Tandem repeats upstream of the Arabidopsis endogene SDC recruit non-CG DNA methylation and initiate siRNA spreading.

Plants use siRNAs to target cytosine DNA methylation to both symmetrical CG and nonsymmetrical (CHG and CHH) sequence contexts. DNA methylation and siRNA clusters most frequently overlap with transposons in the Arabidopsis thaliana genome. However, a significant number of protein-coding genes also show promoter DNA methylation, and this can be used to silence their expression. Loss of the majority of non-CG DNA methylation in drm1 drm2 cmt3 triple mutants leads to developmental phenotypes. We identified the gene responsible for these phenotypes as SUPPRESSOR OF drm1 drm2 cmt3 (SDC), which encodes an F-box protein and possesses seven promoter tandem repeats. The SDC repeats show a unique silencing requirement for non-CG DNA methylation directed redundantly by histone methylation and siRNAs, and display spreading of siRNAs and methylation beyond the repeated region. In addition to revealing the complexity of DNA methylation control in A. thaliana, SDC has important implications for how plant genomes utilize gene silencing to repress endogenous genes.

MicroRNAs and other small RNAs enriched in the Arabidopsis RNA-dependent RNA polymerase-2 mutant.

The Arabidopsis genome contains a highly complex and abundant population of small RNAs, and many of the endogenous siRNAs are dependent on RNA-Dependent RNA Polymerase 2 (RDR2) for their biogenesis. By analyzing an rdr2 loss-of-function mutant using two different parallel sequencing technologies, MPSS and 454, we characterized the complement of miRNAs expressed in Arabidopsis inflorescence to considerable depth. Nearly all known miRNAs were enriched in this mutant and we identified 13 new miRNAs, all of which were relatively low abundance and constitute new families. Trans-acting siRNAs (ta-siRNAs) were even more highly enriched. Computational and gel blot analyses suggested that the minimal number of miRNAs in Arabidopsis is approximately 155. The size profile of small RNAs in rdr2 reflected enrichment of 21-nt miRNAs and other classes of siRNAs like ta-siRNAs, and a significant reduction in 24-nt heterochromatic siRNAs. Other classes of small RNAs were found to be RDR2-independent, particularly those derived from long inverted repeats and a subset of tandem repeats. The small RNA populations in other Arabidopsis small RNA biogenesis mutants were also examined; a dcl2/3/4 triple mutant showed a similar pattern to rdr2, whereas dcl1-7 and rdr6 showed reductions in miRNAs and ta-siRNAs consistent with their activities in the biogenesis of these types of small RNAs. Deep sequencing of mutants provides a genetic approach for the dissection and characterization of diverse small RNA populations and the identification of low abundance miRNAs.

The hemodynamic effects of in-tandem carotid artery stenosis: implications for carotid endarterectomy.

OBJECTIVES: It remains controversial whether patients with severe disease of the internal carotid artery and a coexisting stenotic lesion downstream would benefit from a carotid endarterectomy (CEA) of the proximal lesion. The aim of this study was to simulate the hemodynamic and wall shear effects of in-tandem internal carotid artery stenosis using a computational fluid dynamic (CFD) idealized model to give insight into the possible consequences of CEA on these lesions. METHODS: A CFD model of steady viscous flow in a rigid tube with two asymmetric stenoses was introduced to simulate blood flow in arteries with multiple constrictions. The effect of varying the distance between the two stenoses, and the severity of the upstream stenosis on the pressure and wall shear stress (WSS) distributions on the second plaque, was investigated. The influence of the relative positions of the two stenoses was also assessed. RESULTS: The distance between the plaques was found to have minimal influence on the overall hemodynamic effect except for the presence of a zone of low WSS (range -20 to 30 dyne/cm2) adjacent to both lesions when the two stenoses were sufficiently close (<4 times the arterial diameter). The upstream stenosis was protective if it was larger than the downstream stenosis. The relative positions of the stenoses were found to influence the WSS but not the pressure distribution. CONCLUSIONS: The geometry and positions of the lesions need to be considered when considering the hemodynamic effects of an in-tandem stenosis. Low WSS is thought to cause endothelial dysfunction and initiate atheroma formation. The fact that there was a flow recirculation zone with low WSS in between the two stenoses may demonstrate how two closely positioned plaques may merge into one larger lesion. Decision making for CEA may need to take into account the hemodynamic situation when an in-tandem stenosis is found. CFD may aid in the risk stratification of patients with this problem.