CARPEL FACTORY,a Dicer homolog,and HEN1, a novel protein,act in microRNA metabolism in Arabidopsis thaliana

BACKGROUND: In metazoans, microRNAs, or miRNAs, constitute a growing family of small regulatory RNAs that are usually 19-25 nucleotides in length. They are processed from longer precursor RNAs that fold into stem-loop structures by the ribonuclease Dicer and are thought to regulate gene expression by base pairing with RNAs of protein-coding genes. In Arabidopsis thaliana, mutations in CARPEL FACTORY (CAF), a Dicer homolog, and those in a novel gene, HEN1, result in similar, multifaceted developmental defects, suggesting a similar function of the two genes, possibly in miRNA metabolism.RESULTS: To investigate the potential functions of CAF and HEN1 in miRNA metabolism, we aimed to isolate miRNAs from Arabidopsis and examine their accumulation during plant development in wild-type plants and in hen1-1 and caf-1 mutant plants. We have isolated 11 miRNAs, some of which have potential homologs in tobacco, rice, and maize. The putative precursors of these miRNAs have the capacity to form stable stem-loop structures. The accumulation of these miRNAs appears to be spatially or temporally controlled in plant development, and their abundance is greatly reduced in caf-1 and hen1-1 mutants. HEN1 homologs are found in bacterial, fungal, and metazoan genomes.CONCLUSIONS: miRNAs are present in both plant and animal kingdoms. An evolutionarily conserved mechanism involving a protein, known as Dicer in animals and CAF in Arabidopsis, operates in miRNA metabolism. HEN1 is a new player in miRNA accumulation in Arabidopsis, and HEN1 homologs in metazoans may have a similar function. The developmental defects associated with caf-1 and hen1-1 mutations and the patterns of miRNA accumulation suggest that miRNAs play fundamental roles in plant development.     

Gene Silencing Efficiency and INF-β Induction Effects of Splicing miRNA 155-Based Artificial miRNA with Pre-miRNA Stem-Loop Structures

Artificial microRNA (miRNA) expression vectors have been developed and used for RNA interference. The secondary structure of artificial miRNA is important for RNA interference efficacy. We designed two groups of six artificial splicing miRNA 155-based miRNAs (SM155-based miRNAs) with the same target in the coding region or 3' UTR of a target gene and studied their RNA silencing efficiency and interferon β (IFN-β) induction effects. SM155-based miRNA with a mismatch at the +1 position and a bulge at the +11, +12 positions in a miRNA precursor stem-loop structure showed the highest gene silencing efficiency and lowest IFN-β induction effect (increased IFN-β mRNA level by 10% in both target cases), regardless of the specificity of the target sequence, suggesting that pSM155-based miRNA with this design could be a valuable miRNA expression vector.     

Genome-Wide Identification of Reverse Complementary microRNA Genes in Plants

MicroRNAs (miRNAs) are ∼21-nucleotide small RNAs (sRNAs) with essential regulatory roles in plants. They are generated from stem-loop-structured precursors through two sequential Dicer-like 1 (DCL1)-mediated cleavages. To date, hundreds of plant miRNAs have been uncovered. However, the question, whether the sequences reverse complementary (RC) to the miRNA precursors could form hairpin-like structures and produce sRNA duplexes similar to the miRNA/miRNA* pairs has not been solved yet. Here, we interrogated this possibility in 16 plant species based on sRNA high-throughput sequencing data and secondary structure prediction. A total of 59 RC sequences with great potential to form stem-loop structures and generate miRNA/miRNA*-like duplexes were identified in ten plants, which were named as RC-miRNA precursors. Unlike the canonical miRNAs, only a few cleavage targets of the RC-miRNAs were identified in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), and none in Soybean (Glycine max) based on degradome data. Surprisingly, the genomic regions surrounding some of the RC-miRNA target recognition sites were observed to be specifically methylated in both Arabidopsis and rice. Taken together, we reported a new class of miRNAs, called RC-miRNAs, which were generated from the antisense strands of the miRNA precursors. Based on the results, we speculated that the mature RC-miRNAs might have subtle regulatory activity through target cleavages, but might possess short interfering RNA-like activity by guiding sequence-specific DNA methylation.     

玉米microRNAs及其靶基因的生物信息学预测

microRNAs (miRNAs) 是一类非编码的小分子RNA, 通过碱基互补调控靶基因的表达。鉴定和发现新的miRNAs及其靶基因, 对揭示miRNAs在基因表达调控中的作用至关重要。玉米全基因组测序工作开展较晚, 已经鉴定登记的miRNAs很少, 对靶基因的调控作用尚待解明。文章根据miRNA进化上的保守性, 以已知的植物miRNAs为探针, 与相关数据库中玉米表达序列标签(EST)和基因组序列(GSS)中的非编码序列比对, 共发现11个新的miRNA前体。虽然在序列长度和二级结构方面各有变化, 但这11个前体均可折叠形成miRNA家族的标准二级结构。通过靶基因预测, 找到其中7条miRNAs的26个靶基因, 分别编码与新陈代谢、信号转导、转录调节、跨膜运输、生物和非生物胁迫及叶绿体组装等相关的蛋白。这些miRNAs及其靶基因的鉴定, 补充了miRNA数据库的不足。