CLIP Tool Kit (CTK): a flexible and robust pipeline to analyze CLIP sequencing data

Shah, Ankeeta; Qian, Yingzhi; Weyn-Vanhentenryck, Sebastien M.; Zhang, Chaolin

doi:10.1093/bioinformatics/btw653

Cited by 150 publications

(172 citation statements)

References 12 publications

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“…To this end, we took advantage of the computational approaches we previously developed to infer the precise protein-RNA crosslink sites from CLIP data by identifying crosslink-induced mutation sites (CIMS) and truncation sites (CITS) (Shah et al, 2017; Weyn-Vanhentenryck et al, 2014; Zhang and Darnell, 2011). We applied these methods to two in-depth LIN28 CLIP datasets: LIN28A HITS-CLIP performed in mouse ESCs (Cho et al, 2012), and LIN28B CLIP derived from two human cell lines K562 and HepG2 using a modified CLIP protocol named eCLIP (Van Nostrand et al, 2016) (for this study, we mainly describe results from K562 cells, as the results obtained from HepG2 cells are very similar).…”

Section: Resultsmentioning

confidence: 99%

“…Due to the differences in protocols used to generate these CLIP libraries, we expected HITS-CLIP to capture only CIMS and eCLIP to be enriched in CITS (see Discussion). Following our established pipeline (Shah et al, 2017), we identified 50,292 substitution CIMS from LIN28A HITS-CLIP data and 22,673 CITS from LIN28B eCLIP data in K562 cells (Table S1). Consistent with the previous analysis (Cho et al., 2012), we observed a striking enrichment of the GGAG motif at CIMS inferred from HITS-CLIP data (11.5 fold at position 0), indicating the predominant crosslinking of the first G of the GGAG motif (Figure 1B left panel and Figure 1D).…”

Section: Resultsmentioning

confidence: 99%

“…For each dataset, raw reads were downloaded and processed using our established analysis pipeline CLIP Tool Kit (CTK) (Shah et al, 2017). …”

Section: Star Methodsmentioning

confidence: 99%

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LIN28 Selectively Modulates a Subclass of Let-7 MicroRNAs

et al. 2018

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Summary LIN28 is a bipartite RNA-binding protein that post-transcriptionally inhibits the biogenesis of let-7 microRNAs to regulate development and influence disease states. However, the mechanisms of let-7 suppression remains poorly understood, because LIN28 recognition depends on coordinated targeting by both the zinc knuckle domain (ZKD)—which binds a GGAG-like element in the precursor—and the cold shock domain (CSD), whose binding sites have not been systematically characterized. By leveraging single-nucleotide-resolution mapping of LIN28 binding sites in vivo, we determined that the CSD recognizes a (U)GAU motif. This motif partitions the let-7 microRNAs into two subclasses, precursors with both CSD and ZKD binding sites (CSD+) and precursors with ZKD but no CSD binding sites (CSD−). LIN28 in vivo recognition—and subsequent 3ʹ uridylation and degradation—of CSD+ precursors is more efficient, leading to their stronger suppression in LIN28-activated cells and cancers. Thus, CSD binding sites amplify the regulatory effects of LIN28.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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LIN28 Selectively Modulates a Subclass of Let-7 MicroRNAs

et al. 2018

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“…The adaptor was trimmed by fastx_clipper fastx_toolkit (http://hannonlab.cshl.edu/fastx_tool-kit/). Low quality bases were filtered by fastq_filter.pl, a custom perl script from CLIP Tool Kit (CTK) [65] and reads shorter than 24 nt discarded. After trimming, reads without a barcode marker sequence or within large fragment (length > 60 nt) are discarded to eliminate unspecific RNA tags.…”

Section: Analysis Of Miclip-sequencing Datamentioning

confidence: 99%

Mettl3-mediated m6A regulates spermatogonial differentiation and meiosis initiation

et al. 2017

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METTL3 catalyzes the formation of N6 -methyl-adenosine (m 6 A) which has important roles in regulating various biological processes. However, the in vivo function of Mettl3 remains largely unknown in mammals. Here we generated germ cell-specific Mettl3 knockout mice and demonstrated that Mettl3 was essential for male fertility and spermatogenesis. The ablation of Mettl3 in germ cells severely inhibited spermatogonial differentiation and blocked the initiation of meiosis. Transcriptome and m 6 A profiling analysis revealed that genes functioning in spermatogenesis had altered profiles of expression and alternative splicing. Our findings provide novel insights into the function and regulatory mechanisms of Mettl3-mediated m 6 A modification in spermatogenesis and reproduction in mammals.

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“…Both replicates were concatenated before peak calling. Both traditional peak calling with Clip Tool Kit (CTK) (Shah et al, 2017) [https://zhanglab.c2b2.columbia.edu/index.php/Standard/BrdU-CLIP_data_analysis _using_CTK]as well as CIMS analysis (Moore et al, 2014) were performed.…”

Section: Sequencing and Analysis Of Clip Datamentioning

confidence: 99%

MS2-TRIBE evaluates protein-RNA interactions and nuclear organization of transcription by RNA editing

Biswas

Rahman

Gupta³

et al. 2019

Preprint

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Nearly every step of RNA regulation is mediated by binding proteins (RBPs). The most common method to identify specific RBP target transcripts in vivo is by crosslinking ("CLIP" and its variants), which rely on protein-RNA crosslinking and specific antibodies. Another recently introduced method exploits RNA editing, with the catalytic domain of ADAR covalently attached to a specific RBP ("TRIBE"). Both approaches suffer from difficulties in distinguishing real RNA targets from false negative and especially false positive signals. To critically evaluate this problem, we used fibroblasts from a mouse where every endogenous β -actin mRNA molecule was tagged with the bacteriophage MS2 RNA stem loops; hence there is only a single bona fide target mRNA for the MS2 capsid protein (MCP). CLIP and TRIBE could both detect the single RNA target, albeit with some false positives (transcripts lacking the MS2 stem loops). Consistent false positive CLIP signals could be attributed to nonspecific antibody crosslinking. To our surprise, the supposed false positive TRIBE targets correlated with the location of genes spatially proximal to the β -actin gene. This result indicates that MCP-ADAR bound to β -actin mRNA contacted and edited nearby nascent transcripts, as evidenced by frequent intronic editing. Importantly, nascent transcripts on nearby chromosomes were also edited, agreeing with the interchromosomal contacts observed in chromosome paint and Hi-C. The identification of nascent RNA-RNA contacts imply that RNA-regulatory proteins such as splicing factors can associate with multiple nascent transcripts and thereby form domains of post-transcriptional activity, which increase their local concentrations. These results more generally indicate that TRIBE combined with the MS2 system, MS2-TRIBE, is a new tool to study nuclear RNA organization and regulation.3 Introduction:

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CLIP Tool Kit (CTK): a flexible and robust pipeline to analyze CLIP sequencing data

Abstract: cz2294@columbia.edu.

Cited by 150 publications

References 12 publications

LIN28 Selectively Modulates a Subclass of Let-7 MicroRNAs

LIN28 Selectively Modulates a Subclass of Let-7 MicroRNAs

Mettl3-mediated m6A regulates spermatogonial differentiation and meiosis initiation

MS2-TRIBE evaluates protein-RNA interactions and nuclear organization of transcription by RNA editing

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