Zhao Zhang

UAP56 couples piRNA clusters to the perinuclear transposon silencing machinery

piRNAs silence transposons during germline development. In Drosophila, transcripts from heterochromatic clusters are processed into primary piRNAs in the perinuclear nuage. The nuclear DEAD box protein UAP56 has been previously implicated in mRNA splicing and export, whereas the DEAD box protein Vasa has an established role in piRNA production and localizes to nuage with the piRNA binding PIWI proteins Ago3 and Aub. We show that UAP56 colocalizes with the cluster-associated HP1 variant Rhino, that nuage granules containing Vasa localize directly across the nuclear envelope from cluster foci containing UAP56 and Rhino, and that cluster transcripts immunoprecipitate with both Vasa and UAP56. Significantly, a charge-substitution mutation that alters a conserved surface residue in UAP56 disrupts colocalization with Rhino, germline piRNA production, transposon silencing, and perinuclear localization of Vasa. We therefore propose that UAP56 and Vasa function in a piRNA-processing compartment that spans the nuclear envelope.

Heterotypic piRNA Ping-Pong Requires Qin, a Protein with Both E3 Ligase and Tudor Domains

piRNAs guide PIWI proteins to silence transposons in animal germ cells. Reciprocal cycles of piRNA-directed RNA cleavage--catalyzed by the PIWI proteins Aubergine (Aub) and Argonaute3 (Ago3) in Drosophila melanogaster--expand the population of antisense piRNAs in response to transposon expression, a process called the Ping-Pong cycle. Heterotypic Ping-Pong between Aub and Ago3 ensures that antisense piRNAs predominate. We show that qin, a piRNA pathway gene whose protein product contains both E3 ligase and Tudor domains, colocalizes with Aub and Ago3 in nuage, a perinuclear structure implicated in transposon silencing. In qin mutants, less Ago3 binds Aub, futile Aub:Aub homotypic Ping-Pong prevails, antisense piRNAs decrease, many families of mobile genetic elements are reactivated, and DNA damage accumulates in nurse cells and oocytes. We propose that Qin enforces heterotypic Ping-Pong between Aub and Ago3, ensuring that transposons are silenced and maintaining the integrity of the germline genome.

The HP1 Homolog Rhino Anchors a Nuclear Complex that Suppresses piRNA Precursor Splicing

piRNAs guide an adaptive genome defense system that silences transposons during germline development. The Drosophila HP1 homolog Rhino is required for germline piRNA production. We show that Rhino binds specifically to the heterochromatic clusters that produce piRNA precursors, and that binding directly correlates with piRNA production. Rhino colocalizes to germline nuclear foci with Rai1/DXO-related protein Cuff and the DEAD box protein UAP56, which are also required for germline piRNA production. RNA sequencing indicates that most cluster transcripts are not spliced and that rhino, cuff, and uap56 mutations increase expression of spliced cluster transcripts over 100-fold. LacI::Rhino fusion protein binding suppresses splicing of a reporter transgene and is sufficient to trigger piRNA production from a trans combination of sense and antisense reporters. We therefore propose that Rhino anchors a nuclear complex that suppresses cluster transcript splicing and speculate that stalled splicing differentiates piRNA precursors from mRNAs.

Strand-specific libraries for high throughput RNA sequencing (RNA-Seq) prepared without poly(A) selection.

BackgroundHigh throughput DNA sequencing technology has enabled quantification of all the RNAs in a cell or tissue, a method widely known as RNA sequencing (RNA-Seq). However, non-coding RNAs such as rRNA are highly abundant and can consume >70% of sequencing reads. A common approach is to extract only polyadenylated mRNA; however, such approaches are blind to RNAs with short or no poly(A) tails, leading to an incomplete view of the transcriptome. Another challenge of preparing RNA-Seq libraries is to preserve the strand information of the RNAs.DesignHere, we describe a procedure for preparing RNA-Seq libraries from 1 to 4 μg total RNA without poly(A) selection. Our method combines the deoxyuridine triphosphate (dUTP)/uracil-DNA glycosylase (UDG) strategy to achieve strand specificity with AMPure XP magnetic beads to perform size selection. Together, these steps eliminate gel purification, allowing a library to be made in less than two days. We barcode each library during the final PCR amplification step, allowing several samples to be sequenced in a single lane without sacrificing read length. Libraries prepared using this protocol are compatible with Illumina GAII, GAIIx and HiSeq 2000 platforms.DiscussionThe RNA-Seq protocol described here yields strand-specific transcriptome libraries without poly(A) selection, which provide approximately 90% mappable sequences. Typically, more than 85% of mapped reads correspond to protein-coding genes and only 6% derive from non-coding RNAs. The protocol has been used to measure RNA transcript identity and abundance in tissues from flies, mice, rats, chickens, and frogs, demonstrating its general applicability.

Slicing and Binding by Ago3 or Aub Trigger Piwi-Bound piRNA Production by Distinct Mechanisms

In Drosophila ovarian germ cells, PIWI-interacting RNAs (piRNAs) direct Aubergine and Argonaute3 to cleave transposon transcripts and instruct Piwi to repress transposon transcription, thereby safeguarding the germline genome. Here, we report that RNA cleavage by Argonaute3 initiates production of most Piwi-bound piRNAs. We find that the cardinal function of Argonaute3, whose piRNA guides predominantly correspond to sense transposon sequences, is to produce antisense piRNAs that direct transcriptional silencing by Piwi, rather than to make piRNAs that guide post-transcriptional silencing by Aubergine. We also find that the Tudor domain protein Qin prevents Aubergines cleavage products from becoming Piwi-bound piRNAs, ensuring that antisense piRNAs guide Piwi. Although Argonaute3 slicing is required to efficiently trigger phased piRNA production, an alternative, slicing-independent pathway suffices to generate Piwi-bound piRNAs that repress transcription of a subset of transposon families. This alternative pathway may help flies silence newly acquired transposons for which they lack extensively complementary piRNAs.

14-kDa phosphohistidine phosphatase and its role in human lung cancer cell migration and invasion

14-kDa phosphohistidine phosphatase (PHP14) was the first protein histidine phosphatase to be discovered, but its biological function remains unclear. In our previous study, we found that it was associated with tumor invasion. Here, we investigated its role in lung cancer cell migration and invasion. Knockdown of PHP14 expression in highly metastatic lung cancer CL1-5 cells inhibited migration and invasion in vitro, but did not alter cell proliferation rates. Overexpression of PHP14 in NCI H1299 cells promoted migration and invasion in vitro, but again did not alter cell proliferation. To evaluate the metastatic properties of PHP14 in vivo, an experimental metastasis assay was performed. Experimental metastasis in vivo was extensively inhibited by PHP14 knockdown. To further examine the mechanism underlying the involvement of PHP14 in cell migration, invasion, and metastasis, a comparative proteomics analysis was performed. The differential protein expression profiles revealed that PHP14 was probably involved in cytoskeletal reorganization; this was further supported by actin filament (F-actin) staining. These results demonstrate for the first time that PHP14 may be functionally important in lung cancer cell migration and the invasion of lung cancer cells, mediated partly through modulation of actin cytoskeleton rearrangement.

Is Yield Increase Sufficient to Achieve Food Security in China

Increasing demand for food, driven by unprecedented population growth and increasing consumption, will keep challenging food security in China. Although cereal yields have substantially improved during the last three decades, whether it will keep thriving to meet the increasing demand is not known yet. Thus, an integrated analysis on the trends of crop yield and cultivated area is essential to better understand current state of food security in China, especially on county scale. So far, yield stagnation has extensively dominated the main cereal-growing areas across China. Rice yield is facing the most severe stagnation that 53.9% counties tracked in the study have stagnated significantly, followed by maize (42.4%) and wheat (41.9%). As another important element for production sustainability, but often neglected is the planted area patterns. It has been further demonstrated that the loss in productive arable land for rice and wheat have dramatically increased the pressure on achieving food security. Not only a great deal of the planted areas have stagnated since 1980, but also collapsed. 48.4% and 54.4% of rice- and wheat-growing counties have lost their cropland areas to varying degrees. Besides, 27.6% and 35.8% of them have retrograded below the level of the 1980s. The combined influence (both loss in yield and area) has determined the crop sustainable production in China to be pessimistic for rice and wheat, and consequently no surprise to find that more than half of counties rank a lower level of production sustainability. Therefore, given the potential yield increase in wheat and maize, as well as substantial area loss of rice and wheat, the possible targeted adaptation measures for both yield and cropping area is required at county scale. Moreover, policies on food trade, alongside advocation of low calorie diets, reducing food loss and waste can help to enhance food security.

SnapShot: Fly piRNAs, PIWI Proteins, and the Ping-Pong Cycle

In animals, PIWI-interacting RNAs (piRNAs) repress transposons and other repetitive genomic sequences, ensuring genomic integrity and faithful transmission of the genetic material from one generation to the next. Expression of these 23–30 nucleotide-long small RNAs is generally restricted to the germline cells. They bind to “PIWI” proteins, a specialized subclass of the Argonaute protein family, whose members use small RNA guides to silence gene expression. In zebrafish and

A High-Temporal Resolution Technology for Dynamic Proteomic Analysis Based on 35S Labeling

As more and more research efforts have been attracted to dynamic or differential proteomics, a method with high temporal resolution and high throughput is required. In present study, a 35S in vivo Labeling Analysis for Dynamic Proteomics (SiLAD) was designed and tested by analyzing the dynamic proteome changes in the highly synchronized A549 cells, as well as in the rat liver 2/3 partial hepatectomy surgery. The results validated that SiLAD technique, in combination with 2-Dimensional Electrophoresis, provided a highly sensitivity method to illustrate the non-disturbed endogenous proteins dynamic changes with a good temporal resolution and high signal/noise ratio. A significant number of differential proteins can be discovered or re-categorized by this technique. Another unique feature of SiLAD is its capability of quantifying the rate of protein expression, which reflects the cellular physiological turn points more effectively. Finally, the prescribed SiLAD proteome snapshot pattern could be potentially used as an exclusive symbol for characterizing each stage in well regulated biological processes.

Public perception and responses to environmental pollution and health risks: evaluation and implication from a national survey in China

Concerns have been raised among policy-makers, researchers and Chinese citizens regarding the widespread environmental degradation that has occurred in China in recent decades. Years of environmental education and media coverage on pollution harm and health risks have not only provided information about pollution to the public, but have also strengthened people’s concerns. However, an ‘intense focus’ on pollution is far from sufficient; at present, it is necessary to assess to what extent the public can identify specific environmental conditions and whether they are ready to cope with potential health risks from pollution. Through face-to-face surveys on trains and at railway stations nationwide, we investigated public experiences of environmental pollution accidents, perceptions of local environmental risks (focused on air and water quality) and responses to local environmental conditions. By comparing public perceptions with official environmental monitoring data-sets, we concluded that the accuracy of perceptions related to four environmental factors ranged from 40 to 60% at the individual scale. Furthermore, the accuracies increased approximately 2–10% at the county scale and 10–30% at the city scale, highlighting the possible benefits of collective intelligence in helping the public to identify existing environment conditions more accurately. Additionally, despite great concerns about pollution and health, public attitudes toward coping with the dangers of pollution and health risks were found to be indifferent. Our study revealed factors at the individual, social and governmental levels that led to low levels of perception accuracy and response scores. Thereout, we stressed potential pathways to improve the accuracy of public perception and the positivity of responses. The survey results indicate that there is a long way to go before the public is well prepared to cope with the risks of pollution; as a consequence, it is necessary to improve both personal environmental awareness as well as governmental, social and commercial responses to pollution events.