CLIP and complementary methods
Markus Hafner,Maria Katsantoni,Maria Katsantoni,Tino Köster,James Marks,Joyita Mukherjee,Joyita Mukherjee,Dorothee Staiger,Jernej Ule,Jernej Ule,Mihaela Zavolan,Mihaela Zavolan +11 more
- Vol. 1, Iss: 1, pp 1-23
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TLDR
The prospect of integrating data obtained by CLIP with complementary methods to gain a comprehensive view of RNP assembly and remodelling, unravel the spatial and temporal dynamics of RNPs in specific cell types and subcellular compartments and understand how defects in RNPs can lead to disease are discussed.Abstract:
RNA molecules start assembling into ribonucleoprotein (RNP) complexes during transcription. Dynamic RNP assembly, largely directed by cis-acting elements on the RNA, coordinates all processes in which the RNA is involved. To identify the sites bound by a specific RNA-binding protein on endogenous RNAs, cross-linking and immunoprecipitation (CLIP) and complementary, proximity-based methods have been developed. In this Primer, we discuss the main variants of these protein-centric methods and the strategies for their optimization and quality assessment, as well as RNA-centric methods that identify the protein partners of a specific RNA. We summarize the main challenges of computational CLIP data analysis, how to handle various sources of background and how to identify functionally relevant binding regions. We outline the various applications of CLIP and available databases for data sharing. We discuss the prospect of integrating data obtained by CLIP with complementary methods to gain a comprehensive view of RNP assembly and remodelling, unravel the spatial and temporal dynamics of RNPs in specific cell types and subcellular compartments and understand how defects in RNPs can lead to disease. Finally, we present open questions in the field and give directions for further development and applications. Ule and colleagues discuss cross-linking and immunoprecipitation (CLIP) methods for characterizing the RNA binding partners of RNA-binding proteins and explore the data analysis workflows, best practices and applications for these techniques. The Primer also considers methods for characterizing the protein binding partners of specific RNAs and discusses how data from these complementary methods can be integrated into CLIP workflows.read more
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References
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RNA-binding proteins: modular design for efficient function.
TL;DR: These studies have shown how, for many RNA-binding proteins, multiple modules define the fundamental structural unit that is responsible for biological function.
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UMI-tools: modeling sequencing errors in Unique Molecular Identifiers to improve quantification accuracy
TL;DR: It is shown that errors in the UMI sequence are common and network-based methods to account for these errors when identifying PCR duplicates are introduced, demonstrating the value of properly accounting for errors in UMIs.
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Mapping the human miRNA interactome by CLASH reveals frequent noncanonical binding
TL;DR: A technique for ligation and sequencing of miRNA-target RNA duplexes associated with human AGO1 was developed, showing that miRNA species systematically differ in their target RNA interactions, and strongly overrepresented motifs were found in the interaction sites of several miRNAs.
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CLIP identifies Nova-regulated RNA networks in the brain.
Jernej Ule,Kirk B. Jensen,Kirk B. Jensen,Matteo Ruggiu,Matteo Ruggiu,Aldo Mele,Aldo Mele,Aljaž Ule,Robert B. Darnell,Robert B. Darnell +9 more
TL;DR: CLIP reveals that Nova coordinately regulates a biologically coherent set of RNAs encoding multiple components of the inhibitory synapse, an observation that may relate to the cause of abnormal motor inhibition in POMA.
Journal ArticleDOI
iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution
Julian König,Kathi Zarnack,Gregor Rot,Tomaž Curk,Melis Kayikci,Blaž Zupan,Daniel J. Turner,Nicholas M. Luscombe,Jernej Ule +8 more
TL;DR: Individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) data show that hnRNP C recognizes uridine tracts with a defined long-range spacing consistent with hmRNP particle organization, and integration of transcriptome-wide iCLIP data and alternative splicing profiles into an 'RNA map' indicates how the positioning of hn RNP particles determines their effect on the inclusion of alternative exons.
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