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GoldCLIP: Gel-omitted Ligation-dependent CLIP.

ABSTRACT: Protein-RNA interaction networks are essential to understand gene regulation control. Identifying binding sites of RNA-binding proteins (RBPs) by the UV-crosslinking and immunoprecipitation (CLIP) represents one of the most powerful methods to map protein-RNA interactions in vivo. However, the traditional CLIP protocol is technically challenging, which requires radioactive labeling and suffers from material loss during PAGE-membrane transfer procedures. Here we introduce a super-efficient CLIP method (GoldCLIP) that omits all gel purification steps. This nonisotopic method allows us to perform highly reproducible CLIP experiments with polypyrimidine tract-binding protein (PTB), a classical RBP in human cell lines. In principle, our method guarantees sequencing library constructions, providing the protein of interest can be successfully crosslinked to RNAs in living cells. GoldCLIP is readily applicable to diverse proteins to uncover their endogenous RNA targets.

SUBMITTER: Gu J

PROVIDER: S-EPMC6112358 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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GoldCLIP: Gel-omitted Ligation-dependent CLIP.

Gu Jiaqi J Wang Ming M Yang Yang Y Qiu Ding D Zhang Yiqun Y Ma Jinbiao J Zhou Yu Y Hannon Gregory J GJ Yu Yang Y

Genomics, proteomics & bioinformatics 20180428 2

Protein-RNA interaction networks are essential to understand gene regulation control. Identifying binding sites of RNA-binding proteins (RBPs) by the UV-crosslinking and immunoprecipitation (CLIP) represents one of the most powerful methods to map protein-RNA interactions in vivo. However, the traditional CLIP protocol is technically challenging, which requires radioactive labeling and suffers from material loss during PAGE-membrane transfer procedures. Here we introduce a super-efficient CLIP m ...[more]

PMID: 29709556

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GoldCLIP: Gel-omitted Ligation-dependent CLIP

Project description:Protein-RNA interaction networks are essential to understand gene regulation control. Identifying the binding sites of RNA-binding proteins (RBPs) by CLIP (UV-crosslinking and immunoprecipitation) represents one of the most powerful methods to map in vivo protein-RNA interactions. However, the traditional CLIP protocol is technically challenging, which requires radioactive labeling and suffers from material loss during PAGE-membrane transfer procedures. Here we introduce a super-efficient CLIP method (GoldCLIP) that omits all gel purification steps. This nonisotopic method allowed us to perform highly reproducible CLIP experiments with classical RBP such as PTB in human cell lines. In principle, our method guarantees sequencing library constructions, providing the protein of interest can be successfully crosslinked to RNAs in living cells. GoldCLIP is readily applicable to diverse factors to uncover their endogenous targets.

2018-05-20 | GSE111406 | GEO

GoldCLIP: Gel-omitted Ligation-dependent CLIP

Project description:GoldCLIP: Gel-omitted Ligation-dependent CLIP

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Clip band ligation ablation.

Project description: Not available

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Minimally Invasive Thumb-sized Pterional Craniotomy for Surgical Clip Ligation of Unruptured Anterior Circulation Aneurysms.

Project description:Less invasive surgical approaches for intracranial aneurysm clipping may reduce length of hospital stay, surgical morbidity, treatment cost, and improve patient outcomes. We present our experience with a minimally invasive pterional approach for anterior circulation aneurysms performed in a major tertiary cerebrovascular center and compare the results with an aged matched dataset from the Nationwide Inpatient Sample (NIS). From August 2008 to December 2012, 22 elective aneurysm clippings on patients ≤55 years of age were performed by the same dual fellowship-trained cerebrovascular/endovascular neurosurgeon. One patient (4.5%) experienced transient post-operative complications. 18 of 22 patients returned for follow-up imaging and there were no recurrences through an average duration of 22 months. A search in the NIS database from 2008 to 2010, also for patients aged ≤55 years of age, yielded 1,341 hospitalizations for surgical clip ligation of unruptured cerebral aneurysms. Inpatient length of stay and hospital charges at our institution using the minimally invasive thumb-sized pterional technique were nearly half that of NIS (length of stay: 3.2 vs 5.7 days; hospital charges: $52,779 vs. $101,882). The minimally invasive thumb-sized pterional craniotomy allows good exposure of unruptured small and medium-sized supraclinoid anterior circulation aneurysms. Cerebrospinal fluid drainage from key subarachnoid cisterns and constant bimanual microsurgical techniques avoid the need for retractors which can cause contusions, localized venous infarctions, and post-operative cerebral edema at the retractor sites. Utilizing this set of techniques has afforded our patients with a shorter hospital stay at a lower cost compared to the national average.

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Archaeal RNA ligase from thermoccocus kodakarensis for template dependent ligation.

Project description:Nicking-sealing RNA ligases play a significant biological role in host defense and cellular repair, and have become an important molecular tool in biomedical engineering. Due to the propensity for RNA to form secondary structures, RNA modifying enzymes with elevated optimum temperatures are highly desired. Current characterized double stranded RNA ligases, such as the bacteriophage T4 RNA ligase 2, while possessing good template dependency, are not active at elevated temperatures. The few characterized RNA ligases from thermophiles exhibit high template independency. We synthesize and characterize here, KOD RNA ligase (KOD1Rnl), a thermostable and template dependent RNA ligase from the archaeon, Thermoccocus Kodakarensis. We disclose that a 13 time reduction in template independent ligation can be achieved with the addition of a single stranded DNase, such as RecJ. We also elucidate the effects of the presence of blood proteins on the activity of KOD1Rnl. Template dependent and thermostable RNA ligases, such as KOD RNA ligase, can be utilized in RNA detection, modification and sequencing.

| S-EPMC5270536 | biostudies-literature

Murine chondrocytes cells：stiff gel vs. the soft gel

Project description:Transcriptional profiling of mouse chondrocytes cells comparing control untreated chondrocytes cells with chondrocytes cells plated in stiff and soft ECM. Goal was to determine the effects of ECM stiffness on gene expression.

2016-07-26 | GSE63615 | GEO

CLIP-170-dependent capture of membrane organelles by microtubules initiates minus-end directed transport.

Project description:Cytoplasmic microtubules (MTs) continuously grow and shorten at free plus ends. During mitosis, this dynamic behavior allows MTs to capture chromosomes to initiate their movement to the spindle poles; however, the role of MT dynamics in capturing organelles for transport in interphase cells has not been demonstrated. Here we use Xenopus melanophores to test the hypothesis that MT dynamics significantly contribute to the efficiency of MT minus-end directed transport of membrane organelles. We demonstrate that initiation of transport of membrane-bounded melanosomes (pigment granules) to the cell center involves their capture by MT plus ends, and that inhibition of MT dynamics or loss of the MT plus-end tracking protein CLIP-170 from MT tips dramatically inhibits pigment aggregation. We conclude that MT dynamics are required for the initiation of MT transport of membrane organelles in interphase cells, and that +TIPs such as CLIP-170 play an important role in this process.

| S-EPMC2759999 | biostudies-literature

Dynein, Lis1 and CLIP-170 counteract Eg5-dependent centrosome separation during bipolar spindle assembly.

Project description:Bipolar spindle assembly critically depends on the microtubule plus-end-directed motor Eg5 that binds antiparallel microtubules and slides them in opposite directions. As such, Eg5 can produce the necessary outward force within the spindle that drives centrosome separation and inhibition of this antiparallel sliding activity results in the formation of monopolar spindles. Here, we show that upon depletion of the minus-end-directed motor dynein, or the dynein-binding protein Lis1, bipolar spindles can form in human cells with substantially less Eg5 activity, suggesting that dynein and Lis1 produce an inward force that counteracts the Eg5-dependent outward force. Interestingly, we also observe restoration of spindle bipolarity upon depletion of the microtubule plus-end-tracking protein CLIP-170. This function of CLIP-170 in spindle bipolarity seems to be mediated through its interaction with dynein, as loss of CLIP-115, a highly homologous protein that lacks the dynein-dynactin interaction domain, does not restore spindle bipolarity. Taken together, these results suggest that complexes of dynein, Lis1 and CLIP-170 crosslink and slide microtubules within the spindle, thereby producing an inward force that pulls centrosomes together.

| S-EPMC2609737 | biostudies-literature

PIPE-CLIP: a comprehensive online tool for CLIP-seq data analysis.

Project description:CLIP-seq is widely used to study genome-wide interactions between RNA-binding proteins and RNAs. However, there are few tools available to analyze CLIP-seq data, thus creating a bottleneck to the implementation of this methodology. Here, we present PIPE-CLIP, a Galaxy framework-based comprehensive online pipeline for reliable analysis of data generated by three types of CLIP-seq protocol: HITS-CLIP, PAR-CLIP and iCLIP. PIPE-CLIP provides both data processing and statistical analysis to determine candidate cross-linking regions, which are comparable to those regions identified from the original studies or using existing computational tools. PIPE-CLIP is available at http://pipeclip.qbrc.org/.

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Galaxy CLIP-Explorer: a web server for CLIP-Seq data analysis.

Project description:Post-transcriptional regulation via RNA-binding proteins plays a fundamental role in every organism, but the regulatory mechanisms lack important understanding. Nevertheless, they can be elucidated by cross-linking immunoprecipitation in combination with high-throughput sequencing (CLIP-Seq). CLIP-Seq answers questions about the functional role of an RNA-binding protein and its targets by determining binding sites on a nucleotide level and associated sequence and structural binding patterns. In recent years the amount of CLIP-Seq data skyrocketed, urging the need for an automatic data analysis that can deal with different experimental set-ups. However, noncanonical data, new protocols, and a huge variety of tools, especially for peak calling, made it difficult to define a standard. CLIP-Explorer is a flexible and reproducible data analysis pipeline for iCLIP data that supports for the first time eCLIP, FLASH, and uvCLAP data. Individual steps like peak calling can be changed to adapt to different experimental settings. We validate CLIP-Explorer on eCLIP data, finding similar or nearly identical motifs for various proteins in comparison with other databases. In addition, we detect new sequence motifs for PTBP1 and U2AF2. Finally, we optimize the peak calling with 3 different peak callers on RBFOX2 data, discuss the difficulty of the peak-calling step, and give advice for different experimental set-ups. CLIP-Explorer finally fills the demand for a flexible CLIP-Seq data analysis pipeline that is applicable to the up-to-date CLIP protocols. The article further shows the limitations of current peak-calling algorithms and the importance of a robust peak detection.

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