38 research outputs found
RNA localization is a key determinant of neurite-enriched proteome
Protein subcellular localization is fundamental to the establishment of the body axis, cell migration, synaptic plasticity, and a vast range of other biological processes. Protein localization occurs through three mechanisms: protein transport, mRNA localization, and local translation. However, the relative contribution of each process to neuronal polarity remains unknown. Using neurons differentiated from mouse embryonic stem cells, we analyze protein and RNA expression and translation rates in isolated cell bodies and neurites genome-wide. We quantify 7323 proteins and the entire transcriptome, and identify hundreds of neurite-localized proteins and locally translated mRNAs. Our results demonstrate that mRNA localization is the primary mechanism for protein localization in neurites that may account for half of the neurite-localized proteome. Moreover, we identify multiple neurite-targeted non-coding RNAs and RNA-binding proteins with potential regulatory roles. These results provide further insight into the mechanisms underlying the establishment of neuronal polarity. Subcellular localization of RNAs and proteins is important for polarized cells such as neurons. Here the authors differentiate mouse embryonic stem cells into neurons, and analyze the local transcriptome, proteome, and translated transcriptome in their cell bodies and neurites, providing a unique resource for future studies on neuronal polarity
Kinetic modelling of competition and depletion of shared miRNAs by competing endogenous RNAs
Non-conding RNAs play a key role in the post-transcriptional regulation of
mRNA translation and turnover in eukaryotes. miRNAs, in particular, interact
with their target RNAs through protein-mediated, sequence-specific binding,
giving rise to extended and highly heterogeneous miRNA-RNA interaction
networks. Within such networks, competition to bind miRNAs can generate an
effective positive coupling between their targets. Competing endogenous RNAs
(ceRNAs) can in turn regulate each other through miRNA-mediated crosstalk.
Albeit potentially weak, ceRNA interactions can occur both dynamically,
affecting e.g. the regulatory clock, and at stationarity, in which case ceRNA
networks as a whole can be implicated in the composition of the cell's
proteome. Many features of ceRNA interactions, including the conditions under
which they become significant, can be unraveled by mathematical and in silico
models. We review the understanding of the ceRNA effect obtained within such
frameworks, focusing on the methods employed to quantify it, its role in the
processing of gene expression noise, and how network topology can determine its
reach.Comment: review article, 29 pages, 7 figure
microRNA-Mediated Messenger RNA Deadenylation Contributes to Translational Repression in Mammalian Cells
Animal microRNAs (miRNAs) typically regulate gene expression by binding to partially complementary target sites in the 3′ untranslated region (UTR) of messenger RNA (mRNA) reducing its translation and stability. They also commonly induce shortening of the mRNA 3′ poly(A) tail, which contributes to their mRNA decay promoting function. The relationship between miRNA-mediated deadenylation and translational repression has been less clear. Using transfection of reporter constructs carrying three imperfectly matching let-7 target sites in the 3′ UTR into mammalian cells we observe rapid target mRNA deadenylation that precedes measureable translational repression by endogenous let-7 miRNA. Depleting cells of the argonaute co-factors RCK or TNRC6A can impair let-7-mediated repression despite ongoing mRNA deadenylation, indicating that deadenylation alone is not sufficient to effect full repression. Nevertheless, the magnitude of translational repression by let-7 is diminished when the target reporter lacks a poly(A) tail. Employing an antisense strategy to block deadenylation of target mRNA with poly(A) tail also partially impairs translational repression. On the one hand, these experiments confirm that tail removal by deadenylation is not strictly required for translational repression. On the other hand they show directly that deadenylation can augment miRNA-mediated translational repression in mammalian cells beyond stimulating mRNA decay. Taken together with published work, these results suggest a dual role of deadenylation in miRNA function: it contributes to translational repression as well as mRNA decay and is thus critically involved in establishing the quantitatively appropriate physiological response to miRNAs
Dre-miR-2188 Targets Nrp2a and Mediates Proper Intersegmental Vessel Development in Zebrafish Embryos
BACKGROUND: MicroRNAs (miRNAs) are a class of small RNAs that are implicated in the control of eukaryotic gene expression by binding to the 3'UTR of target mRNAs. Several algorithms have been developed for miRNA target prediction however, experimental validation is still essential for the correct identification of miRNA targets. We have recently predicted that Neuropilin2a (Nrp2a), a vascular endothelial growth factor receptor which is essential for normal developmental angiogenesis in zebrafish, is a dre-miR-2188 target. METHODOLOGY: Here we show that dre-miR-2188 targets the 3'-untranslated region (3'UTR) of Nrp2a mRNA and is implicated in proper intersegmental vessel development in vivo. Over expression of miR-2188 in zebrafish embryos down regulates Nrp2a expression and results in intersegmental vessel disruption, while its silencing increases Nrp2a expression and intersegmental vessel sprouting. An in vivo GFP sensor assay based on a fusion between the GFP coding region and the Nrp2a 3'UTR confirms that miR-2188 binds to the 3'UTR of Nrp2a and inhibits protein translation. CONCLUSIONS: We demonstrate that miR-2188 targets Nrp2a and affects intersegmental vessel development in zebrafish embryos
Genome-Wide Analysis of GLD-1–Mediated mRNA Regulation Suggests a Role in mRNA Storage
Translational repression is often accompanied by mRNA degradation. In contrast, many mRNAs in germ cells and neurons are “stored" in the cytoplasm in a repressed but stable form. Unlike repression, the stabilization of these mRNAs is surprisingly little understood. A key player in Caenorhabditis elegans germ cell development is the STAR domain protein GLD-1. By genome-wide analysis of mRNA regulation in the germ line, we observed that GLD-1 has a widespread role in repressing translation but, importantly, also in stabilizing a sub-population of its mRNA targets. Additionally, these mRNAs appear to be stabilized by the DDX6-like RNA helicase CGH-1, which is a conserved component of germ granules and processing bodies. Because many GLD-1 and CGH-1 stabilized mRNAs encode factors important for the oocyte-to-embryo transition (OET), our findings suggest that the regulation by GLD-1 and CGH-1 serves two purposes. Firstly, GLD-1–dependent repression prevents precocious translation of OET–promoting mRNAs. Secondly, GLD-1– and CGH-1–dependent stabilization ensures that these mRNAs are sufficiently abundant for robust translation when activated during OET. In the absence of this protective mechanism, the accumulation of OET–promoting mRNAs, and consequently the oocyte-to-embryo transition, might be compromised
The impact of transposable element activity on therapeutically relevant human stem cells
Human stem cells harbor significant potential for basic and clinical translational research as well as regenerative
medicine. Currently ~ 3000 adult and ~ 30 pluripotent stem cell-based, interventional clinical trials are ongoing
worldwide, and numbers are increasing continuously. Although stem cells are promising cell sources to treat a
wide range of human diseases, there are also concerns regarding potential risks associated with their clinical use,
including genomic instability and tumorigenesis concerns. Thus, a deeper understanding of the factors and
molecular mechanisms contributing to stem cell genome stability are a prerequisite to harnessing their therapeutic
potential for degenerative diseases. Chemical and physical factors are known to influence the stability of stem cell
genomes, together with random mutations and Copy Number Variants (CNVs) that accumulated in cultured human
stem cells. Here we review the activity of endogenous transposable elements (TEs) in human multipotent and
pluripotent stem cells, and the consequences of their mobility for genomic integrity and host gene expression. We
describe transcriptional and post-transcriptional mechanisms antagonizing the spread of TEs in the human genome,
and highlight those that are more prevalent in multipotent and pluripotent stem cells. Notably, TEs do not only
represent a source of mutations/CNVs in genomes, but are also often harnessed as tools to engineer the stem cell
genome; thus, we also describe and discuss the most widely applied transposon-based tools and highlight the
most relevant areas of their biomedical applications in stem cells. Taken together, this review will contribute to the
assessment of the risk that endogenous TE activity and the application of genetically engineered TEs constitute for
the biosafety of stem cells to be used for substitutive and regenerative cell therapiesS.R.H. and P.T.R. are funded by the Government of Spain (MINECO, RYC-2016-
21395 and SAF2015–71589-P [S.R.H.]; PEJ-2014-A-31985 and SAF2015–71589-
P [P.T.R.]). GGS is supported by a grant from the Ministry of Health of the
Federal Republic of Germany (FKZ2518FSB403)
Measurement of the cross-section for b-jets produced in association with a Z boson at root s=7 TeV with the ATLAS detector ATLAS Collaboration
A measurement is presented of the inclusive cross-section for b-jet production in association with a Z boson in pp collisions at a centre-of-mass energy of root s = 7 TeV. The analysis uses the data sample collected by the ATLAS experiment in 2010, corresponding to an integrated luminosity of approximately 36 pb(-1). The event selection requires a Z boson decaying into high P-T electrons or muons, and at least one b-jet, identified by its displaced vertex, with transverse momentum p(T) > 25 GeV and rapidity vertical bar y vertical bar < 2.1. After subtraction of background processes, the yield is extracted from the vertex mass distribution of the candidate b-jets. The ratio of this cross-section to the inclusive Z cross-section (the average number of b-jets per Z event) is also measured. Both results are found to be in good agreement with perturbative QCD predictions at next-to-leading order
Identification and rejection of pile-up jets at high pseudorapidity with the ATLAS detector
The rejection of forward jets originating from
additional proton–proton interactions (pile-up) is crucial for
a variety of physics analyses at the LHC, including Standard
Model measurements and searches for physics beyond
the Standard Model. The identification of such jets is challenging
due to the lack of track and vertex information in
the pseudorapidity range |η| > 2.5. This paper presents a
novel strategy for forward pile-up jet tagging that exploits
jet shapes and topological jet correlations in pile-up interactions.
Measurements of the per-jet tagging efficiency are
presented using a data set of 3.2 fb−1 of proton–proton collisions
at a centre-of-mass energy of 13 TeV collected with the
ATLAS detector. The fraction of pile-up jets rejected in the
range 2.5 < |η| < 4.5 is estimated in simulated events with
an average of 22 interactions per bunch-crossing. It increases
with jet transverse momentum and, for jets with transverse
momentum between 20 and 50 GeV, it ranges between 49%
and 67% with an efficiency of 85% for selecting hard-scatter
jets. A case study is performed in Higgs boson production
via the vector-boson fusion process, showing that these techniques
mitigate the background growth due to additional
proton–proton interactions, thus enhancing the reach for such
signatures