MIR166基因家族在陆生植物中的进化模式分析

MicroRNA（miRNA）是一类广泛存在于真核生物中的具有转录后水平调控功能的内源非编码小分子RNA。在植物中．miRNA通过对靶基因的剪切或沉默来实现对植物生命活动的调控,它是基因表达调控网络的重要组成部分。miR165／166（miR166）是陆生植物中最为古老的MIRNA家族之一,它通过对3型同源异域型-亮氨酸拉链（1id—ZIPⅢ）等靶标的调控,在植物的众多发育时期起着关键的调控作用。本文分析了MIR166基因在陆生植物中的进化关系,并对MIR166在基部陆生植物小立碗藓（Physcomitrella patens）中的复制及进化进行了研究。此外,HD—ZIPⅢ蛋白是植物中重要的一类转录因子,miR166对HD-ZIP Ⅲ基因的调控作用在陆地植物保守的存在,本文对HD—ZIP Ⅲ基因和miR166在进化中的相互作用进行了初步的探讨。     

Identification of small RNAs in late developmental stage of rice anthers

Small RNAs including microRNA (miRNA) and small interfering RNA (siRNA) are known as repressors of gene expression. There are many plant proteins involved in small RNA-mediated gene silencing, such as Dicer ribonucleases and RNA-dependent RNA polymerases. However, most of these proteins have been reported to be absent in the late developmental stage of the plant male gamete, pollen. In order to clarify the existence of the small RNAs during maturation of pollen, we cloned and sequenced small RNAs from rice anthers including tricellular pollen. From fifty six candidates of small RNAs, we identified two known miRNAs (miR166 and miR167), eight potential miRNAs, and ten putative heterochromatic siRNAs (hc-siRNAs). RNA gel blot analyses clearly showed that miR166 and miR167 were accumulated in the uninuclear pollen stage of anther development and remained until the tricellular pollen stage. Our cloning and RNA gel blot analyses of small RNAs led us to propose a possible function of small RNA-mediated gene regulation for the development of male gametes in rice.     

A microRNA derived from an apparent canonical biogenesis pathway regulates variant surface protein gene expression in Giardia lamblia

We have previously shown that a snoRNA-derived microRNA, miR2, in Giardia lamblia potentially regulates the expression of 22 variant surface protein (VSP) genes. Here, we identified another miRNA, miR4, also capable of regulating the expression of several VSPs but derived from an unannotated open reading frame (ORF) rather than a snoRNA, suggesting a canonical miRNA biogenesis pathway in Giardia. miR4 represses expression of a reporter containing two miR4 antisense sequences at the 3' UTR without causing a corresponding decrease in the mRNA level. This repression requires the presence of the Giardia Argonaute protein (GlAgo) and is reversed by 2' O-methylated antisense oligo to miR4, suggesting an RNA-induced silencing complex (RISC)-mediated mechanism. Furthermore, in vivo and in vitro evidence suggested that the Giardia Dicer protein (GlDcr) is required for miR4 biogenesis. Coimmunoprecipitation of miR4 with GlAgo further verified miR4 as a miRNA. A total of 361 potential target sites for miR4 were bioinformatically identified in Giardia, out of which 69 (32.7%) were associated with VSP genes. miR4 reduces the expression of a reporter containing two copies of the target site from VSP (GL50803_36493) at the 3' UTR. Sixteen of the 69 VSP genes were further found to contain partially overlapping miR2 and miR4 targeting sites. Expression of a reporter carrying the two overlapping sites was inhibited by either miR2 or miR4, but the inhibition was neither synergistic nor additive, suggesting a complex mechanism of miRNA regulation of VSP expression and the presence of a rich miRNAome in Giardia.     

miRNA profiling in leaf and cork tissues of Quercus suber reveals novel miRNAs and tissue-specific expression patterns

The differentiation of cork (phellem) cells from the phellogen (cork cambium) is a secondary growth process observed in the cork oak tree conferring a unique ability to produce a thick layer of cork. At present, the molecular regulators of phellem differentiation are unknown. The previously documented involvement of microRNAs (miRNAs) in the regulation of developmental processes, including secondary growth, motivated the search for these regulators in cork oak tissues. We performed deep sequencing of the small RNA fraction obtained from cork oak leaves and differentiating phellem. RNA sequences with lengths of 19–25 nt derived from the two libraries were analysed, leading to the identification of 41 families of conserved miRNAs, of which the most abundant were miR167, miR165/166, miR396 and miR159. Thirty novel miRNA candidates were also unveiled, 11 of which were unique to leaves and 13 to phellem. Northern blot detection of a set of conserved and novel miRNAs confirmed their differential expression profile. Prediction and analysis of putative miRNA target genes provided clues regarding processes taking place in leaf and phellem tissues, but further experimental work will be needed for functional characterization. In conclusion, we here provide a first characterization of the miRNA population in a Fagacea species, and the comparative analysis of miRNA expression in leaf and phellem libraries represents an important step to uncovering specific regulatory networks controlling phellem differentiation.     

Recovery of dicer-like 1-late flowering phenotype by miR172 expressed by the noncanonical DCL4-dependent biogenesis pathway

MicroRNAs (miRNAs) act as down-regulators of gene expression, and play a dominant role in eukaryote development. In Arabidopsis thaliana, DICER-LIKE 1 (DCL1) is the main processor in miRNA biogenesis, and dcl1 mutants show various developmental defects at the early stage of embryogenesis or at gamete formation. However, miRNAs responsible for the respective developmental stages of the dcl1 defects have not been identified. Here, we developed a DCL1-independent miRNA expression system using the unique DCL4-dependent miRNA, miR839. By replacing the mature sequence in the miR839 precursor sequence with that of miR172, one of the most widely conserved miRNAs in angiosperms, we succeeded in expressing miR172 from a chimeric miR839 precursor in dcl1-7 plants and observed the repression of miR172 target gene expression. In parallel, the DCL4-dependent miR172 expression rescued the late flowering phenotype of dcl1-7 by acceleration of flowering. We established the DCL1-independent miRNA expression system, and revealed that the reduction of miR172 expression is responsible for the dcl1-7 late flowering phenotype.     

A non-canonical plant microRNA target site

Plant microRNAs (miRNAs) typically form near-perfect duplexes with their targets and mediate mRNA cleavage. Here, we describe an unconventional miRNA target of miR398 in Arabidopsis, an mRNA encoding the blue copper-binding protein (BCBP). BCBP mRNA carries an miR398 complementary site in its 5′-untranslated region (UTR) with a bulge of six nucleotides opposite to the 5′ region of the miRNA. Despite the disruption of a target site region thought to be especially critical for function, BCBP mRNAs are cleaved by ARGONAUTE1 between nucleotides 10th and 11th, opposite to the miRNA, like conventional plant target sites. Levels of BCBP mRNAs are inversely correlated to levels of miR398 in mutants lacking the miRNA, or transgenic plants overexpressing it. Introducing two mutations that disrupt the miRNA complementarity around the cleavage site renders the target cleavage-resistant. The BCBP site functions outside of the context of the BCBP mRNA and does not depend on 5′-UTR location. Reducing the bulge does not interfere with miR398-mediated regulation and completely removing it increases the efficiency of the slicing. Analysis of degradome data and target predictions revealed that the miR398-BCBP interaction seems to be rather unique. Nevertheless, our results imply that functional target sites with non-perfect pairings in the 5′ region of an ancient conserved miRNA exist in plants.