Structural features of microRNA (miRNA) precursors and their relevance to miRNA biogenesis and small interfering RNA/short hairpin RNA design

We have established the structures of 10 human microRNA (miRNA) precursors using biochemical methods. Eight of these structures turned out to be different from those that were computer-predicted. The differences localized in the terminal loop region and at the opposite side of the precursor hairpin stem. We have analyzed the features of these structures from the perspectives of miRNA biogenesis and active strand selection. We demonstrated the different thermodynamic stability profiles for pre-miRNA hairpins harboring miRNAs at their 5'- and 3'-sides and discussed their functional implications. Our results showed that miRNA prediction based on predicted precursor structures may give ambiguous results, and the success rate is significantly higher for the experimentally determined structures. On the other hand, the differences between the predicted and experimentally determined structures did not affect the stability of termini produced through "conceptual dicing." This result confirms the value of thermodynamic analysis based on mfold as a predictor of strand section by RNAi-induced silencing complex (RISC).     

A miRNA involved in phosphate-starvation response in Arabidopsis

Although microRNAs (miRNAs) have been documented to regulate development in plants and animals , the function of miRNAs in physiology is unclear. miR399 has multiple target sites in the 5' untranslated region (UTR) of a gene encoding a putative ubiquitin-conjugating enzyme (UBC) in Arabidopsis thaliana. We report here that miR399 was highly induced, whereas the target UBC mRNA was reduced by low-phosphate (Pi) stress. In transgenic plants with constitutive expression of miR399, UBC mRNA accumulation was suppressed even under high Pi. The expression of transgene UBC mRNA with 5' UTR miR399 target sites, but not the one without 5' UTR, was reduced under low-Pi condition. Furthermore, transgenic Arabidopsis plants with constitutive expression of miR399 accumulated more Pi than the wild-type, and transgenic plants expressing the UBC mRNA without 5' UTR (miRNA-deregulated) showed less inhibition of primary root growth and less induction of a Pi transporter gene by low-Pi stress than those of wild-type plants. We conclude that miR399 downregulates UBC mRNA accumulation by targeting the 5' UTR, and this regulation is important for plant responses to Pi starvation. The results suggest that miRNAs have functional roles for plants to cope with fluctuations in mineral-nutrient availability in the soil.     

拟南芥基因组中新的microRNA预测及分析

MicroRNA（miRNA）是一类存在于动植物体内、长度为21～25nt的内源性小RNA,对生物体的转录后基因调控起着关键作用,但一些低丰度的miRNA和组织特异性miRNA往往很难发现。为了系统识别拟南芥基因组中新的非同源miRNA,首先基于已报道的拟南芥miRNA的特征,从全基因组范围中筛选出453条可能的miRNA前体;其次,为了进一步对上述miRNA前体进行筛选,利用人的miRNA前体数据构建了支持向量机模型GenomicSVM,该模型对人测试集的敏感性和特异性分别为86.3％和98．1％（30个人miRNA前体和1000个阴性miRNA前体）,对拟南芥测试集的正确率为93．6％（78个miRNA前体）;最后,利用GenomicSVM预测上述453条miRNA前体序列,得到了37条候选的新的拟南芥miRNA前体,为进一步的miRNA实验发现研究提供了指导。     

The Influence of Secondary Processing on the Structural Relaxation Dynamics of Fluticasone Propionate

This study investigated the structural relaxation of micronized fluticasone propionate (FP) under different lagering conditions and its influence on aerodynamic particle size distribution (APSD) of binary and tertiary carrier-based dry powder inhaler (DPI) formulations. Micronized FP was lagered under low humidity (LH 25 C, 33% RH [relative humidity]), high humidity (HH 25°C, 75% RH) for 30, 60, and 90 days, respectively, and high temperature (HT 60°C, 44% RH) for 14 days. Physicochemical, surface interfacial properties via cohesive-adhesive balance (CAB) measurements and amorphous disorder levels of the FP samples were characterized. Particle size, surface area, and rugosity suggested minimal morphological changes of the lagered FP samples, with the exception of the 90-day HH (HH90) sample. HH90 FP samples appeared to undergo surface reconstruction with a reduction in surface rugosity. LH and HH lagering reduced the levels of amorphous content over 90-day exposure, which influenced the CAB measurements with lactose monohydrate and salmeterol xinafoate (SX). CAB analysis suggested that LH and HH lagering led to different interfacial interactions with lactose monohydrate but an increasing adhesive affinity with SX. HT lagering led to no detectable levels of the amorphous disorder, resulting in an increase in the adhesive interaction with lactose monohydrate. APSD analysis suggested that the fine particle mass of FP and SX was affected by the lagering of the FP. In conclusion, environmental conditions during the lagering of FP may have a profound effect on physicochemical and interfacial properties as well as product performance of binary and tertiary carrier-based DPI formulations.

Electronic supplementary material

The online version of this article (doi:10.1208/s12249-014-0222-8) contains supplementary material, which is available to authorized users.KEY WORDS: cohesive-adhesive balance, laagering, mechanical activation, particle adhesion, process-induced structural disorder     

Determinants of simulated RNA evolution

Models of RNA secondary structure folding are widely used to study evolution in theory and simulation. However, systematic studies of the parameters involved are rare. In this paper, we study by simulation how RNA evolution is influenced by three different factors, namely the mutation rate, scaling of the fitness function, and distance measure. We found that for low mutation rates the qualitative evolutionary behavior is robust with respect to the scaling of the fitness function. For efficient mutation rates, which are close to the error threshold, scaling and distance measure have a strong influence on the evolutionary behavior. A global distance measure that takes sequence information additively into account lowers the error threshold. When using a local sequence-structure alignment for the distance, we observed a smoother evolution of the fitness over time. Finally, in addition to the well known error threshold, we identify another threshold of the mutation rate, called divergence threshold, where the qualitative transient behavior changes from a localized to an exploratory search.     

Instability of miRNA and cDNAs derivatives in RNA preparations

Micro RNAs (miRNAs) are small RNA molecules, which function as important regulators of gene expression. We found that RNA preparation methods commonly utilized for miRNA expression studies yield highly unstable miRNAs. We studied the stability of four miRNAs belonging to different miRNAs families. A significant degradation of these molecules may be observed already three days after RNA isolation. Moreover, the respective cDNAs are highly unstable as well. Our findings indicate that instability of miRNAs and their cDNAs should be considered when designing miRNA expression studies.     

A Secondary Processing Site in the Precursor of the Small Subunit of Ribulose Bisphosphate Carboxylase of Chlamydomonas reinhardtii y-1

When the precursor of ribulose bisphosphate carboxylase of Chlamydomonas reinhardtii y-1 is bound to antibodies and treated with the soluble cell fraction, it is cleaved to the mature form (M_r 16,500) via an intermediate of M_r 18,500. Although this intermediate has only been observed in vitro, it may be produced during processing of the precursor in vivo.     

Cellular Membrane Fluidity in Amyloid Precursor Protein Processing

The senile plaque is a pathologic hallmark of Alzheimer's disease (AD). Amyloid-β peptide (Aβ), the main constituent of senile plaques, is neurotoxic especially in its oligomeric form. Aβ is derived from the sequential cleavage of amyloid precursor protein (APP) by β- and γ-secretases in the amyloidogenic pathway. Alternatively, APP can be cleaved by α-secretases within the Aβ domain to produce neurotrophic and neuroprotective α-secretase-cleaved soluble APP (sAPPα) in the nonamyloidogenic pathway. Since APP and α-, β-, and γ-secretases are membrane proteins, APP processing should be highly dependent on the membrane composition and the biophysical properties of cellular membrane. In this review, we discuss the role of the biophysical properties of cellular membrane in APP processing, especially the effects of phospholipases A₂ (PLA₂s), fatty acids, cholesterol, and Aβ on membrane fluidity in relation to their effects on APP processing.