Guiliang Tang

MicroRNA control of PHABULOSA in leaf development: importance of pairing to the microRNA 5¿ region

MicroRNAs (miRNAs) are ∼22‐nucleotide noncoding RNAs that can regulate gene expression by directing mRNA degradation or inhibiting productive translation. Dominant mutations in PHABULOSA (PHB) and PHAVOLUTA (PHV) map to a miR165/166 complementary site and impair miRNA‐guided cleavage of these mRNAs in vitro. Here, we confirm that disrupted miRNA pairing, not changes in PHB protein sequence, causes the developmental defects in phb‐d mutants. In planta, disrupting miRNA pairing near the center of the miRNA complementary site had far milder developmental consequences than more distal mismatches. These differences correlated with differences in miRNA‐directed cleavage efficiency in vitro, where mismatch scanning revealed more tolerance for mismatches at the center and 3′ end of the miRNA compared to mismatches to the miRNA 5′ region. In this respect, miR165/166 resembles animal miRNAs in its pairing requirements. Pairing to the 5′ portion of the small silencing RNA appears crucial regardless of the mode of post‐transcriptional repression or whether it occurs in plants or animals, supporting a model in which this region of the silencing RNA nucleates pairing to its target.

The Expression of MicroRNA miR-107 Decreases Early in Alzheimer's Disease and May Accelerate Disease Progression through Regulation of β-Site Amyloid Precursor Protein-Cleaving Enzyme 1

MicroRNAs (miRNAs) are small regulatory RNAs that participate in posttranscriptional gene regulation in a sequence-specific manner. However, little is understood about the role(s) of miRNAs in Alzheimers disease (AD). We used miRNA expression microarrays on RNA extracted from human brain tissue from the University of Kentucky Alzheimers Disease Center Brain Bank with near-optimal clinicopathological correlation. Cases were separated into four groups: elderly nondemented with negligible AD-type pathology, nondemented with incipient AD pathology, mild cognitive impairment (MCI) with moderate AD pathology, and AD. Among the AD-related miRNA expression changes, miR-107 was exceptional because miR-107 levels decreased significantly even in patients with the earliest stages of pathology. In situ hybridization with cross-comparison to neuropathology demonstrated that particular cerebral cortical laminas involved by AD pathology exhibit diminished neuronal miR-107 expression. Computational analysis predicted that the 3′-untranslated region (UTR) of β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) mRNA is targeted multiply by miR-107. From the same RNA material analyzed on miRNA microarrays, mRNA expression profiling was performed using Affymetrix Exon Array microarrays on nondemented, MCI, and AD patients. BACE1 mRNA levels tended to increase as miR-107 levels decreased in the progression of AD. Cell culture reporter assays performed with a subset of the predicted miR-107 binding sites indicate the presence of at least one physiological miR-107 miRNA recognition sequence in the 3′-UTR of BACE1 mRNA. Together, the coordinated application of miRNA profiling, Affymetrix microarrays, new bioinformatics predictions, in situ hybridization, and biochemical validation indicate that miR-107 may be involved in accelerated disease progression through regulation of BACE1.

Identification of glucose-regulated miRNAs from pancreatic β cells reveals a role for miR-30d in insulin transcription

MicroRNAs (miRNAs) are small noncoding ribonucleotides that bind mRNAs and function mainly as translational repressors in mammals. MicroRNAs have been implicated to play a role in many diseases, including diabetes. Several reports indicate an important function for miRNAs in insulin production as well as insulin secretion. We have recently carried out a screen in the pancreatic beta-cell line MIN6 to identify miRNAs with altered abundance in response to changes in glucose concentrations. This screen resulted in identification of 61 glucose-regulated miRNAs from a total of 108 miRNAs detectable in MIN6 cells. Many of the identified miRNAs, including miR-124a, miR-107, and miR-30d were up-regulated in the presence of high glucose. Only a few of the miRNAs, including miR-296, miR-484, and miR-690 were significantly down-regulated by high glucose treatment. Interestingly, we found that overexpression of miR-30d, one of the miRNAs up-regulated by glucose, increased insulin gene expression, while inhibition of miR-30d abolished glucose-stimulated insulin gene transcription. Overexpression or inhibition of miR-30d did not have any effect on insulin secretion. These data suggest that the putative target genes of miR-30d may be negative regulators of insulin gene expression.

Effective Small RNA Destruction by the Expression of a Short Tandem Target Mimic in Arabidopsis

This work presents a technology for effectively silencing endogenous small RNAs by expressing a small tandem target mimic (STTM) composed of two noncleavable small RNA binding sites linked by an empirically determined spacer. Expression of STTM in Arabidopsis thaliana leads to the specific degradation of endogenous small RNAs by small RNA degrading nuclease family enzymes. MicroRNAs (miRNAs) and other endogenous small RNAs act as sequence-specific regulators of the genome, transcriptome, and proteome in eukaryotes. The interrogation of small RNA functions requires an effective, widely applicable method to specifically block small RNA function. Here, we report the development of a highly effective technology that targets specific endogenous miRNAs or small interfering RNAs for destruction in Arabidopsis thaliana. We show that the expression of a short tandem target mimic (STTM), which is composed of two short sequences mimicking small RNA target sites, separated by a linker of an empirically determined optimal size, leads to the degradation of targeted small RNAs by small RNA degrading nucleases. The efficacy of the technology was demonstrated by the strong and specific developmental defects triggered by STTMs targeting three miRNAs and an endogenous siRNA. In summary, we developed an effective approach for the destruction of endogenous small RNAs, thereby providing a powerful tool for functional genomics of small RNA molecules in plants and potentially animals.

ARGONAUTE10 and ARGONAUTE1 Regulate the Termination of Floral Stem Cells Through Two MicroRNAs in Arabidopsis

Stem cells are crucial in morphogenesis in plants and animals. Much is known about the mechanisms that maintain stem cell fates or trigger their terminal differentiation. However, little is known about how developmental time impacts stem cell fates. Using Arabidopsis floral stem cells as a model, we show that stem cells can undergo precise temporal regulation governed by mechanisms that are distinct from, but integrated with, those that specify cell fates. We show that two microRNAs, miR172 and miR165/166, through targeting APETALA2 and type III homeodomain-leucine zipper (HD-Zip) genes, respectively, regulate the temporal program of floral stem cells. In particular, we reveal a role of the type III HD-Zip genes, previously known to specify lateral organ polarity, in stem cell termination. Both reduction in HD-Zip expression by over-expression of miR165/166 and mis-expression of HD-Zip genes by rendering them resistant to miR165/166 lead to prolonged floral stem cell activity, indicating that the expression of HD-Zip genes needs to be precisely controlled to achieve floral stem cell termination. We also show that both the ubiquitously expressed ARGONAUTE1 (AGO1) gene and its homolog AGO10, which exhibits highly restricted spatial expression patterns, are required to maintain the correct temporal program of floral stem cells. We provide evidence that AGO10, like AGO1, associates with miR172 and miR165/166 in vivo and exhibits “slicer” activity in vitro. Despite the common biological functions and similar biochemical activities, AGO1 and AGO10 exert different effects on miR165/166 in vivo. This work establishes a network of microRNAs and transcription factors governing the temporal program of floral stem cells and sheds light on the relationships among different AGO genes, which tend to exist in gene families in multicellular organisms.

In vitro analysis of RNA interference in Drosophila melanogaster

Double-stranded RNA (dsRNA) triggers the destruction of mRNA sharing sequence with the dsRNA, a phenomenon termed RNA interference (RNAi). The dsRNA is converted by endonucleolytic cleavage into 21- to 23-nt small interfering RNAs (siRNAs), which direct a multiprotein complex, the RNA-induced silencing complex to cleave RNA complementary to the siRNA. RNAi can be recapitulated in vitro in lysates of syncytial blastoderm Drosophila embryos. These lysates reproduce all of the known steps in the RNAi pathway in flies and mammals. Here we explain how to prepare and use Drosophila embryo lysates to dissect the mechanism of RNAi.

Lysine catabolism: a stress and development super-regulated metabolic pathway

Lysine is a nutritionally important essential amino acid whose level in plants is largely regulated by the rate of its synthesis. In some plant tissues and under some stress conditions, however, lysine is also efficiently catabolized into glutamate and several other stress-related metabolites by novel mechanisms of metabolic regulation. Lysine catabolism is important for mammalian brain function; it is possible that the generation of glutamate regulates nerve transmission signals via glutamate receptors. Plants also possess homologues of animal glutamate receptors. It is thus likely that lysine catabolism also regulates various plant processes via these receptors.

Role of microRNAs in diabetes.

Diabetes is one of the most common chronic diseases in the world. Multiple and complex factors including various genetic and physiological changes can lead to type 1 and type 2 diabetes. However, the major mechanisms underlying the pathogenesis of diabetes remain obscure. With the recent discovery of microRNAs (miRNAs), these small ribonucleotides have been implicated as new players in the pathogenesis of diabetes and diabetes-associated complications. MiRNAs have been shown to regulate insulin production, insulin secretion, and insulin action. This review summarizes the recent progress in the cutting-edge research of miRNAs involved in diabetes and diabetes related complications.

Expression analysis suggests potential roles of microRNAs for phosphate and arbuscular mycorrhizal signaling in Solanum lycopersicum

MicroRNAs (miRNAs) have emerged as a class of gene expression regulators that play crucial roles in many biological processes. Recently, several reports have revealed that micoRNAs participate in regulation of symbiotic interaction between plants and nitrogen-fixing rhizobia bacteria. However, the role of miRNAs in another type of plant-microbe interaction, arbuscular mycorrhizal (AM) symbiosis, has not been documented. We carried out a microarray screen and poly(A)-tailed reverse transcriptase-polymerase chain reaction (RT-PCR) validation for miRNA expression in tomato (Solanum lycopersicum) under varying phosphate (Pi) availability and AM symbiosis conditions. In roots, miRNA158, miRNA862-3p, miRNA319, miRNA394 and miR399 were differentially regulated under three different treatments, Pi sufficient (+P ), Pi deficient (-P) and AM symbiosis (+M ). In leaves, up to 14 miRNAs were up- or down-regulated under either or both of the Pi treatments and AM symbiosis, of which miR158, miR319 and miR399 were responsive to the treatments in both roots and leaves. We detected that miR395, miR779.1, miR840 and miR867 in leaves were specifically responsive to AM symbiosis, which is independent of Pi availability, whereas miR398 in leaves and miR399 in both roots and leaves were Pi starvation induced. Furthermore, miR158 in roots as well as miR837-3p in leaves were responsive to both Pi deprivation and AM colonization. In contrast, miR862-3p in roots was responsive to Pi nutrition, but not to AM symbiosis. Moreover, the group of miRNA consisting miR319 and miR394 in roots and miR158, miR169g*, miR172, miR172b*, miR319, miR771 and miR775 in leaves were up- and down-regulated by Pi starvation, respectively. The data suggest that altered expression of distinct groups of miRNA is an essential component of Pi starvation-induced responses and AM symbiosis.

UV-B-responsive microRNAs in Populus tremula

MicroRNAs (miRNAs) play vital roles in down-regulating gene expression at the post-transcriptional level. A set of 24 UV-B stress-responsive miRNAs (13 up-regulated and 11 down-regulated) was identified in Populus tremula plantlet by expression profiling with our in-house miRNA filter array. Six of the UV-B-responsive miRNA and their corresponding target genes were verified for their expressions by RNA blotting and quantitative reverse transcription PCR (qRT-PCR), respectively. The predicted target genes for these miRNAs encode diverse proteins including transcription factors and phytohormone signal-related proteins. Promoter analysis of the UV-B-responsive miRNAs revealed the presence of many light-relevant cis-elements. However, these cis-elements were not necessarily specific to the promoters of UV-responsive miRNAs, indicating that other machinery may be involved in the regulation of UV-responsive miRNAs. Finally, a model was developed to describe the potential regulatory networks mediated by the UV-B-responsive miRNAs in P. tremula. These results provide new insights into the understanding of miRNAs as ubiquitous regulators in plant response to UV-B and other stresses.