG-1 is one of two Caenorhabditis elegans Argonautes (ALG-1 and ALG-2) that together are essential for microRNA biogenesis and function. Here, we report the identification of novel antimorphic (anti) alleles of ALG-1 as suppressors of lin-28(lf) precocious developmental phenotypes. The alg-1(anti) mutations broadly impair the function of many microRNAs and cause dosage-dependent phenotypes that are more severe than the complete loss of ALG-1. ALG-1(anti) mutant proteins are competent for promoting Dicer cleavage of microRNA precursors and for associating with and stabilizing microRNAs. However, our results suggest that ALG-1(anti) proteins may sequester microRNAs in immature and functionally deficient microRNA Induced Silencing Complexes (miRISCs), and hence compete with ALG-2 for access to functional microRNAs. Immunoprecipitation experiments show that ALG-1(anti) proteins display an increased association with Dicer and a decreased association with AIN-1/GW182. These findings suggest that alg-1(anti) mutations impair the ability of ALG-1 miRISC to execute a transition from Dicer-associated microRNA processing to AIN-1/GW182 associated effector function, and indicate an active role for ALG/Argonaute in mediating this transition.

microRNAs are small non-coding RNAs that function in diverse processes by post-transcriptionally regulating gene expression. Argonautes form the core of the microRNA Induced Silencing Complex (miRISC) and are required for microRNA biogenesis and function. Here we describe the identification and characterization of a novel set of mutations in alg-1, a Caenorhabditis elegans microRNA specific Argonaute. This new class of alg-1 mutations causes phenotypes more severe than the complete loss of alg-1. Interestingly, the mutant ALG-1 proteins are able to promote microRNA biogenesis, but are defective in mediating microRNA target gene repression. We found that mutant ALG-1 associates more with Dicer, but less with miRISC effector AIN-1, compared to wild type ALG-1. We propose that these mutant ALG-1 proteins assemble nonfunctional complexes that effectively compete with the paralogous ALG-2 for critical miRISC components, including mature microRNAs. This new class of Argonaute mutants highlights the role of Argonaute in mediating a functional transition for miRISC from microRNA processing phase to target repression phase.

• Alleles
• Amino Acid Sequence
• Animals
• Argonaute Proteins/genetics
• Caenorhabditis elegans
• Caenorhabditis elegans Proteins/genetics
• Carrier Proteins/genetics
• Conserved Sequence
• Gene Expression Regulation, Developmental/genetics
• MicroRNAs/genetics
• Molecular Sequence Data
• Mutation/genetics
• Repressor Proteins/genetics
• Sequence Alignment

• P40 OD010440 NIH HHS
• MOP-81186 CIHR
• NIH R01-GM34028 NIGMS NIH HHS
• P30 CA045508 NCI NIH HHS
• R01 GM034028 NIGMS NIH HHS

• Calcium Signaling
• Cell Signaling
• Dicer
• Protein-Protein Interactions
• TRP Channels