<i>In vivo</i>
            identification of ribonucleoprotein-RNA interactions

Zielinski, Jennifer; Kilk, Kalle; Peritz, Tiina; Kannanayakal, Theresa; Miyashiro, Kevin; Eiríksdóttir, Emelía; Jochems, Jeanine; Langel, Ülo; Eberwine, James

doi:10.1073/pnas.0510611103

Cited by 69 publications

(83 citation statements)

References 53 publications

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“…These results are compatible with the antagonistic role widely described for both SR protein family members on the regulation of alternative splicing in different genes (Tacke & Manley, 1995), such as the growth hormone gene (Solis et al, 2008) and the glutamate receptor subunit 2 gene (GluR2) (Crovato & Egebjerg, 2005). Trans-elements, or splicing factors, act by means of binding to short degenerate sequences of low complexity, and these protein ⁄ RNA interactions can be studied by RNA immunoprecipitation (RIP) (Zielinski et al, 2006). Thus, we found that both SC35 and ASF ⁄ SF2 co-immunoprecipitate with the mRNA corresponding to the long variant of the MinigEND.…”

Section: Discussionsupporting

confidence: 82%

Alternative splicing factor or splicing factor‐2 plays a key role in intron retention of the endoglin gene during endothelial senescence

Blanco

Bernabeu

2011

Aging Cell

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SummaryAlternative splicing involving intron retention plays a key role in the regulation of gene expression. We previously reported that the alternatively spliced short isoform of endoglin (S-endoglin) is induced during the aging or senescence of endothelial cells by a mechanism of intron retention. In this work, we demonstrate that the alternative splicing factor or splicing factor-2 (ASF ⁄ SF2) is involved in the synthesis of endoglin. Overexpression of ASF ⁄ SF2 in endothelial cells switched the balance between the two endoglin isoforms, favoring the synthesis of S-endoglin. Using a minigene reporter vector and RNA immunoprecipitation experiments, it was shown that ASF ⁄ SF2 interacts with the nucleotide sequence of the endoglin minigene, suggesting the direct involvement of ASF ⁄ SF2. Accordingly, the sequence recognized by ASF ⁄ SF2 in the endoglin gene was identified inside the retained intron near the consensus branch point. Finally, the ASF ⁄ SF2 subcellular localization during endothelial senescence showed a preferential scattered distribution throughout the cytoplasm, where it interferes with the activity of the minor spliceosome, leading to an increased expression of S-endoglin mRNA. In summary, we report for the first time the molecular mechanisms by which ASF ⁄ SF2 regulates the alternative splicing of endoglin in senescent endothelial cells, as well as the involvement of ASF ⁄ SF2 in the minor spliceosome.

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Section: Discussionsupporting

confidence: 82%

Alternative splicing factor or splicing factor‐2 plays a key role in intron retention of the endoglin gene during endothelial senescence

Blanco

Bernabeu

2011

Aging Cell

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“…Unlike bandshift assays with cell extracts, the identity of the RBP can be assessed in a hypothesis-free way. Furthermore, there is no length limitation of the nucleic acid probe as is the case in EMSA or peptide-nucleic acid (PNA)-assisted identification of RBPs (34). Quantitative, high-resolution MS results in high confidence identification of specific RNA-protein interactions from crude cell extracts in a single-step affinity purification at near-physiological conditions.…”

Section: Discussionmentioning

confidence: 99%

Unbiased RNA–protein interaction screen by quantitative proteomics

Butter

Scheibe

Mörl

et al. 2009

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

127

115

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Mass spectrometry (MS)-based quantitative interaction proteomics has successfully elucidated specific protein-protein, DNA-protein, and small molecule-protein interactions. Here, we developed a gelfree, sensitive, and scalable technology that addresses the important area of RNA-protein interactions. Using aptamer-tagged RNA as bait, we captured RNA-interacting proteins from stable isotope labeling by amino acids in cell culture (SILAC)-labeled mammalian cell extracts and analyzed them by high-resolution, quantitative MS. Binders specific to the RNA sequence were distinguished from background by their isotope ratios between bait and control. We demonstrated the approach by retrieving known and novel interaction partners for the HuR interaction motif, H4 stem loop, ''zipcode'' sequence, tRNA, and a bioinformatically-predicted RNA fold in DGCR-8/Pasha mRNA. In all experiments we unambiguously identified known interaction partners by a single affinity purification step. The 5 region of the mRNA of DGCR-8/Pasha, a component of the microprocessor complex, specifically interacts with components of the translational machinery, suggesting that it contains an internal ribosome entry site.quantitative mass spectrometry ͉ ribonucleoprotein ͉ RNA-binding proteins ͉ HuR ͉ internal ribosome entry site R ibonucleic acid (RNA) is increasingly recognized for its diversity of cellular functions ranging from its classical roles as structural component in complexes such as the ribosome and its role as the central intermediate in gene expression to recentlydiscovered, critical roles in gene regulation (1). Analysis of RNA can efficiently be performed by hybridization or sequencing-based methods; however, in the cellular environment RNA is associated with RNA-binding proteins (RBPs) forming functional ribonucleoprotein (RNP) complexes. These proteins are essential to the function of RNPs but have been studied much less.Mass spectrometry (MS)-based proteomics has become a widespread tool for studying complex mixtures of proteins at high sensitivity (2, 3). High-resolution and high-accuracy technologies have recently been introduced at a large scale (4), and entire proteomes can now be quantified (5). Quantitative proteomics has also emerged as a powerful tool for detecting specific binding of proteins to baits by an unbiased proteome-wide screen using peptides and proteins (reviewed in ref. 6) and DNA (7, 8) as baits. These screens have provided candidates that subsequently proved to be of physiological importance in vivo (7, 9, 10). However, application of this technique to detect RBPs has not been reported to our knowledge.In one technology of quantitative proteomics, stable isotope labeling of amino acids in cell culture (SILAC), 2 cell populations are metabolically encoded with either 13 C 6 (heavy) amino acids or 12 C 6 (light) amino acids (11,12). Background proteins occur equally in control and bait eluate, and the SILAC peptide pairs consequently have a 1:1 intensity ratio. Specific binders to the bait have heavy/light ratio si...

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“…The PNA-assisted identification of RBPs (PAIR) method developed by the Eberwine laboratory (42) was designed to identify the RBPs that associate with a particular mRNA, to determine where they bind on the message and whether this association changes over time or following receptor-mediated stimulation (Fig. 3).…”

Section: Pairmentioning

confidence: 99%

“…Essential elements of PAIR as defined by Zielinski et al (42) includes (1) CPP-BPA-PNA intracellular delivery, dissociation of the BPA-PNA from the CPP and its hybridization to a complementary sequence on the target RNA, (2) UV crosslinking, (3-4) cell lysis and RNase treatment, (5) isolation of the PNA-RBP complexes by hybridizing to a sense oligo coupled to magnetic beads, followed by (6) proteolysis and mass spectrometry. Use of a PNA -BPA probe targeted to nucleotides upstream of the BPA-PNA probe (box) such that the BPA adduct queries the same sequence space could be used to assess whether local RNA remodeling occurs as a result of probe hybridization.…”

Section: Trimolecular Fluorescencementioning

confidence: 99%

“…These include the crosslinking immuoprecipitation assay (CLIP) developed by the Darnell laboratory, (40) the trimolecular fluorescence assay of Rackam et al (41) and, most recently, the peptide nucleic acid (PNA)-assisted identification of RBPs (PAIR) method designed in the Eberwine laboratory. (42) This article will be devoted to setting these new methods in the context of the ones that have been traditionally used to establish RNA-RBP interactions.…”

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confidence: 99%

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mRNPs take shape by CLIPPING and PAIRING

Denman¹

2006

Bioessays

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The interaction of RNA-binding proteins (RBPs) with RNA is a crucial aspect of normal cellular metabolism. Yet, the diverse number of RBPs and RNA motifs to which they bind, the wide range of interaction strengths and the fact that RBPs associate in dynamic complexes have made it challenging to determine whether a particular RNA-binding protein binds a particular RNA. Recent work by three different laboratories has led to the development of new tools to query such interactions in the more physiological environs of cultured cells. The use of these methods has led to insights into (1) the networks of RNAs regulated by a particular protein, (2) the identification of new protein partners within messenger ribonucleoprotein particles and (3) the flux of RNA-binding proteins on an mRNA throughout its lifecycle. Here, I examine these new methods and discuss their relative strengths and current limitations.

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In vivo identification of ribonucleoprotein-RNA interactions

Cited by 69 publications

References 53 publications

Alternative splicing factor or splicing factor‐2 plays a key role in intron retention of the endoglin gene during endothelial senescence

Alternative splicing factor or splicing factor‐2 plays a key role in intron retention of the endoglin gene during endothelial senescence

Unbiased RNA–protein interaction screen by quantitative proteomics

mRNPs take shape by CLIPPING and PAIRING

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