Interaction cloning and characterization of RoBPI, a novel protein binding to human Ro ribonucleoproteins

Bouffard, Pascal; Barbar, Élie; Brière, Francis; Boire, Gilles

doi:10.1017/s1355838200990277

Cited by 49 publications

(41 citation statements)

References 22 publications

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“…2B, lanes 10,11), only Y5 remained substantially associated with RoBPI. In sum, our results indicate that RoBPI associates with Y5 as described previously (Bouffard et al 2000) and also binds Y1 and Y3 RNPs. We conclude that RoBPI is not a unique binding partner of Y5 but does engage in specific interactions with a subset of Y RNAs.…”

Section: Rna-binding Specificity Of Robpi and Ifit5supporting

confidence: 88%

“…Together, the results above suggest that particular features of the Y5 internal loop confer to Y5 RNP the ability to recognize variant 5S RNA in RNP. The Y5 internal loop has also been proposed to determine the specificity of RoBPI interaction (Bouffard et al 2000). We were unable to detect copurification of variant 5S RNA with RoBPI (data not shown), consistent with a mutually exclusive interaction of Y5 RNP with either RoBPI or 5S RNP.…”

Section: Y Rna Sequence Requirements For 5s Rna Associationsupporting

confidence: 57%

“…Proteins suggested to interact with Y RNPs include nucleolin and hnRNP I/PTB, both of which are reported to bind only Y1 and Y3 (Fabini et al 2000;Fouraux et al 2002), and Ro RNP-Binding Protein I (RoBPI/FIR/PUF60), a protein identified from diverse assays, including a yeast three-hybrid screen with the combination of Y5 RNA and Ro as bait (Page-McCaw et al 1999;Bouffard et al 2000;Liu et al 2000). To investigate the existence and specificity of substoichiometric interaction partners of an individual human Y RNA, we selected Y5 RNA for additional study.…”

Section: Rna-based Affinity Purification Of Individual Y Rnpsmentioning

confidence: 99%

“…Two additional proteins specific to the Y5-RAT sample were represented by five unique peptides each. One of these was RoBPI, isolated previously as an interaction partner of Y5 RNA and Ro (Bouffard et al 2000). The second was interferon-induced protein with tetratricopeptide repeats 5 (IFIT5), a protein of unknown function.…”

Section: Rna-based Affinity Purification Of Individual Y Rnpsmentioning

confidence: 99%

“…RoBPI has been suggested to bind Y5 but not Y1, Y3, or Y4 RNP based on assays in yeast and human cell extract (Bouffard et al 2000). In order to establish the Y RNAbinding specificity of RoBPI in vivo, we used formaldehyde cross-linking prior to cell lysis to trap endogenous interactions.…”

Section: Rna-binding Specificity Of Robpi and Ifit5mentioning

confidence: 99%

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Human Y5 RNA specializes a Ro ribonucleoprotein for 5S ribosomal RNA quality control

Hogg

Collins²

2007

Genes Dev.

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Humans express four distinct non-protein-coding Y RNAs (ncRNAs). To investigate Y RNA functional diversification, we exploited an RNA-based affinity purification method to isolate ribonucleoproteins (RNPs) assembled on individual human Y RNAs. Silver staining and mass spectrometry revealed that the Ro and La proteins assemble with all Y RNAs, while additional proteins associate with specific Y RNAs. Unexpectedly, Y5 RNA uniquely copurified ribosomal protein L5 and its binding partner 5S RNA. These findings reveal a contribution of Y5 to 5S surveillance and suggest that interactions between Ro-Y5 and L5-5S RNPs establish 5S RNA as a target of quality control.Supplemental material is available at http://www.genesdev.org.Received August 13, 2007; revised version accepted October 11, 2007. Empirical and computational approaches have revealed a dramatically expanded complexity of non-protein-coding RNA (ncRNA) in higher eukaryotes (Taft et al. 2007). Here we show that individual human Y RNAs have unique protein and ncRNA interaction specificities. We demonstrate that all four human Y RNAs (Y1, Y3, Y4, and Y5) assemble with the core Y RNP proteins Ro and La, and additional proteins exhibit specific interactions with subsets of Y RNPs. Of the human Y RNAs, only Y5 copurifies ribosomal protein L5 and an extensively characterized target of Ro-mediated quality control, 5S ribosomal RNA. We propose that Y RNAs play both positive and negative roles in establishing the target specificity of ncRNA surveillance by Ro. Our findings provide new insights about the functions of ncRNA and the significance of expansion of vertebrate ncRNA diversity. Results and Discussion RNA-based affinity purification of individual Y RNPsAn RNA-based method of affinity purification was required to separate the RNPs endogenously assembled on human Y1, Y3, Y4, and Y5 RNAs. For this purpose, we developed an RNP purification method termed RNA Affinity in Tandem (RAT). Each Y RNA was expressed in vivo as a fusion to an affinity tag composed of two hairpins: one binding the Pseudomonas phage 7 (PP7) coat protein (CP) and one binding the antibiotic tobramycin (Hogg and Collins 2007). Each Y RNA was tagged at the 5Ј or 3Ј end with one or both orientations of the tag hairpins and expressed under the control of the commonly exploited human U6 RNA Polymerase III promoter by transient transfection of human 293T cells. The most uniform Y RNA expression levels were obtained using a 3Ј tag with the PP7 hairpin followed by the tobramycin hairpin (data not shown). Cells expressing the entire panel of Y RNAs with this tag configuration and a parallel empty vector control were lysed to generate whole-cell extracts. Recombinant PP7CP tagged with the two Protein A domains (ZZ) and Tobacco Etch Virus (TEV) protease cleavage site of the protein tandem affinity purification (TAP) tag (Puig et al. 2001) was added to cell extract, and complexes harboring tagged RNA were recovered on IgG agarose. Following elution by TEV protease, tagged RNPs were bound to tobramycin re...

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Section: Rna-binding Specificity Of Robpi and Ifit5supporting

confidence: 88%

Section: Y Rna Sequence Requirements For 5s Rna Associationsupporting

confidence: 57%