RNA干涉在提高植物营养价值上的应用

RNA干涉(RNAi)是由具同源性的外源dsRNA引起的序列特异性转录后基因沉默现象,广泛存在于各种动、植物中。人类大多数疾病,像心脏病和癌症,都能够通过食用添加具有特殊营养成分养的饮食来预防。应用RNAi技术可为人和动物提高植物的营养价值。     

Bifunctional small molecule-oligonucleotide hybrid as microRNA inhibitor

miRNAs are key regulators of various biological processes. Dysregulation of miRNA is linked to many diseases. Development of miRNA inhibitor has implication in disease therapy and study of miRNA function. The biogenesis pathway of miRNA involves the processing of pre-miRNA into mature miRNA by Dicer enzyme. We previously reported a proximity enabled approach that employs bifunctional small molecules to regulate miRNA maturation through inhibiting the enzymatic activity of Dicer. By conjugating to an RNA targeting unit, an RNase inhibitor could be delivered to the cleavage site of specific pre-miRNA to deactivate the complexed Dicer enzyme. Herein, we expanded this bifunctional strategy by showing that antisense oligonucleotides (ASOs), including morpholinos and γPNAs, could be readily used as the RNA recognition unit to generate bifunctional small molecule-oligonucleotide hybrids as miRNA inhibitors. A systematic comparison revealed that the potency of these hybrids is mainly determined by the RNA binding of the targeting ASO molecules. Since the lengths of the ASO molecules used in this approach were much shorter than commonly used anti-miRNA ASOs, this may provide benefits to the specificity and cellular delivery of these hybrids. We expect that this approach could be complementary to traditional ASO and small molecule based miRNA inhibition and contribute to the study of miRNA.     

Probing the shRNA characteristics that hinder Dicer recognition and consequently allow Ago-mediated processing and AgoshRNA activity

Recent evidence indicates the presence of alternative pathways for microRNA (miRNA) and short hairpin (shRNA) processing. Specifically, some of these molecules are refractory to Dicer-mediated processing, which allows alternative processing routes via the Ago2 endonuclease. The resulting RNA molecules differ in size and sequence and will thus trigger the silencing of different target RNAs. It is, therefore, important to understand these processing routes in mechanistic detail such that one can design exclusive RNA reagents for a specific processing route. The exact sh/miRNA properties that determine this routing toward Dicer or Ago2 are incompletely understood. The size of the base-paired stem seems an important determinant, but other RNA elements may contribute as well. In this study, we document the importance of a weak G-U or U-G base pair at the top of the hairpin stem.     

Structural analysis of RIG-I-like receptors reveals ancient rules of engagement between diverse RNA helicases and TRIM ubiquitin ligases

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Filtering of deep sequencing data reveals the existence of abundant Dicer-dependent small RNAs derived from tRNAs

Deep sequencing technologies such as Illumina, SOLiD, and 454 platforms have become very powerful tools in discovering and quantifying small RNAs in diverse organisms. Sequencing small RNA fractions always identifies RNAs derived from abundant RNA species such as rRNAs, tRNAs, snRNA, and snoRNA, and they are widely considered to be random degradation products. We carried out bioinformatic analysis of deep sequenced HeLa RNA and after quality filtering, identified highly abundant small RNA fragments, derived from mature tRNAs that are likely produced by specific processing rather than from random degradation. Moreover, we showed that the processing of small RNAs derived from tRNA^Gln is dependent on Dicer in vivo and that Dicer cleaves the tRNA in vitro.     

Processing of Drosophila endo-siRNAs depends on a specific Loquacious isoform

Drosophila melanogaster expresses three classes of small RNAs, which are classified according to their mechanisms of biogenesis. MicroRNAs are ∼22–23 nucleotides (nt), ubiquitously expressed small RNAs that are sequentially processed from hairpin-like precursors by Drosha/Pasha and Dcr-1/Loquacious complexes. MicroRNAs usually associate with AGO1 and regulate the expression of protein-coding genes. Piwi-interacting RNAs (piRNAs) of ∼24–28 nt associate with Piwi-family proteins and can arise from single-stranded precursors. piRNAs function in transposon silencing and are mainly restricted to gonadal tissues. Endo-siRNAs are found in both germline and somatic tissues. These ∼21-nt RNAs are produced by a distinct Dicer, Dcr-2, and do not depend on Drosha/Pasha complexes. They predominantly bind to AGO2 and target both mobile elements and protein-coding genes. Surprisingly, a subset of endo-siRNAs strongly depend for their production on the dsRNA-binding protein Loquacious (Loqs), thought generally to be a partner for Dcr-1 and a cofactor for miRNA biogenesis. Endo-siRNA production depends on a specific Loqs isoform, Loqs-PD, which is distinct from the one, Loqs-PB, required for the production of microRNAs. Paralleling their roles in the biogenesis of distinct small RNA classes, Loqs-PD and Loqs-PB bind to different Dicer proteins, with Dcr-1/Loqs-PB complexes and Dcr-2/Loqs-PD complexes driving microRNA and endo-siRNA biogenesis, respectively.     

Molecular mechanisms that funnel RNA precursors into endogenous small-interfering RNA and microRNA biogenesis pathways in Drosophila

In Drosophila, three types of endogenous small RNAs—microRNAs (miRNAs), PIWI-interacting RNAs (piRNAs), and endogenous small-interfering RNAs (endo-siRNAs or esiRNAs)—function as triggers in RNA silencing. Although piRNAs are produced independently of Dicer, miRNA and esiRNA biogenesis pathways require Dicer1 and Dicer2, respectively. Recent studies have shown that among the four isoforms of Loquacious (Loqs), Loqs-PB and Loqs-PD are involved in miRNA and esiRNA processing pathways, respectively. However, how these Loqs isoforms function in their respective small RNA biogenesis pathways remains elusive. Here, we show that Loqs-PD associates specifically with Dicer2 through its C-terminal domain. The Dicer2–Loqs-PD complex contains R2D2, another known Dicer2 partner, and excises both exogenous siRNAs and esiRNAs from their corresponding precursors in vitro. However, Loqs-PD, but not R2D2, enhanced Dicer2 activity. The Dicer2–Loqs-PD complex processes esiRNA precursor hairpins with long stems, which results in the production of AGO2-associated small RNAs. Interestingly, however, small RNAs derived from terminal hairpins of esiRNA precursors are loaded onto AGO1; thus, they are classified as a new subset of miRNAs. These results suggest that the precursor RNA structure determines the biogenesis mechanism of esiRNAs and miRNAs, thereby implicating hairpin structures with long stems as intermediates in the evolution of Drosophila miRNA.     

Role of Dicer1 in thyroid cell proliferation and differentiation

DICER1 plays a central role in the biogenesis of microRNAs and it is important for normal development. Altered microRNA expression and DICER1 dysregulation have been described in several types of tumors, including thyroid carcinomas. Recently, our group identified a new somatic mutation (c.5438A>G; E1813G) within DICER1 gene of an unknown function. Herein, we show that DICER1 is overexpressed, at mRNA level, in a significant-relative number of papillary (70%) and anaplastic (42%) thyroid carcinoma samples, whereas is drastically downregulated in all the analyzed human thyroid carcinoma cell lines (TPC-1, BCPAP, FRO and 8505c) in comparison with normal thyroid tissue samples. Conversely, DICER1 is downregulated, at protein level, in PTC in comparison with normal thyroid tissues. Our data also reveals that DICER1 overexpression positively regulates thyroid cell proliferation, whereas its silencing impairs thyroid cell differentiation. The expression of DICER1 gene mutation (c.5438A>G; E1813G) negatively affects the microRNA machinery and cell proliferation as well as upregulates DICER1 protein levels of thyroid cells but has no impact on thyroid differentiation. In conclusion, DICER1 protein is downregulated in papillary thyroid carcinomas and affects thyroid proliferation and differentiation, while DICER1 gene mutation (c.5438A>G; E1813G) compromises the DICER1 wild-type-mediated microRNA processing and cell proliferation.