12 research outputs found
In situ functional dissection of RNA cis-regulatory elements by multiplex CRISPR-Cas9 genome engineering.
RNA regulatory elements (RREs) are an important yet relatively under-explored facet of gene regulation. Deciphering the prevalence and functional impact of this post-transcriptional control layer requires technologies for disrupting RREs without perturbing cellular homeostasis. Here we describe genome-engineering based evaluation of RNA regulatory element activity (GenERA), a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 platform for in situ high-content functional analysis of RREs. We use GenERA to survey the entire regulatory landscape of a 3'UTR, and apply it in a multiplex fashion to analyse combinatorial interactions between sets of miRNA response elements (MREs), providing strong evidence for cooperative activity. We also employ this technology to probe the functionality of an entire MRE network under cellular homeostasis, and show that high-resolution analysis of the GenERA dataset can be used to extract functional features of MREs. This study provides a genome editing-based multiplex strategy for direct functional interrogation of RNA cis-regulatory elements in a native cellular environment
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A transgenic resource for conditional competitive inhibition of conserved Drosophila microRNAs
Although the impact of microRNAs (miRNAs) in development and disease is well established, understanding the function of individual miRNAs remains challenging. Development of competitive inhibitor molecules such as miRNA sponges has allowed the community to address individual miRNA function in vivo. However, the application of these loss-of-function strategies has been limited. Here we offer a comprehensive library of 141 conditional miRNA sponges targeting well-conserved miRNAs in Drosophila. Ubiquitous miRNA sponge delivery and consequent systemic miRNA inhibition uncovers a relatively small number of miRNA families underlying viability and gross morphogenesis, with false discovery rates in the 4â8% range. In contrast, tissue-specific silencing of muscle-enriched miRNAs reveals a surprisingly large number of novel miRNA contributions to the maintenance of adult indirect flight muscle structure and function. A strong correlation between miRNA abundance and physiological relevance is not observed, underscoring the importance of unbiased screens when assessing the contributions of miRNAs to complex biological processes
Progress with the Prime Focus Spectrograph for the Subaru Telescope: a massively multiplexed optical and near-infrared fiber spectrograph
The Prime Focus Spectrograph (PFS) is an optical/near-infrared multi-fiber
spectrograph with 2394 science fibers, which are distributed in 1.3 degree
diameter field of view at Subaru 8.2-meter telescope. The simultaneous wide
wavelength coverage from 0.38 um to 1.26 um, with the resolving power of 3000,
strengthens its ability to target three main survey programs: cosmology,
Galactic archaeology, and galaxy/AGN evolution. A medium resolution mode with
resolving power of 5000 for 0.71 um to 0.89 um also will be available by simply
exchanging dispersers. PFS takes the role for the spectroscopic part of the
Subaru Measurement of Images and Redshifts project, while Hyper Suprime-Cam
works on the imaging part. To transform the telescope plus WFC focal ratio, a
3-mm thick broad-band coated glass-molded microlens is glued to each fiber tip.
A higher transmission fiber is selected for the longest part of cable system,
while one with a better FRD performance is selected for the fiber-positioner
and fiber-slit components, given the more frequent fiber movements and tightly
curved structure. Each Fiber positioner consists of two stages of
piezo-electric rotary motors. Its engineering model has been produced and
tested. Fiber positioning will be performed iteratively by taking an image of
artificially back-illuminated fibers with the Metrology camera located in the
Cassegrain container. The camera is carefully designed so that fiber position
measurements are unaffected by small amounts of high special-frequency
inaccuracies in WFC lens surface shapes. Target light carried through the fiber
system reaches one of four identical fast-Schmidt spectrograph modules, each
with three arms. Prototype VPH gratings have been optically tested. CCD
production is complete, with standard fully-depleted CCDs for red arms and
more-challenging thinner fully-depleted CCDs with blue-optimized coating for
blue arms.Comment: 14 pages, 12 figures, submitted to "Ground-based and Airborne
Instrumentation for Astronomy V, Suzanne K. Ramsay, Ian S. McLean, Hideki
Takami, Editors, Proc. SPIE 9147 (2014)
In situ functional dissection of RNA cis-regulatory elements by multiplex CRISPR-Cas9 genome engineering
RNA regulatory elements (RREs) are an important yet relatively under-explored facet of gene regulation. Deciphering the prevalence and functional impact of this post-transcriptional control layer requires technologies for disrupting RREs without perturbing cellular homeostasis. Here we describe genome-engineering based evaluation of RNA regulatory element activity (GenERA), a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 platform for in situ high-content functional analysis of RREs. We use GenERA to survey the entire regulatory landscape of a 3âČUTR, and apply it in a multiplex fashion to analyse combinatorial interactions between sets of miRNA response elements (MREs), providing strong evidence for cooperative activity. We also employ this technology to probe the functionality of an entire MRE network under cellular homeostasis, and show that high-resolution analysis of the GenERA dataset can be used to extract functional features of MREs. This study provides a genome editing-based multiplex strategy for direct functional interrogation of RNA cis-regulatory elements in a native cellular environment. © 2017 The Author(s)1
miR-Blood â a small RNA atlas of human blood components
Abstract miR-Blood is a high-quality, small RNA expression atlas for the major components of human peripheral blood (plasma, erythrocytes, thrombocytes, monocytes, neutrophils, eosinophils, basophils, natural killer cells, CD4+ T cells, CD8+ T cells, and B cells). Based on the purified blood components from 52 individuals, the dataset provides a comprehensive repository for the expression of 4971 small RNAs from eight non-coding RNA classes