iCLIP: Protein–RNA interactions at nucleotide resolution

Huppertz, Ina; Attig, Jan; D’Ambrogio, Andrea; Easton, Laura E.; Sibley, Christopher R.; Sugimoto, Yoichiro; Tajnik, Mojca; König, Julian; Ule, Jernej

doi:10.1016/j.ymeth.2013.10.011

Cited by 402 publications

(465 citation statements)

References 25 publications

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“…cDNA fragments were amplified by PCR and further purified with Ampure XP beads (Beckman Coulter). The iCLIP libraries were quantified using the D1000 ScreenTape and reagents (Agilent) on the 2200 Tapestation System (Agilent) and sequenced using the standard Illumina protocol for 50‐bp single‐end sequencing (Huppertz et al , 2014). …”

Section: Methodsmentioning

confidence: 99%

Mice with endogenous TDP ‐43 mutations exhibit gain of splicing function and characteristics of amyotrophic lateral sclerosis

et al. 2018

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TDP‐43 (encoded by the gene TARDBP) is an RNA binding protein central to the pathogenesis of amyotrophic lateral sclerosis (ALS). However, how TARDBP mutations trigger pathogenesis remains unknown. Here, we use novel mouse mutants carrying point mutations in endogenous Tardbp to dissect TDP‐43 function at physiological levels both in vitro and in vivo. Interestingly, we find that mutations within the C‐terminal domain of TDP‐43 lead to a gain of splicing function. Using two different strains, we are able to separate TDP‐43 loss‐ and gain‐of‐function effects. TDP‐43 gain‐of‐function effects in these mice reveal a novel category of splicing events controlled by TDP‐43, referred to as “skiptic” exons, in which skipping of constitutive exons causes changes in gene expression. In vivo, this gain‐of‐function mutation in endogenous Tardbp causes an adult‐onset neuromuscular phenotype accompanied by motor neuron loss and neurodegenerative changes. Furthermore, we have validated the splicing gain‐of‐function and skiptic exons in ALS patient‐derived cells. Our findings provide a novel pathogenic mechanism and highlight how TDP‐43 gain of function and loss of function affect RNA processing differently, suggesting they may act at different disease stages.

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Section: Methodsmentioning

confidence: 99%

Mice with endogenous TDP ‐43 mutations exhibit gain of splicing function and characteristics of amyotrophic lateral sclerosis

et al. 2018

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“…To test if the identified, differentially spliced, transcripts in PSIΔAB fly heads are direct targets bound by PSI, we performed PSI iCLIP (individual-nucleotide resolution cross-linking and immunoprecipitation) experiments (20) with nuclear extracts prepared from Drosophila Schneider Line-2 (S2) cells. Using a statistical method adapted from previous applications of HITS-CLIP (21) and iCLIP (22) (Materials and Methods), we identified 4,937 PSI binding sites in transcripts from 1,628 genes (Table S1 and Dataset S5).…”

Section: Targeting Of Psi To Key Courtship Behavior Regulatory Gene Tmentioning

confidence: 99%

The PSI–U1 snRNP interaction regulates male mating behavior in Drosophila

Wang

Taliaferro

Klibaite

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Alternative pre-mRNA splicing (AS) is a critical regulatory mechanism that operates extensively in the nervous system to produce diverse protein isoforms. Fruitless AS isoforms have been shown to influence male courtship behavior, but the underlying mechanisms are unknown. Using genome-wide approaches and quantitative behavioral assays, we show that the P-element somatic inhibitor (PSI) and its interaction with the U1 small nuclear ribonucleoprotein complex (snRNP) control male courtship behavior. PSI mutants lacking the U1 snRNP-interacting domain (PSIΔAB mutant) exhibit extended but futile mating attempts. The PSIΔAB mutant results in significant changes in the AS patterns of ∼1,200 genes in the Drosophila brain, many of which have been implicated in the regulation of male courtship behavior. PSI directly regulates the AS of at least one-third of these transcripts, suggesting that PSI-U1 snRNP interactions coordinate the behavioral network underlying courtship behavior. Importantly, one of these direct targets is fruitless, the master regulator of courtship. Thus, PSI imposes a specific mode of regulatory control within the neuronal circuit controlling courtship, even though it is broadly expressed in the fly nervous system. This study reinforces the importance of AS in the control of gene activity in neurons and integrated neuronal circuits, and provides a surprising link between a pleiotropic pre-mRNA splicing pathway and the precise control of successful male mating behavior.PSI | U1 snRNP | alternative pre-mRNA splicing | male courtship behavior H ow gene regulation modulates neuronal activities leading to cognition and behavior is an important question in biology. Although many behavior-associated genes and neuronal cell types have been identified, a detailed understanding that links the molecular events of gene regulation to specific behaviors is still lacking. Alternative pre-mRNA splicing (AS) is a crucial gene regulatory mechanism that enables a single gene to generate functionally distinct messenger RNA transcripts (mRNAs) and protein products (1). The nervous system makes extensive use of AS to generate diverse and complex neural mRNA expression patterns that determine numerous neuronal cell types and functions (2). AS is regulated by the small nuclear ribonucleoprotein complexes (snRNPs) that compose the spliceosome for intron recognition and removal, as well as a large repertoire of non-snRNP RNA-binding proteins that affect decisions on splice site use (3). This dynamic and complex AS regulatory network modulates diverse neuronal functions, like synaptic transmission and signal processing, hence further impacting higher brain functions, such as cognition and behavioral control (4).The Drosophila KH-domain RNA binding splicing factor P-element somatic inhibitor (PSI) is best known for regulating tissue-specific AS of the Drosophila P-element transposon transcripts to restrict transposition activity to germ-line tissues (5, 6). PSI directly interacts with the U1 snRNP through a 70-aa tandem direct ...

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“…Hence, it becomes important to understand the structural and functional characteristics of RBPs in humans. Increasing interest in RBPs has led to the development of various experimental protocols like SELEX (systematic evolution of ligands by exponential enrichment) [9,10], CLIP [11], PAR-CLIP [12,13], iCLIP [14] and RNA compete [15], to identify the binding specificities of RBPs; thus adding context and dynamics to the regulation of gene expression at post-transcriptional level.…”

Section: Introductionmentioning

confidence: 99%