Changes in transcript architecture can have powerful effects on protein expression. Regulation of the transcriptome is often dramatically revealed during dynamic conditions such as development. To examine changes in transcript architecture we analyzed the expression and transcript boundaries of protein-coding and noncoding RNAs over the developmental process of meiosis in Saccharomyces cerevisiae. Custom-designed, high-resolution tiling arrays were used to define the time-resolved transcriptome of cells undergoing meiosis and sporulation. These arrays were specifically designed for the S. cerevisiae strain SK1 that sporulates with high efficiency and synchrony. In addition, new methods were created to define transcript boundaries and to identify dynamic changes in transcript expression and architecture over time. Of 8407 total segments, 699 (8.3%) were identified by our algorithm as regions containing potential transcript architecture changes. Our analyses reveal extensive changes to both the coding and noncoding transcriptome, including altered 59 ends, 39 ends, and splice sites. Additionally, 3910 (46.5%) unannotated expressed segments were identified. Interestingly, subsets of unannotated RNAs are located across from introns (anti-introns) or across from the junction between two genes (anti-intergenic junctions). Many of these unannotated RNAs are abundant and exhibit sporulation-specific changes in expression patterns. All work, including heat maps of the tiling array, annotation for the SK1 strain, and phastCONS conservation analysis, is available at http://groups.molbiosci.northwestern. edu/sontheimer/sk1meiosis.php. Our high-resolution transcriptome analyses reveal that coding and noncoding transcript architectures are exceptionally dynamic in S. cerevisiae and suggest a vast array of novel transcriptional and post-transcriptional control mechanisms that are activated upon meiosis and sporulation.
The Drosophila melanogaster RNA-induced silencing complex (RISC) forms a large ribonucleoprotein particle on small interfering RNAs (siRNAs) and catalyzes target mRNA cleavage during RNA interference (RNAi). Dicer-2, R2D2, Loquacious, and Argonaute-2 are examples of RISC-associated factors that are involved in RNAi. Holo-RISC is an ϳ80 S small interfering ribonucleoprotein, which suggests that there are many additional proteins that participate in the RNAi pathway. In this study, we used siRNA affinity capture combined with mass spectrometry to identify novel components of the Drosophila RNAi machinery. Our study identified both established RISC components and novel siRNAassociated factors, many of which contain domains that are consistent with potential roles in RNAi. Functional analysis of these novel siRNA-associated proteins suggests that these factors may play an important role in RNAi. Molecular & Cellular Proteomics 9:1866 -1872, 2010.Small RNAs can regulate gene expression through a collection of mechanisms broadly termed RNA silencing. Small RNA-mediated silencing mechanisms occur in most species (1-5). The ability to silence the expression of specific genes using small RNAs via RNA interference (RNAi) 1 has greatly facilitated our understanding of gene function in eukaryotes. In addition, small RNA-mediated gene silencing has therapeutic potential and holds promise for the treatment of specific diseases (6). Understanding the mechanism of RNAi and identifying the components of the RNAi machinery are essential for harnessing its full potential in both genome-wide screens and therapeutic applications.Recently, high throughput sequencing technology has revealed the presence of endogenous siRNAs in plant, fly, worm, and mammalian cells (7-16). These endogenous siRNAs target transposable element RNAs, pseudogene RNAs, and protein-coding mRNAs (17). Therefore, the endogenous siRNA pathway seems to have evolved as a mechanism of cellular defense against selfish genetic elements. The roles of these siRNAs in development and cell physiology are poorly understood.Drosophila melanogaster is a well characterized model system for studying RNAi. In Drosophila, long double-stranded RNAs (dsRNAs) are processed by the endonuclease Dicer-2 into 21-nucleotide siRNAs (18). After processing, these siRNAs form an initiator complex with Dicer-2 and the dsRNA-binding domain (dsRBD)-containing protein R2D2 (19 -23). This R2D2-Dicer-2 Initiator (RDI) complex transitions to a larger siRNP called the RISC loading complex (21,22,24,25) and then to pre-RISC (26). Subsequently, pre-RISC matures into holo-RISC, which includes the catalytic activity necessary for target mRNA cleavage (21,25,27). The endonuclease subunit responsible for target cleavage in holo-RISC is Argonaute-2 (Ago2) (28, 29), which uses the guide strand of the siRNA duplex to target complementary mRNA sequences for cleavage and degradation.Studies of the RDI complex strongly suggest that it includes no other proteins besides . Additional proteins such as Ago2 are p...
In Saccharomyces cerevisiae, meiosis and sporulation are highly regulated responses that are driven in part by changes in RNA expression. Alternative mRNA forms with extended 5′ UTRs are atypical in S. cerevisiae, and 5′ extensions with upstream open reading frames (uORFs) are even more unusual. Here we characterize the gene YPR036W-A, now renamed SPO24, which encodes a very small (67-amino-acid) protein. This gene gives rise to two mRNA forms: a shorter form throughout meiosis and a longer, 5′-extended form in mid-late meiosis. The latter form includes a uORF for a 14-amino-acid peptide (Spo24u14). Deletion of the downstream ORF (dORF) leads to sporulation defects and the appearance of pseudohyphae-like projections. Experiments with luciferase reporters indicate that the uORF does not downregulate dORF translation. The protein encoded by the dORF (Spo24d67) localizes to the prospore membrane and is differentially phosphorylated during meiosis. Transcription of the 5′-extended mRNA in mid-meiosis depends upon the presence of two middle sporulation elements (MSEs). Removal of the MSEs severely inhibits the mid-meiotic appearance of the 5′-extended mRNA and limits the ability of plasmid-borne SPO24 to rescue the sporulation defect of a spo24Δ mutant, suggesting that the 5′-extended mRNA is functionally important. These results reveal Spo24d67 as a sporulation-related factor that is encoded by a transcriptionally dynamic, uORF-containing locus.
The Institute for In Vitro Sciences (IIVS) is sponsoring a series of workshops to identify, discuss and develop recommendations for optimal scientific and technical approaches for conducting in vitro assays, to assess potential toxicity within and across tobacco and various next generation nicotine and tobacco products (NGPs), including heated tobacco products (HTPs) and electronic nicotine delivery systems (ENDS). The third workshop (24–26 February 2020) summarised the key challenges and made recommendations concerning appropriate methods of test article generation and cell exposure from combustible cigarettes, HTPs and ENDS. Expert speakers provided their research, perspectives and recommendations for the three basic types of tobacco-related test articles: i) pad-collected material (PCM); ii) gas vapour phase (GVP); and iii) whole smoke/aerosol. These three types of samples can be tested individually, or the PCM and GVP can be combined. Whole smoke/aerosol can be bubbled through media or applied directly to cells at the air–liquid interface. Summaries of the speaker presentations and the recommendations developed by the workgroup are presented. Following discussion, the workshop concluded the following: that there needs to be greater standardisation in aerosol generation and collection processes; that methods for testing the NGPs need to be developed and/or optimised, since simply mirroring cigarette smoke testing approaches may be insufficient; that understanding and quantitating the applied dose is fundamental to the interpretation of data and conclusions from each study; and that whole smoke/aerosol approaches must be contextualised with regard to key information, including appropriate experimental controls, environmental conditioning, analytical monitoring, verification and performance criteria.
3,4 Methylenedioxymethamphetamine (MDMA)-assisted therapy has been recently found to be highly effective for treatment of posttraumatic stress disorder (PTSD). Previous studies have been inconclusive in elucidating potential MDMA genotoxicity. We performed three regulatory compliant studies to investigate the potential of genotoxic effects of MDMA treatment in humans: (1) an in vitro bacterial reverse mutation (Ames) assay, (2) an in vitro chromosome aberration test in Chinese hamster ovary cells, and (3) an in vivo micronucleus study in male Sprague Dawley rats. MDMA was found to not have genotoxic effects in any of the assays at or above clinically relevant concentrations.
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