The cytoplasmic-localized, cytoskeletal-associated RNA binding protein OsTudor-SN: evidence for an essential role in storage protein RNA transport and localization

Previous studies have demonstrated that the major storage protein RNAs found in the rice endosperm are transported as particles via actomyosin to specific subdomains of the cortical endoplasmic reticulum. In this study, we examined the potential role of Os Tudor-SN, a major cytoskeletal-associated RNA binding protein, in RNA transport and localization. Os Tudor-SN molecules occur as high-molecular-weight forms, the integrity of which are sensitive to RNase. Immunoprecipitation followed by RT-PCR showed that Os Tudor-SN binds prolamine and glutelin RNAs. Immunofluorescence studies using affinity-purified antibodies show that Os Tudor-SNs exists as particles in the cytoplasm, and are distributed to both the protein body endoplasmic reticulum (ER) and cisternal ER. Examination of Os Tudor-SN particles in transgenic rice plants expressing GFP-tagged prolamine RNA transport particles showed co-localization of Os Tudor-SN and GFP, suggesting a role in RNA transport. Consistent with this view, GFP-tagged Os Tudor-SN is observed in living endosperm sections as moving particles, a property inhibited by microfilament inhibitors. Downregulation of Os Tudor-SN by antisense and RNAi resulted in a decrease in steady state prolamine RNA and protein levels, and a reduction in the number of prolamine protein bodies. Collectively, these results show that Os Tudor-SN is a component of the RNA transport particle, and may control storage protein biosynthesis by regulating one or more processes leading to the transport, localization and anchoring of their RNAs to the cortical ER.     

Cytopathological evaluations combined RNA and protein analyses on defined cell regions using single frozen tissue block

The co-existence of multiple cell components in tissue samples is the main obstacle for precise molecular evaluation on defined cell types. Based on morphological examination, we developed an efficient approach for paralleled RNA and protein isolations from an identical histological region in frozen tissue section. The RNA and protein samples prepared were sufficient for RT-PCR and Western blot analyses, and the results obtained were well coincident each other as well as with the corresponding parameters revealed from immunohistochemical examinations. By this way, the sampling problem caused by cell-cross contamination can be largely avoided, committing the experimental data more specific to a denned cell type. These novel methods thus allow us to use single tissue block for a comprehensive study by integration of conventional cytological evaluations with nucleic acid and protein analyses.     

Mixed effects of long-term frozen storage on cord tissue stem cells

Background aimsCord tissue (CT) storage is promoted as an opportunity to preserve a source of mesenchymal stromal cells (MSCs) for future use. We analyzed maximal MSC yields from fresh and frozen CT including functional capacity after long-term cryopreservation as a means of assessing potential utility.MethodsCT was evaluated immediately upon harvest or frozen and banked for 5 years before analysis. Upon thawing, cell viability and yield were determined, as were growth characteristics and the ability to differentiate into various tissues. After thawing, enzymatic digestion of CT to release MSCs resulted in poor cell recoveries and few viable cells, requiring explant cultures to recover sufficient cell numbers for analysis. Upon expansion of surviving cells, fluorescence-activated cell sorter analysis showed the cells to be MSCs based on phenotype (CD34–, 45–, 44+, 90+, 105+) and function (ability to form adipocytes and osteoblasts). Frozen CT, however, exhibited decreased plating efficiency, increased doubling times but near equivalent maximum cell expansion, compared with fresh CT.ConclusionsPoor cell yields and recoveries, along with slower growth characteristics, make frozen CT a less-than-optimal choice for MSC banking, despite good functional recovery. In addition, because the amount of fresh CT available at birth is limited and total MSC yields are low, even fresh CT-MSC requires extensive in vitro expansion before clinical use, which limits it application.     

Mitochondrial dysfunction in human skeletal muscle biopsies of lipid storage disorder

Mitochondria regulate the balance between lipid metabolism and storage in the skeletal muscle. Altered lipid transport, metabolism and storage influence the bioenergetics, redox status and insulin signalling, contributing to cardiac and neurological diseases. Lipid storage disorders (LSD s) are neurological disorders which entail intramuscular lipid accumulation and impaired mitochondrial bioenergetics in the skeletal muscle causing progressive myopathy with muscle weakness. However, the mitochondrial changes including molecular events associated with impaired lipid storage have not been completely understood in the human skeletal muscle. We carried out morphological and biochemical analysis of mitochondrial function in muscle biopsies of human subjects with LSD s (n  = 7), compared to controls (n  = 10). Routine histology, enzyme histochemistry and ultrastructural analysis indicated altered muscle cell morphology and mitochondrial structure. Protein profiling of the muscle mitochondria from LSD samples (n  = 5) (vs. control, n  = 5) by high‐throughput mass spectrometric analysis revealed that impaired metabolic processes could contribute to mitochondrial dysfunction and ensuing myopathy in LSD s. We propose that impaired fatty acid and respiratory metabolism along with increased membrane permeability, elevated lipolysis and altered cristae entail mitochondrial dysfunction in LSD s. Some of these mechanisms were unique to LSD apart from others that were common to dystrophic and inflammatory muscle pathologies. Many differentially regulated mitochondrial proteins in LSD are linked with other human diseases, indicating that mitochondrial protection via targeted drugs could be a treatment modality in LSD and related metabolic diseases.

Cover Image for this Issue: doi: 10.1111/jnc.14177 .

     

植物组织RNA提取的难点及对策

酚类化合物、多糖、蛋白质和未知次级代谢产物是影响植物组织ＲＮＡ提取的几个主要干扰因素。本文对它们在植物ＲＮＡ提取过程中的干扰作用、现象以及消除它们的一些方法进行了综述。     

Comparison of frozen and RNALater solid tissue storage methods for use in RNA expression microarrays

Background  

Primary human tissues are an invaluable widely used tool for discovery of gene expression patterns which characterize disease States. Tissue processing methods remain unstandardized, leading to unanswered concerns of how to best store collected tissues and maintain reproducibility between laboratories. We subdivided uterine myometrial tissue specimens and stored split aliquots using the most common tissue processing methods (fresh, frozen, RNALater) before comparing quantitative RNA expression profiles on the Affymetrix U133 human expression array. Split samples and inclusion of duplicates within each processing group allowed us to undertake a formal genome-wide analysis comparing the magnitude of result variation contributed by sample source (different patients), processing protocol (fresh vs. frozen vs. 24 or 72 hours RNALater), and random background (duplicates). The dataset was randomly permuted to define a baseline pattern of ANOVA test statistic values against which the observed results could be interpreted.     

Biphasic folding kinetics of RNA pseudoknots and telomerase RNA activity

Using a combined master equation and kinetic cluster approach, we investigate RNA pseudoknot folding and unfolding kinetics. The energetic parameters are computed from a recently developed Vfold model for RNA secondary structure and pseudoknot folding thermodynamics. The folding kinetics theory is based on the complete conformational ensemble, including all the native-like and non-native states. The predicted folding and unfolding pathways, activation barriers, Arrhenius plots, and rate-limiting steps lead to several findings. First, for the PK5 pseudoknot, a misfolded 5' hairpin emerges as a stable kinetic trap in the folding process, and the detrapping from this misfolded state is the rate-limiting step for the overall folding process. The calculated rate constant and activation barrier agree well with the experimental data. Second, as an application of the model, we investigate the kinetic folding pathways for human telomerase RNA (hTR) pseudoknot. The predicted folding and unfolding pathways not only support the proposed role of conformational switch between hairpin and pseudoknot in hTR activity, but also reveal molecular mechanism for the conformational switch. Furthermore, for an experimentally studied hTR mutation, whose hairpin intermediate is destabilized, the model predicts a long-lived transient hairpin structure, and the switch between the transient hairpin intermediate and the native pseudoknot may be responsible for the observed hTR activity. Such finding would help resolve the apparent contradiction between the observed hTR activity and the absence of a stable hairpin.     

High‐resolution mapping of function and protein binding in an RNA nuclear enrichment sequence

The functions of long RNAs, including mRNAs and long noncoding RNAs (lncRNAs), critically depend on their subcellular localization. The identity of the sequences that dictate subcellular localization and their high‐resolution anatomy remain largely unknown. We used a suite of massively parallel RNA assays and libraries containing thousands of sequence variants to pinpoint the functional features within the SIRLOIN element, which dictates nuclear enrichment through hnRNPK recruitment. In addition, we profiled the endogenous SIRLOIN RNA‐nucleoprotein complex and identified the nuclear RNA‐binding proteins SLTM and SNRNP70 as novel SIRLOIN binders. Taken together, using massively parallel assays, we identified the features that dictate binding of hnRNPK, SLTM, and SNRNP70 to SIRLOIN and found that these factors are jointly required for SIRLOIN activity. Our study thus provides a roadmap for high‐throughput dissection of functional sequence elements in long RNAs.     

Effect of frozen storage and aging on the Kashkaval cheese starter culture

Summary The changes in the number of the starter microorganisms Lb. delbrueckii subsp. bulgaricus and Str. thermophiluswere followed in frozen-stored Kashkaval cheese made from cow’s milk. Kashkaval samples of various aging times were produced industrially, frozen at T=−16 °C and stored at T=−10 to −12 °C for 12 months. It was found that the number of Lb. delbrueckiisubsp. bulgaricus and Str. thermophilusdecreased considerably during frozen storage. The decrease was more substantial for Lb. delbrueckiisubsp. bulgaricus, which was evidence for its greater sensitivity to the impact of low temperatures. The aging time of Kashkaval did not influence the changes in the starter culture during frozen storage but is important for its amount in the product aged after defrosting. There was an increase in the Str. thermophilus: Lb. delbrueckiisubsp. bulgaricus ratio in samples with shorter aging time subjected to frozen storage and aged after defrosting. The changes in the starter culture in frozen stored Kashkaval cheese can be controlled by an appropriate combination of the two factors: aging time and period of frozen storage.     

Correlation between seed longevity and RNA integrity in the embryos of rice seeds

The mature embryos of rice seeds contain translatable mRNAs required for the initial phase of germination. To clarify the relationship between seed longevity and RNA integrity in embryos, germinability and stability of embryonic RNAs were analyzed using the seeds of japonica rice cultivars subjected to controlled deterioration treatment (CDT) or long periods of storage. Degradation of RNA from embryos of a japonica rice cultivar “Nipponbare” was induced by CDT before the decline of the germination rate and we observed a positive relationship between seed germinability and integrity of embryonic RNAs. Moreover, this relationship was confirmed in the experiments using aged seeds from the “Nipponbare”, “Sasanishiki” and “Koshihikari” rice cultivars. In addition, the RNA integrity number (RIN) values, calculated using electrophoresis data and Agilent Bioanalyzer software, had a positive correlation with germinability (R²=0.75). Therefore, the stability of embryonic RNAs required for germination is involved in maintaining seed longevity over time and RIN values can serve as a quantitative indicator to evaluate germinability in rice.     

Packing them up and dusting them off: RNA helicases and mRNA storage

Cytoplasmic mRNA can be translated, translationally repressed, localized or degraded. Regulation of translation is an important step in control of gene expression and the cell can change whether and to what extent an mRNA is translated. If an mRNA is not translating, it will associate with translation repression factors; the mRNA can be stored in these non-translating states. The movement of mRNA into storage and back to translation is dictated by the recognition of the mRNA by trans factors. So, remodeling the factors that bind mRNA is critical for changing the fate of mRNA. RNA helicases, which have the ability to remodel RNA or RNA–protein complexes, are excellent candidates for facilitating such rearrangements. This review will focus on the RNA helicases implicated in translation repression and/or mRNA storage and how their study has illuminated mechanisms of mRNA regulation. This article is part of a Special Issue entitled: The Biology of RNA helicases — Modulation for life.     

Cycling of the Sm-like protein Hfq on the DsrA small regulatory RNA

Small RNAs (sRNAs) regulate bacterial genes involved in environmental adaptation. This RNA regulation requires Hfq, a bacterial Sm-like protein that stabilizes sRNAs and enhances RNA-RNA interactions. To understand the mechanism of target recognition by sRNAs, we investigated the interactions between Hfq, the sRNA DsrA, and its regulatory target rpoS mRNA, which encodes the stress response sigma factor. Nuclease footprinting revealed that Hfq recognized multiple sites in rpoS mRNA without significantly perturbing secondary structure in the 5' leader that inhibits translation initiation. Base-pairing with DsrA, however, made the rpoS ribosome binding site fully accessible, as predicted by genetic data. Hfq bound DsrA four times more tightly than the DsrA.rpoS RNA complex in gel mobility-shift assays. Consequently, Hfq is displaced rapidly from its high-affinity binding site on DsrA by conformational changes in DsrA, when DsrA base-pairs with rpoS mRNA. Hfq accelerated DsrA.rpoS RNA association and stabilized the RNA complex up to twofold. Hybridization of DsrA and rpoS mRNA was optimal when Hfq occupied its primary binding site on free DsrA, but was inhibited when Hfq associated with the DsrA.rpoS RNA complex. We conclude that recognition of rpoS mRNA is stimulated by binding of Hfq to free DsrA sRNA, followed by release of Hfq from the sRNA.mRNA complex.     

基于细菌体系生产双链RNA的条件优化

化学杀虫剂是农业害虫防治的主要手段,然而农业害虫已对化学杀虫剂产生了抗药性。RNA农药的应用将是减少使用化学杀虫剂的一种途径。RNA农药具有专一、高效、易降解和对环境友好等优点而受到了关注,但在靶标分子、靶标分子的双链RNA（dsRNA）生产工艺和应用制剂等方面仍需进一步研究。本研究以褐飞虱Nilaparvata lugens蛋白激酶B基因的dsRNA（dsNlAKT）为例,对利用细菌体系生产dsRNA的方法进行了探究。将NlAKT构建进L4440载体后,将其转化到缺乏核酸酶（RNase Ⅲ）的大肠杆菌Escherichia coli HT115（DE3）中,进行了dsRNA生产条件的测试。结果表明：（1）当异丙基-β-D-硫代半乳糖苷（IPTG）工作浓度为0.1 mM或0.5 mM时dsRNA的产量没有明显变化,而当其工作浓度增加至1 mM时dsRNA产量明显减少;（2）在IPTG诱导时间为2~8 h范围内,添加IPTG后诱导6 h获得的dsRNA产量最多;（3）在实验室常规提取RNA方法中,增加低剂量溶菌酶预处理这一步骤可增加提取产物中dsRNA的含量。试验结果证明了采用工作浓度为0.1 mM的IPTG诱导6 h的生产条件,并在提取过程中增加溶菌酶的预处理步骤有助于获得较高的dsRNA含量。研究结果为RNA农药的生产条件提供了实验依据。     

Lectin histochemistry of glycolipid storage diseases on frozen and paraffin-embedded tissue sections

Lectin histochemical studies were performed on frozen and paraffin-embedded brain tissue sections from six cases of galactosylceramide lipidosis (i.e., globoid cell leukodystrophy, or Krabbe's disease) in Twitcher mice and one case of canine infantile GM1-gangliosidosis. The globoid cells in Krabbe's disease stained with Ricinus communis agglutinin-I (RCA-I), peanut agglutinin (PNA), and Bandeirea simplicifolia agglutinin-I (BS-I) in frozen sections. However, paraffin sections and frozen sections pretreated with chloroform-methanol or xylene, from the same animals, stained with Concanavlia ensiformis agglutinin (ConA), wheat germ agglutinin (WGA), and succinylated-WGA (S-WGA), in addition to staining with RCA-I, PNA, and BS-I. The affected neurons of canine infantile GM1-gangliosidosis stained only with RCA-I in frozen sections. In paraffin sections, however, these cells were negative with RCA-I but positive with BS-I, ConA, Dolichos biflorus agglutinin (DBA), soybean agglutinin (SBA) and Ulex europaeus agglutinin (UEA-I) in paraffin sections. These results indicate that in paraffin processing of glycolipid storage disease tissue, some lectin receptors are lost and others are unmasked. The retained receptors can be stained with specific lectins and could serve as markers to characterize and differentiate among the various glycolipid storage diseases.     

The origin of life: RNA and protein co-evolution on the ancient Earth

How life emerged from simple non-life chemicals on the ancient Earth is one of the greatest mysteries in biology. The gene expression system of extant life is based on the interdependence between multiple molecular species (DNA, RNA, and proteins). While DNA is mainly used as genetic material and proteins as functional molecules in modern biology, RNA serves as both genetic material and enzymes (ribozymes). Thus, the evolution of life may have begun with the birth of a ribozyme that replicated itself (the RNA world hypothesis), and proteins and DNA joined later. However, the complete self-replication of ribozymes from monomeric substrates has not yet been demonstrated experimentally, due to their limited activity and stability. In contrast, peptides are more chemically stable and are considered to have existed on the ancient Earth, leading to the hypothesis of RNA–peptide co-evolution from the very beginning. Our group and collaborators recently demonstrated that (1) peptides with both hydrophobic and cationic moieties (e.g., KKVVVVVV) form β-amyloid aggregates that adsorb RNA and enhance RNA synthesis by an artificial RNA polymerase ribozyme and (2) a simple peptide with only seven amino acid types (especially rich in valine and lysine) can fold into the ancient β-barrel conserved in various enzymes, including the core of cellular RNA polymerases. These findings, together with recent reports from other groups, suggest that simple prebiotic peptides could have supported the ancient RNA-based replication system, gradually folded into RNA-binding proteins, and eventually evolved into complex proteins like RNA polymerase.