Localization and translocation of mature miRNAs

The scientific review shows the ways of nuclear import and export of miRNAs in the cell. The authors present a clear and accessible scheme of microRNA translocation in the cell. The article shows that the main site of localization in the cytoplasm of cells of the RISC complex and its components, including miRNAs, are processing P-cells. The authors cite the fact that Argonaute proteins — signature components of the effector complex of RISC RNA interference — are localized in mammalian P-bodies. It is shown that proteins of the karyopherin family mediate the translocation of miRISC into the cell nucleus. These proteins recognize nuclear localization sequences (NLS) in the amino acid sequences of proteins and actively transport these proteins through the pores of the cell’s nuclear membrane. It is emphasized that in addition to non-selective mechanisms of nuclear import of miRNAs, there are transport mechanisms that carry certain miRNAs across the cell membrane. Some miRNAs are presented, which are mainly loca­lized in the nucleus of a certain type of cell. Scientists believe that much of the nucleus miRNA is concentrated in polysomes. Export of nuclear pool microRNA into the cytoplasm of the cell occurs with the help of export 1. Thus, in the cytoplasm of the cell, mature forms of microRNA accumulate, some of which are translocated to the cell nucleus or the extracellular space. Assembly of the miRISC complex is carried out in the cytoplasm of the cell, and only after the formation of the complex, it is imported into the cell nucleus. The spectrum of exosome-associated miRNAs can be a highly important diagnostic criterion for some nosologies, and exosomes containing certain miRNAs can be used for targeted therapy of specific diseases. To write the article, information was searched using databases Scopus, Web of Science, MedLine, PubMed, Google Scholar, EMBASE, Global Health, The Cochrane Library, CyberLeninka.

MicroRNA biogenesis. Part 2. Formation of mature miRNAs. Maturation of non-canonical miRNAs

The scientific review presents the biogenesis of miRNAs. To write the article, information was searched using databases Scopus, Web of Science, MedLine, PubMed, Google Scholar, EMBASE, Global Health, The Cochrane Library, CyberLeninka. The article shows the stages of formation of mature miRNAs. It is noted that duplex RNAs resulting from DICER-mediated cleavage interact with Argonaute (AGO) proteins to form an effector RNA-induced silencing complex (RISC). It is shown that the deficiency of AGO proteins leads to a significant decrease in the amount of miRs, and overexpression of AGO proteins is accompanied by an increase in the level of miRs. The main stages of assembling a fully functional RISC are presented. The first stage is the loading of duplex miRs on AGO proteins. The second stage is the promotion of duplex miRs. Human diseases associated with processing disorders in the cytoplasm of the cell are presented. Numerous alternative mechanisms involved in the formation of functionally active miRs are is characterized. There are three classes of mirtrons: typical mirtrons, 5’-tailed mirtrons and 3’-tailed mirtrons. Endogenous csRNAs resemble Drosha-independent synthetic csRNAs used to experimentally induce gene knockout. Chimeric hairpins of non-canonical miR genes are transcribed in tandem or as a part of another type of small RNA gene. Thus, the formation of mature miRs occurs due to the formation of the RISC complex. The core of the RISC complex consists of microRNA, AGO and protein with a trinucleotide repeat 6. Loading dsRNA on AGO proteins and subsequent promotion of duplex RNA are the main stages of assembly of a fully functional RISC. Disorders of processing in the cytoplasm of the cell are associated with the development of some human diseases. There are alternative mechanisms involved in the formation of functionally active miRs: mirtrons, endogenous short RNAs containing hairpins, chimeric hairpins.

CRL4-DCAF12 Ubiquitin Ligase Controls MOV10 RNA Helicase during Spermatogenesis and T Cell Activation

Multisubunit cullin-RING ubiquitin ligase 4 (CRL4)-DCAF12 recognizes the C-terminal degron containing acidic amino acid residues. However, its physiological roles and substrates are largely unknown. Purification of CRL4-DCAF12 complexes revealed a wide range of potential substrates, including MOV10, an “ancient” RNA-induced silencing complex (RISC) complex RNA helicase. We show that DCAF12 controls the MOV10 protein level via its C-terminal motif in a proteasome- and CRL-dependent manner. Next, we generated Dcaf12 knockout mice and demonstrated that the DCAF12-mediated degradation of MOV10 is conserved in mice and humans. Detailed analysis of Dcaf12-deficient mice revealed that their testes produce fewer mature sperms, phenotype accompanied by elevated MOV10 and imbalance in meiotic markers SCP3 and γ-H2AX. Additionally, the percentages of splenic CD4+ T and natural killer T (NKT) cell populations were significantly altered. In vitro, activated Dcaf12-deficient T cells displayed inappropriately stabilized MOV10 and increased levels of activated caspases. In summary, we identified MOV10 as a novel substrate of CRL4-DCAF12 and demonstrated the biological relevance of the DCAF12-MOV10 pathway in spermatogenesis and T cell activation.

To “Z” or not to “Z”: Z-RNA, self-recognition, and the MDA5 helicase

Double-stranded RNA (dsRNA) is produced both by virus and host. Its recognition by the melanoma differentiation–associated gene 5 (MDA5) initiates type I interferon responses. How can a host distinguish self-transcripts from nonself to ensure that responses are targeted correctly? Here, I discuss a role for MDA5 helicase in inducing Z-RNA formation by Alu inverted repeat (AIR) elements. These retroelements have highly conserved sequences that favor Z-formation, creating a site for the dsRNA-specific deaminase enzyme ADAR1 to dock. The subsequent editing destabilizes the dsRNA, ending further interaction with MDA5 and terminating innate immune responses directed against self. By enabling self-recognition, Alu retrotransposons, once invaders, now are genetic elements that keep immune responses in check. I also discuss the possible but less characterized roles of the other helicases in modulating innate immune responses, focusing on DExH-box helicase 9 (DHX9) and Mov10 RISC complex RNA helicase (MOV10). DHX9 and MOV10 function differently from MDA5, but still use nucleic acid structure, rather than nucleotide sequence, to define self. Those genetic elements encoding the alternative conformations involved, referred to as flipons, enable helicases to dynamically shape a cell’s repertoire of responses. In the case of MDA5, Alu flipons switch off the dsRNA-dependent responses against self. I suggest a number of genetic systems in which to study interactions between flipons and helicases further.

Structure of the RPAP3:TRBP interaction reveals an involvement of the HSP90/R2TP chaperone complex in dsRNA pathways

ABSTRACTMicroRNAs silence mRNAs by guiding the RISC complex. RISC assembly requires cleavage of pre-miRNAs by Dicer, assisted by TRBP or PACT, and the transfer of miRNAs to AGO proteins. The R2TP complex is an HSP90 cochaperone involved in the assembly of ribonucleoprotein particles. Here, we show that the R2TP component RPAP3 binds TRBP but not PACT. Specifically, the RPAP3-TPR1 domain interacts with the TRBP-dsRBD3 and the 1.5 Å resolution crystal structure of this complex is presented. We identify key residues involved in the interaction and show that binding of TRBP to RPAP3 or Dicer is mutually exclusive. In contrast, RPAP3 can simultaneously bind TRBP and HSP90. Interestingly, AGOs and Dicer are sensitive to HSP90 inhibition and TRBP becomes sensitive in absence of RPAP3. These data indicate that the HSP90/R2TP chaperone is an important cofactor of proteins involved in dsRNA pathways.

LncRNA CRNDE Acts as a ceRNA and Regulates AML Cell Proliferation and Apoptosis via miR136-5p/MCM5 Axis

Background: Increasing evidence demonstrated that long noncoding RNAs (lncRNAs) act as important factors in the regulation of cell processes and tumorigenesis. The long-noncoding RNA colorectal neoplasia differentially expressed (CRNDE) gene has been found to be related to several types of cancer. Although CRNDE is highly expressed in AML, its mechanism of action in acute myeloid leukemia (AML) is unknown.Methods: The expression levels of CRNDE and miR-136-5p mRNAs were measured by quantitative real-time PCR. The effects of CRNDE knockdown on cell proliferation was assessed by the CCK8 assay, while apoptosis and cell cycle distribution were analyzed by flow cytometry. The expression of proteins related to cell cycle, cell apoptosis and MCM5 were analyzed by Western blotting. The luciferase reporter assay was used to confirm the interaction between CRNDE and miR-136-5p and between MCM5 and miR-136-5p in AML. The RNA immunoprecipitation assay was used to verify whether CRNDE exists in the miRNA mediated RISC complex.Results: In this study, we used GEPIA database to confirm that CRNDE expression was significantly upregulated in AML samples. The silencing of CRNDE inhibited AML cells’ proliferation ability, increased AML cells’ apoptotic rate and arrested AML cells at the G1 phase of the cell cycle. Mechanistically, CRNDE served as a competing endogenous RNA (ceRNA) for miR-136-5p and upregulated MCM5 expression by sponging miR-136-5p. In addition, rescue assays revealed that the effects of CRNDE knockdown could be reversed by miR-136-5p inhibitors in AML cells. Conclusion: Our results demonstrate that the CRNDE-miR-136-5p-MCM5 axis modulates AML progression and provide a new regulatory network of CRNDE in AML.

LncRNA CRNDE acts as a ceRNA and regulates AML cell proliferation and apoptosis via miR136-5p/MCM5 axis

Background Increasing evidence demonstrated that long noncoding RNAs (lncRNAs) act as important factors in the regulation of cell processes and tumorigenesis. The long-noncoding RNA colorectal neoplasia differentially expressed (CRNDE) gene has been found to be related to several types of cancer. Although CRNDE is highly expressed in AML, its mechanism of action in acute myeloid leukemia (AML) is unknown. Methods The expression levels of CRNDE and miR-136-5p mRNAs were measured by quantitative real-time PCR. The effects of CRNDE knockdown on cell proliferation was assessed by the CCK8 assay, while apoptosis and cell cycle distribution were analyzed by flow cytometry. The expression of proteins related to cell cycle, cell apoptosis and MCM5 were analyzed by Western blotting. The luciferase reporter assay was used to confirm the interaction between CRNDE and miR-136-5p and between MCM5 and miR-136-5p in AML. The RNA immunoprecipitation assay was used to verify whether CRNDE exists in the miRNA mediated RISC complex. Results In this study, we used GEPIA database to confirm that CRNDE expression was significantly upregulated in AML samples. The silencing of CRNDE inhibited AML cells’ proliferation ability, increased AML cells’ apoptotic rate and arrested AML cells at the G1 phase of the cell cycle. Mechanistically, CRNDE served as a competing endogenous RNA (ceRNA) for miR-136-5p and upregulated MCM5 expression by sponging miR-136-5p. In addition, rescue assays revealed that the effects of CRNDE knockdown could be reversed by miR-136-5p inhibitors in AML cells. Conclusion Our results demonstrate that the CRNDE-miR-136-5p-MCM5 axis modulates AML progression and provide a new regulatory network of CRNDE in AML.

LncRNA CRNDE Acts as a ceRNA and Regulates AML Cell Proliferation and Apoptosis via miR136-5p/MCM5 Axis

Background: Increasing evidence demonstrated that long noncoding RNAs (lncRNAs) act as important factors in the regulation of cell processes and tumorigenesis. The long-noncoding RNA colorectal neoplasia differentially expressed (CRNDE) gene has been found to be related to several types of cancer. Although CRNDE is highly expressed in AML, its mechanism of action in acute myeloid leukemia (AML) is unknown.Methods: The expression levels of CRNDE and miR-136-5p mRNAs were measured by quantitative real-time PCR. The effects of CRNDE knockdown on cell proliferation was assessed by the CCK8 assay, while apoptosis and cell cycle distribution were analyzed by flow cytometry. The expression of proteins related to cell cycle, cell apoptosis and MCM5 were analyzed by Western blotting. The luciferase reporter assay was used to confirm the interaction between CRNDE and miR-136-5p and between MCM5 and miR-136-5p in AML. The RNA immunoprecipitation assay was used to verify whether CRNDE exists in the miRNA mediated RISC complex.Results: In this study, we used GEPIA database to confirm that CRNDE expression was significantly upregulated in AML samples. The silencing of CRNDE inhibited AML cells’ proliferation ability, increased AML cells’ apoptotic rate and arrested AML cells at the G1 phase of the cell cycle. Mechanistically, CRNDE served as a competing endogenous RNA (ceRNA) for miR-136-5p and upregulated MCM5 expression by sponging miR-136-5p. In addition, rescue assays revealed that the effects of CRNDE knockdown could be reversed by miR-136-5p inhibitors in AML cells. Conclusion: Our results demonstrate that the CRNDE-miR-136-5p-MCM5 axis modulates AML progression and provide a new regulatory network of CRNDE in AML.

Loss of CNOT9 begets impairment in gastrulation leading to embryonic lethality

The multi-subunit eukaryotic CCR4-NOT complex imparts gene expression control primarily via messenger RNA (mRNA) decay. Here, we present the role of subunit CNOT9 in target mRNA decay during embryonic development. CNOT9 null mice appear normal by the onset of gastrulation (E7.0), however, exhibit growth and differentiation defects accompanied by extensive cell death by embryonic day 9.5 (E9.5). Sox-2 Cre conditional CNOT9 knockout mice show almost identical phenotype with brief delay in onset and progression, suggesting defects to be epiblast-dominant. Among various identified targets, we show that Lefty2 mRNA expression is post-transcriptionally regulated by CNOT9. Lefty2 3’-UTR containing mRNA has significantly higher stability in cells expressing mutant form of CNOT9, relative to cells expressing wild-type CNOT9. In addition, CNOT9 primarily localizes within the cytoplasm and bridges interactions between the CCR4-NOT complex and miRNA-RISC complex in gastrulating embryos.