1,720,965 research outputs found
Next-generation libraries for robust RNA interference-based genome-wide screens
No full textGenetic screening based on loss-of-function phenotypes is a powerful discovery tool in biology. Although the recent development of clustered regularly interspaced short palindromic repeats (CRISPR)-based screening approaches in mammalian cell culture has enormous potential, RNA interference (RNAi)-based screening remains the method of choice in several biological contexts. We previously demonstrated that ultracomplex pooled short-hairpin RNA (shRNA) libraries can largely overcome the problem of RNAi off-target effects in genome-wide screens. Here, we systematically optimize several aspects of our shRNA library, including the promoter and microRNA context for shRNA expression, selection of guide strands, and features relevant for postscreen sample preparation for deep sequencing. We present next-generation high-complexity libraries targeting human and mouse protein-coding genes, which we grouped into 12 sublibraries based on biological function. A pilot screen suggests that our next-generation RNAi library performs comparably to current CRISPR interference (CRISPRi)-based approaches and can yield complementary results with high sensitivity and high specificity131351sciescopu
Uridylation by TUT4 and TUT7 marks mRNA for degradation
No full textUridylation occurs pervasively on mRNAs, yet its
mechanism and significance remain unknown. By
applying TAIL-seq, we identify TUT4 and TUT7
(TUT4/7), also known as ZCCHC11 and ZCCHC6,
respectively, as mRNA uridylation enzymes. Uridylation
readily occurs on deadenylated mRNAs in cells.
Consistently, purified TUT4/7 selectively recognize
and uridylate RNAs with short A-tails (less than
25 nt) in vitro. PABPC1 antagonizes uridylation of
polyadenylated mRNAs, contributing to the specificity
for short A-tails. In cells depleted of TUT4/7,
the vast majority of mRNAs lose the oligo-U-tails,
and their half-lives are extended. Suppression of
mRNA decay factors leads to the accumulation of
oligo-uridylated mRNAs. In line with this, microRNA
induces uridylation of its targets, and TUT4/7 are
required for enhanced decay of microRNA targets.
Our study explains the mechanism underlying
selective uridylation of deadenylated mRNAs and
demonstrates a fundamental role of oligo-U-tail as
a molecular mark for global mRNA decay.170701sciescopu
Structure of human DROSHA
No full textMicroRNA maturation is initiated by RNase III
DROSHA that cleaves the stem loop of primary
microRNA. DROSHA functions together with its
cofactor DGCR8 in a heterotrimeric complex known
as Microprocessor. Here, we report the X-ray structure
of DROSHA in complex with the C-terminal helix
of DGCR8. We find that DROSHA contains two
DGCR8-binding sites, one on each RNase III domain
(RIIID), which mediate the assembly of Microprocessor.
The overall structure of DROSHA is surprisingly
similar to that of Dicer despite no sequence
homology apart from the C-terminal part, suggesting
that DROSHA may have evolved from a Dicer homolog.
DROSHA exhibits unique features, including
non-canonical zinc-finger motifs, a long insertion in
the first RIIID, and the kinked link between Connector
helix and RIIID, which explains the 11-bp-measuring
‘‘ruler’’ activity of DROSHA. Our study implicates the
evolutionary origin of DROSHA and elucidates the
molecular basis of Microprocessor assembly and
primary microRNA processing. ª2016 Elsevier Inc.151511sciescopu
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
The RNA-binding protein repertoire of embryonic stem cells
No full textRNA-binding proteins (RBPs) have essential roles in RNA-mediated gene regulation, and yet annotation of RBPs is limited mainly to those with known RNA-binding domains. To systematically identify the RBPs of embryonic stem cells (ESCs), we here employ interactome capture, which combines UV cross-linking of RBP to RNA in living cells, oligo(dT) capture and MS. From mouse ESCs (mESCs), we have defined 555 proteins constituting the mESC mRNA interactome, including 283 proteins not previously annotated as RBPs. Of these, 68 new RBP candidates are highly expressed in ESCs compared to differentiated cells, implicating a role in stem-cell physiology. Two well-known E3 ubiquitin ligases, Trim25 (also called Efp) and Trim71 (also called Lin41), are validated as RBPs, revealing a potential link between RNA biology and protein-modification pathways. Our study confirms and expands the atlas of RBPs, providing a useful resource for the study of the RNA-RBP network in stem cells. © 2013 Nature America, Inc. All rights reserved.11771801sciescopu
Mono-Uridylation of Pre-MicroRNA as a Key Step in the Biogenesis of Group II let-7 MicroRNAs
No full textRNase III Drosha initiates microRNA (miRNA) maturation by cleaving a primary miRNA transcript and releasing a pre-miRNA with a 2 nt 3` overhang. Dicer recognizes the 2 nt 3` overhang structure to selectively process pre-miRNAs. Here, we find that, unlike prototypic pre-miRNAs #group I#, group II pre-miRNAs acquire a shorter #1 nt# 3` overhang from Drosha processing and therefore require a 3`-end mono-uridylation for Dicer processing. The majority of let-7 and miR-105 belong to group II. We identify TUT7/ZCCHC6, TUT4/ZCCHC11, and TUT2/PAPD4/GLD2 as the terminal uridylyl transferases responsible for pre-miRNA mono-uridylation. The TUTs act specifically on dsRNAs with a 1 nt 3` overhang, thereby creating a 2 nt 3` overhang. Depletion of TUTs reduces let-7 levels and disrupts let-7 function. Although the let-7 suppressor, Lin28, induces inhibitory oligo-uridylation in embryonic stem cells, mono-uridylation occurs in somatic cells lacking Lin28 to promote let-7 biogenesis. Our study reveals functional duality of uridylation and introduces TUT7/4/2 as components of the miRNA biogenesis pathway.12812911sciescopu
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
LIN28A Is a Suppressor of ER-Associated Translation in Embryonic Stem Cells
No full textLIN28 plays a critical role in developmental transition, glucose metabolism, and tumorigenesis. At the molecular level, LIN28 is known to repress maturation of let-7 microRNAs and enhance translation of certain mRNAs. In this study, we obtain a genome-wide view of the molecular function of LIN28A in mouse embryonic stem cells by carrying out RNA crosslinking-immunoprecipitation-sequencing (CLIP-seq) and ribosome footprinting. We find that, in addition to let-7 precursors, LIN28A binds to a large number of spliced mRNAs. LIN28A recognizes AAGNNG, AAGNG, and less frequently UGUG, which are located in the terminal loop of a small hairpin. LIN28A is localized to the periendo-plasmic reticulum (ER) area and inhibits translation of mRNAs that are destined for the ER, reducing the synthesis of transmembrane proteins, ER or Golgi lumen proteins, and secretory proteins. Our study suggests a selective regulatory mechanism for ER-associated translation and reveals an unexpected role of LIN28A as a global suppressor of genes in the secretory pathway.11211111sciescopu
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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