The MuvB complex sequentially recruits B-Myb and FoxM1 to promote mitotic gene expression.

Cell cycle progression is dependent on two major waves of gene expression. Early cell cycle gene expression occurs during G1/S to generate factors required for DNA replication, while late cell cycle gene expression begins during G2 to prepare for mitosis. Here we demonstrate that the MuvB complex-comprised of LIN9, LIN37, LIN52, LIN54, and RBBP4-serves an essential role in three distinct transcription complexes to regulate cell cycle gene expression. The MuvB complex, together with the Rb-like protein p130, E2F4, and DP1, forms the DREAM complex during quiescence and represses expression of both early and late genes. Upon cell cycle entry, the MuvB complex dissociates from p130/DREAM, binds to B-Myb, and reassociates with the promoters of late genes during S phase. MuvB and B-Myb are required for the subsequent recruitment of FoxM1 to late gene promoters during G2. The MuvB complex remains bound to FoxM1 during peak late cell cycle gene expression, while B-Myb binding is lost when it undergoes phosphorylation-dependent, proteasome-mediated degradation during late S phase. Our results reveal a novel role for the MuvB complex in recruiting B-Myb and FoxM1 to promote late cell cycle gene expression and in regulating cell cycle gene expression from quiescence through mitosis.

Distinct argonaute-mediated 22G-RNA pathways direct genome surveillance in the C. elegans germline.

Endogenous small RNAs (endo-siRNAs) interact with Argonaute (AGO) proteins to mediate sequence-specific regulation of diverse biological processes. Here, we combine deep-sequencing and genetic approaches to explore the biogenesis and function of endo-siRNAs in C. elegans. We describe conditional alleles of the Dicer-related helicase, drh-3, that abrogate both RNA interference and the biogenesis of endo-siRNAs, called 22G-RNAs. DRH-3 is a core component of RNA-dependent RNA polymerase (RdRP) complexes essential for several distinct 22G-RNA systems. We show that, in the germline, one system is dependent on worm-specific AGOs, including WAGO-1, which localizes to germline nuage structures called P granules. WAGO-1 silences certain genes, transposons, pseudogenes, and cryptic loci. Finally, we demonstrate that components of the nonsense-mediated decay pathway function in at least one WAGO-mediated surveillance pathway. These findings broaden our understanding of the biogenesis and diversity of 22G-RNAs and suggest additional regulatory functions for small RNAs.

PRG-1 and 21U-RNAs interact to form the piRNA complex required for fertility in C. elegans.

In metazoans, Piwi-related Argonaute proteins have been linked to germline maintenance, and to a class of germline-enriched small RNAs termed piRNAs. Here we show that an abundant class of 21 nucleotide small RNAs (21U-RNAs) are expressed in the C. elegans germline, interact with the C. elegans Piwi family member PRG-1, and depend on PRG-1 activity for their accumulation. The PRG-1 protein is expressed throughout development and localizes to nuage-like structures called P granules. Although 21U-RNA loci share a conserved upstream sequence motif, the mature 21U-RNAs are not conserved and, with few exceptions, fail to exhibit complementarity or evidence for direct regulation of other expressed sequences. Our findings demonstrate that 21U-RNAs are the piRNAs of C. elegans and link this class of small RNAs and their associated Piwi Argonaute to the maintenance of temperature-dependent fertility.

Next-generation sequencing and microarray-based interrogation of microRNAs from formalin-fixed, paraffin-embedded tissue: preliminary assessment of cross-platform concordance.

Next-generation sequencing is increasingly employed in biomedical investigations. Strong concordance between microarray and mRNA-seq levels has been reported in high quality specimens but information is lacking on formalin-fixed, paraffin-embedded (FFPE) tissues, and particularly for microRNA (miRNA) analysis. We conducted a preliminary examination of the concordance between miRNA-seq and cDNA-mediated annealing, selection, extension, and ligation (DASL) miRNA assays. Quantitative agreement between platforms is moderate (Spearman correlation 0.514-0.596) and there is discordance of detection calls on a subset of miRNAs. Quantitative PCR (q-RT-PCR) performed for several discordant miRNAs confirmed the presence of most sequences detected by miRNA-seq but not by DASL but also that miRNA-seq did not detect some sequences, which DASL confidently detected. Our results suggest that miRNA-seq is specific, with few false positive calls, but it may not detect certain abundant miRNAs in FFPE tissue. Further work is necessary to fully address these issues that are pertinent for translational research.

Research on the localized lightfield features of metallic nano-cone-tip optical antenna via investigating near-field lightwave and correlated net-charge distribution.

In this paper, the near-field lightwave characteristics of an arrayed silicon nano-cone-tip optical antenna (NOA) covered by a common metal film, which can be viewed as a featured quasi quantum dot (QD), are carefully investigated. A dipole net-charge distribution closely correlated with the surface lightwaves excited over the antennas by incident lasers with a central wavelength of 633 nm, is clearly observed. An obvious Coulomb-like blockade from the apex apparently influencing the net-charge converging over the surface of NOA, is verified, which can also be predicted by the simulations according to surface standing waves across the apex node. The antinodes of the surface net-charge instantaneous distribution are already pushed away from the normal location owing to the apex Coulomb-like blockade, so as to present a distorted waveform different from traditional standing wave modes. The tip proximity effect leading to a relatively weak net-charge converging over surrounding planar facet and adjacent NOAs, is also discovered.

Expression of bone morphogenetic protein 10 and its role in biomineralization in Hyriopsis cumingii.

Shells and pearls are the products of biomineralization of shellfish after ingesting external mineral ions. Bone morphogenetic proteins (BMPs) play a role in a variety of biological function, and the genes that encode them, are considered important shell-forming genes in mollusks and are associated with shell and pearl formation, embryonic development, and other functions, but bone morphogenetic protein 10 (BMP10) is poorly understood in Hyriopsis cumingii. In this study, we cloned Hc-BMP10 and obtained a 2477 bp full-length sequence encoding 460 amino acids with a conserved TGF-ß structural domain. During the embryonic developmental stages, the cleavage stage had the highest expression of Hc-BMP10, followed by juvenile clams; the expression in the mantle gradually decreased with increasing mussel age. A strong signal was detected on epidermal cells on the mantle edge by in situ hybridization. In both the shell notching and inserting operations of the pearl fragment assay, we found that the expression of Hc-BMP10 increased after the above treatments. RNA interference assays showed that the silencing of Hc-BMP10 resulted in a change in the morphology of the prismatic layer and nacreous layer, with the prismatic layer less closely aligned and the disordered aragonite flakes in the nacreous layer. These findings indicate that Hc-BMP10 is involved in the growth and development of H. cumingii, as well as the formation of shells and pearls. Therefore, this study provides some reference for selecting superior species for growth and pearl breeding of H. cumingii at a molecular level and further investigation of the molecular mechanism for biomineralization of Hc-BMP10.

Ovarian reserve status in young women is associated with altered gene expression in membrana granulosa cells.

Diminished ovarian reserve (DOR) is a challenging diagnosis of infertility, as there are currently no tests to predict who may become affected with this condition, or at what age. We designed the present study to compare the gene expression profile of membrana granulosa cells from young women affected with DOR with those from egg donors of similar age and to determine if distinct genetic patterns could be identified to provide insight into the etiology of DOR. Young women with DOR were identified based on FSH level in conjunction with poor follicular development during an IVF cycle (n = 13). Egg donors with normal ovarian reserve (NOR) comprised the control group (n = 13). Granulosa cells were collected following retrieval, RNA was extracted and microarray analysis was conducted to evaluate genetic differences between the groups. Confirmatory studies were undertaken with quantitative RT-PCR (qRT-PCR). Multiple significant differences in gene expression were observed between the DOR patients and egg donors. Two genes linked with ovarian function, anti-Mullerian hormone (AMH) and luteinizing hormone receptor (LHCGR), were further analyzed with qRT-PCR in all patients. The average expression of AMH was significantly higher in egg donors (adjusted P-value = 0.01), and the average expression of LHCGR was significantly higher in DOR patients (adjusted P-value = 0.005). Expression levels for four additional genes, progesterone receptor membrane component 2 (PGRMC2), prostaglandin E receptor 3 (subtype EP3) (PTGER3), steroidogenic acute regulatory protein (StAR), and StAR-related lipid transfer domain containing 4 (StarD4), were validated in a group consisting of five NOR and five DOR patients. We conclude that gene expression analysis has substantial potential to determine which young women may be affected with DOR. More importantly, our analysis suggests that DOR patients fall into two distinct subgroups based on gene expression profiles, indicating that different mechanisms may be involved during development of this pathology.

Integrated mRNA and miRNA expression profile analysis of female and male gonads in Hyriopsis cumingii.

Hyriopsis cumingii is an important species for freshwater pearl cultivation in China. In terms of pearl production, males have larger pearls and better glossiness than females, but there are few reports focusing on the sex of H. cumingii. In this study, six mRNA and six microRNA (miRNA) libraries were prepared from ovaries and testes. Additionally, 28,502 differentially expressed genes (DEGs) and 32 differentially expressed miRNAs (DEMs) were identified. Compared with testis, 14,360 mRNAs and 20 miRNAs were up-regulated in ovary, 14,142 mRNAs and 12 miRNAs were down-regulated. In DEGs, the known genes related to sex determinism and/or differentiation were also identified, such as DMRT1, SOX9, SF1 for males, FOXL2 for females, and other potentially significant candidate genes. Three sex-related pathways have also been identified, which are Wnt, Notch, and TGF-beta. In 32 DEMs, the three miRNAs (miR-9-5p, miR-92, miR-184) were paid more attention, they predicted 28 target genes, which may also be candidates for sex-related miRNAs and genes. Differential miRNAs target genes analysis reveals the pathway associated with oocyte meiosis and spermatogenesis. Overall, the findings of the study provide significant insights to enhance our understanding of sex differentiation and/or sex determination mechanisms for H. cumingii.

Identification of Foxl2 in freshwater mussel Hyriopsis cumingii and its involvement in sex differentiation.

The freshwater mussel Hyriopsis cumingii, is the most important species for pearl culture in China. At present, the mechanisms underlying sex differentiation and determination remain unclear in this species. Herein the open reading frame (ORF) of Foxl2 from H. cumingii (Hc-Foxl2) was cloned, and Hc-Foxl2 expression levels in six tissues (the gonad, gill, adductor muscle, foot, mantle, and kidney) were determined. Further, we performed quantitative real-time PCR to compare expressions levels between 1 and 8 months of age and 1-, 2-, and 3-year-old H. cumingii. The localization of Hc-Foxl2 expression in the ovary was analyzed by in situ hybridization, and its function was explored using RNA interference. We found that the ORF region of Hc-Foxl2 was 1215 bp in length, encoded 404 amino acids, and contained conserved FH domains. Hc-Foxl2 was expressed in the male and female tissues, with the expression levels being significantly higher in the ovary than in the testis. In 1-8-month-old H. cumingii, Hc-Foxl2 was expressed at the highest level at 5 months of age, and the gonads began to differentiate at the same time. Moreover, in 1-, 2-, and 3-year-old individuals, Hc-Foxl2 expression levels in the ovaries gradually decreased, but they were higher than those in the testis. Strong hybridization signals for Hc-Foxl2 were detected on the oocyte membrane in 3-year-old female mussels. We also performed double-stranded RNA (dsRNA) interference experiments using three dsRNA strands, which were injected into 5-month-old H. cumingii; the interference effects were the best at 12 h and 48 h post-injection. After interference with Hc-Foxl2, the expression levels of Wnt4, which has an antagonistic relationship with Foxl2 during ovarian development, were slightly increased. Thus, we speculate that Hc-Foxl2 is a female-related gene in H. cumingii and that it is involved in sex differentiation and ovarian development.

Selective quenching of fluorescence from unbound oligonucleotides by gold nanoparticles as a probe of RNA structure.

Binding of small oligonucleotides to the periphery of folded RNA can provide insight into the secondary structure of complex RNA in solution. To discriminate between bound and unbound fluorescein-labeled 2'-O-methyl RNA probes, we use ionically coated gold nanoparticles to selectively adsorb unbound probes and quench their fluorescence. The target is the 3' untranslated region of Bombyx mori R2 RNA. Fluorescence indicates that R2 sequences complementary to some of the probes are accessible for binding in the three-dimensional structure. Hybridization occurs under homogeneous conditions in the absence of the gold nanoparticles so that steric issues associated with chip-based assays are avoided. The assay is compatible with well plate formats, takes less than 5 min, and requires only 2 pmol or less of unlabeled target RNA per probe sequence tested.

Interpreting oligonucleotide microarray data to determine RNA secondary structure: application to the 3' end of Bombyx mori R2 RNA.

A method to deduce RNA secondary structure on the basis of data from microarrays of 2'-O-methyl RNA 9-mers immobilized in agarose film on glass slides is tested with a 249 nucleotide RNA from the 3' end of the R2 retrotransposon from Bombyx mori. Various algorithms incorporating binding data and free-energy minimization calculations were compared for interpreting the data to provide possible secondary structures. Two different methods give structures with 100 and 87% of the base pairs determined by sequence comparison. In contrast, structures predicted by free-energy minimization alone by Mfold and RNAstructure contain 52 and 72% of the known base pairs, respectively. This combination of high throughput microarray techniques with algorithms using free-energy calculations has potential to allow for fast determination of RNA secondary structure. It should also facilitate the design of antisense and siRNA oligonucleotides.