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.

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 b-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.

The insulin-regulated glucose transporter GLUT4 is a key modulator of whole body glucose homeostasis, and its selective loss in adipose tissue or skeletal muscle causes insulin resistance and diabetes. Here we report an RNA interference-based screen of protein kinases expressed in adipocytes and identify four negative regulators of insulin-responsive glucose transport: the protein kinases PCTAIRE-1 (PCTK1), PFTAIRE-1 (PFTK1), I B kinase ␣, and MAP4K4͞NIK. Integrin-linked protein kinase was identified as a positive regulator of this process. We characterized one of these hits, MAP4K4͞NIK, and found that it is unique among mitogenactivated protein (MAP) kinases expressed in cultured adipocytes in attenuating hexose transport. Remarkably, MAP4K4͞NIK suppresses expression of the adipogenic transcription factors C͞EBP␣, C͞EBP␤, and PPAR␥ and of GLUT4 itself in these cells. RNA interference-mediated depletion of MAP4K4͞NIK early in differentiation enhances adipogenesis and triglyceride deposition, and even in fully differentiated adipocytes its loss up-regulates GLUT4. Conversely, conditions that inhibit adipogenesis such as TNF-␣ treatment or depletion of PPAR␥ markedly up-regulate MAP4K4͞ NIK expression in cultured adipocytes. Furthermore, TNF-␣ signaling to down-regulate GLUT4 is impaired in the absence of MAP4K4͞NIK, indicating that MAP4K4 expression is required for optimal TNF-␣ action. These results reveal a MAP4K4͞NIK-dependent signaling pathway that potently inhibits PPAR␥-responsive gene expression, adipogenesis, and insulin-stimulated glucose transport.GLUT4 function ͉ adipocyte differentiation ͉ protein kinase screening

MicroRNAs (miRNAs) are endogenous small RNAs of ~22 nucleotides (nt) that play a key role in down regulation of gene expression at the post-transcriptional level in plants and animals. Various studies have identified numerous miRNAs that were either up regulated or down regulated upon stress treatment. Here, we sought to understand the temporal regulation of miRNAs in different plant species under abscisic acid (ABA) and salt (NaCl) stress. Our results showed that the regulation of miR398 in response to ABA and salt stress was more dynamic in plants than previously reported. In poplars, miR398 was first induced upon 3-4 h of ABA or salt stress. However, this induction declined after 48 h and finally accumulated again over a prolonged stress (72 h). We referred to this kind of regulation as dynamic regulation. In contrast, such dynamic regulation of miR398 under salt stress was completely absent in Arabidopsis, in which miR398 was steadily and unidirectionally suppressed. Interestingly, ABA treatment caused a deviate dynamic regulation of miR398 in Arabidopsis, showing an opposite response as compared to that in poplars. We referred to the difference in regulation between Arabidopsis and poplars as differential regulation. Furthermore, the expression of the miR398 target, copper superoxide dismutase1 (CSD1), was in reverse correlation with the miR398 level, suggesting a control of this specific target expression predominantly by miR398 under abiotic stress. Together, these data consistently show a correlated regulation between miR398 and its representative target, CSD1, by ABA and salt stresses, and raise the possibility that regulation of miRNAs in plants is twofold: a dynamic regulation within a plant species and a differential regulation between different plant species.

Small RNAs are widespread in plants and animals. They largely include microRNAs (miRNAs) and short interfering RNAs (siRNAs), and they play key roles in gene and chromatin regulations. Here we describe in detail the method for an effective construction of the recently developed short tandem target mimic (STTM) technology to block small RNA functions in plants and animals. STTM is a powerful technology complementing the previous target mimic (TM) in plants and the miRNA sponge, as well as the recently defined endogenous competing RNA (CeRNA) in animals. We expect STTM will not only be effective in blocking small RNA functions in plants but will also become a popular approach in animals.