Functional parameters of Dicer-independent microRNA biogenesis

Until recently, a Dicer-class RNase III enzyme was believed to be essential for microRNA (miRNA) biogenesis in all animals. The conserved vertebrate locus mir-451 defies this expectation and instead matures by direct cleavage of its pre-miRNA hairpin via the Slicer activity of Argonaute2 (Ago2). In this study, we used structure-function analysis to define the functional parameters of Ago2-mediated miRNA biogenesis. These include (1) the requirement for base-pairing at most, but not all, positions within the pre-mir-451 stem; (2) surprisingly little influence of the 5'-nucleotide on Ago sorting; (3) substantial influence of Ago protein stoichiometry on mir-451 maturation; (4) strong influence of G:C content in the distal stem on 3' resection of cleaved mir-451 substrates; and (5) the influence of hairpin length on substrate utilization by Ago2 and Dicer. Unexpectedly, we find that certain hairpin lengths confer competence to mature via both Dicer-mediated and Ago2-mediated pathways, and we show, in fact, that a conventional shRNA can traverse the Dicer-independent pathway. Altogether, these data inform the design of effective Dicer-independent substrates for gene silencing and reveal novel aspects of substrate handling by Ago proteins.     

microRNA研究进展

小分子RNA家族中的一员——microRNA,是一段非常短的非编码RNA序列,对多种生物学过程起调控作用。本文试从microRNA的结构特点、合成及作用机制和功能等方面对microRNA的研究进展作一个简单回顾。     

RNase III-independent microRNA biogenesis in mammalian cells

RNase III enzymes are fundamental to the biogenesis of microRNAs (miRNAs) and small interfering RNAs (siRNAs) in all species studied. Although alternative miRNA pathways independent of Drosha or Dicer exist, each still requires one RNase III-type enzyme. Here, we describe two strategies that marry either RNase Z or the Integrator complex with the slicing activity of Argonaute2 to generate highly functional mature miRNAs. We provide stringent validation of their RNase III independence by demonstrating efficient miRNA biogenesis and activity in Drosha and Dicer knockout cells. These data provide proof-of-principle evidence for additional mechanistic possibilities for efficient generation of small regulatory RNAs, and represent novel silencing triggers that may be exploited for technical purposes.     

Structural basis of microRNA biogenesis by Dicer-1 and its partner protein Loqs-PB

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MicroRNA-independent roles of the RNase III enzymes Drosha and Dicer

The ribonuclease III enzymes Drosha and Dicer are renowned for their central roles in the biogenesis of microRNAs (miRNAs). For many years, this has overshadowed the true versatility and importance of these enzymes in the processing of other RNA substrates. For example, Drosha also recognizes and cleaves messenger RNAs (mRNAs), and potentially ribosomal RNA. The cleavage of mRNAs occurs via recognition of secondary stem-loop structures similar to miRNA precursors, and is an important mechanism of repressing gene expression, particularly in progenitor/stem cell populations. On the other hand, Dicer also has critical roles in genome regulation and surveillance. These include the production of endogenous small interfering RNAs from many sources, and the degradation of potentially harmful short interspersed element and viral RNAs. These findings have sparked a renewed interest in these enzymes, and their diverse functions in biology.     

Analogues of Uridinediphosphatehexoses. A New Type of Protein Glycosylation Inhibitors That Show Antiviral Activity

Abstract

A series of analogues of UDP-Glc and UDP-GlcNAc prepared by reaction of protected hexoses with ClSO₂NCO and 2′3′-O-isopropylideneuridine, inhibited glycosylation of proteins in HSV-1 infected HeLa cells and were active against several enveloped viruses.     

Molecular Basis for the Single-Nucleotide Precision of Primary microRNA Processing

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Dicer结构和功能研究进展

摘要: Dicer蛋白是RNA干扰机制的关键组分, 负责siRNA和miRNA的产生。它主要由RNA解旋酶结构域、PAZ结构域、RNaseⅢ结构域和双链RNA结合结构域构成。Dicer的结构特点决定了它所产生的小RNA的结构特点。不同生物体具有不同数量的Dicer, 各Dicer既有功能上各自独立的特点, 同时又有功能的冗余和交叉, 而在进化过程中, Dicer的数量逐渐减少, 功能却逐步整合从而表现出多功能的特点。对Dicer结构和功能进行深入研究, 有助于了解Dicer乃至整个RNAi及相关途径的作用机制, 也有助于揭示它们在进化过程中所表现出的规律和特点。文章对上述Dicer结构及功能特点作简要综述。     

Reactive oligonucleotide derivatives as gene-targeted biologically active compounds and affinity probes

Development of efficient methods for synthesis of oligonucleotides and oligonucleotide analogs has opened up the possibility of designing a broad spectrum of affinity reagents for specific modification of nucleic acids and proteins. These affinity reagents are used for investigation of the topology of ribosomes and nucleic acid polymerases. Oligonucleotides and their analogs are already used for suppression of specific gene expression and for elucidation of the physiological role of their products. Oligonucleotide derivatives appear to offer considerable promise as potential gene-targeted drugs such as antivirals and specific inhibitors of oncogene expression.     

Tranylcypromine and 6-trifluoroethyl thienopyrimidine hybrid as LSD1 inhibitor

Tranylcypromine moiety extracted from LSD1 inhibitors and 6-trifluoroethyl thienopyrimidine moiety from menin-MLL1 PPI inhibitors were merged to give new chemotypes for medicinal chemistry study. Among 15 new compounds prepared in this work, some exhibited nanomolar LSD1 activity and good selectivity over MAO-A/B, low micromolar menin-MLL1 PPI inhibitory activity, as well as submicromolar MV4-11 antiprofilative activities. Intracellular LSD1 engagement of compounds with higher enzymatic and antiproliferative activities was confirmed by CD86 mRNA up-regulation experiments.     

Establishment of cells to monitor Microprocessor through fusion genes of microRNA and GFP

Microprocessor, the complex of Drosha and DGCR8, promotes the processing of primary microRNA to precursor microRNA, which is a crucial step for microRNA maturation. So far, no convenient assay systems have been developed for observing this step in vivo.Here we report the establishment of highly sensitive cellular systems where we can visually monitor the function of Microprocessor. During a series of screening of transfectants with fusion genes of the EGFP cDNA and primary microRNA genes, we have obtained certain cell lines where introduction of siRNA against DGCR8 or Drosha strikingly augments GFP signals. In contrast, these cells have not responded to Dicer siRNA; thus they have a unique character that GFP signals should be negatively and specifically correlated to the action of Microprocessor among biogenesis of microRNA.These cell lines can be useful tools for real-time analysis of Microprocessor action in vivo and identifying its novel modulators.     

Dysregulation of microRNA biogenesis machinery in cancer

MicroRNAs (miRNAs) are integral to the gene regulatory network. A single miRNA is capable of controlling the expression of hundreds of protein coding genes and modulate a wide spectrum of biological functions, such as proliferation, differentiation, stress responses, DNA repair, cell adhesion, motility, inflammation, cell survival, senescence and apoptosis, all of which are fundamental to tumorigenesis. Overexpression, genetic amplification, and gain-of-function mutation of oncogenic miRNAs (“onco-miRs”) as well as genetic deletion and loss-of-function mutation of tumor suppressor miRNAs (“suppressor-miRs”) are linked to human cancer. In addition to the dysregulation of a specific onco-miR or suppressor-miRs, changes in global miRNA levels resulting from a defective miRNA biogenesis pathway play a role in tumorigenesis. The function of individual onco-miRs and suppressor-miRs and their target genes in cancer has been described in many different articles elsewhere. In this review, we primarily focus on the recent development regarding the dysregulation of the miRNA biogenesis pathway and its contribution to cancer.     

小分子RNA

小分子RNA(miRNA)是继siRNA之后发现的调节mRNA稳定和mRNA翻译并普遍存在于动植物体内的一类内源非编码RNA。这类非编码RNAs长约22 nt,在动植物的基因调控中起着重要的作用。它是近几年生命科学研究的热点之一。在2002年,miRNA被Science杂志评为十大科技突破之一。     

Combined small RNA and degradome sequencing reveals microRNA regulation during immature maize embryo dedifferentiation

Genetic transformation of maize is highly dependent on the development of embryonic calli from the dedifferentiated immature embryo. To better understand the regulatory mechanism of immature embryo dedifferentiation, we generated four small RNA and degradome libraries from samples representing the major stages of dedifferentiation. More than 186 million raw reads of small RNA and degradome sequence data were generated. We detected 102 known miRNAs belonging to 23 miRNA families. In total, we identified 51, 70 and 63 differentially expressed miRNAs (DEMs) in the stage I, II, III samples, respectively, compared to the control. However, only 6 miRNAs were continually up-regulated by more than fivefold throughout the process of dedifferentiation. A total of 87 genes were identified as the targets of 21 DEM families. This group of targets was enriched in members of four significant pathways including plant hormone signal transduction, antigen processing and presentation, ECM-receptor interaction, and alpha-linolenic acid metabolism. The hormone signal transduction pathway appeared to be particularly significant, involving 21 of the targets. While the targets of the most significant DEMs have been proved to play essential roles in cell dedifferentiation. Our results provide important information regarding the regulatory networks that control immature embryo dedifferentiation in maize.