Mass-spectrometric analysis of proteins cross-linked to 4-thio-uracil- and 5-bromo-uracil-substituted RNA

Kramer, Katharina; Hummel, Petra; Hsiao, He-Hsuan; Luo, Xiao; Wahl, Markus; Urlaub, Henning

doi:10.1016/j.ijms.2010.10.009

Cited by 32 publications

(70 citation statements)

References 34 publications

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“…In addition, we also took an unbiased approach using UV-cross-linking followed by MS to identify protein-RNA contact sites (Luo et al 2008;Kramer et al 2011). …”

Section: Genes and Development 755mentioning

confidence: 99%

The NHL domain of BRAT is an RNA-binding domain that directly contacts the hunchback mRNA for regulation

et al. 2014

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The Drosophila protein brain tumor (Brat) forms a complex with Pumilio (Pum) and Nanos (Nos) to repress hunchback (hb) mRNA translation at the posterior pole during early embryonic development. It is currently thought that complex formation is initiated by Pum, which directly binds the hb mRNA and subsequently recruits Nos and Brat. Here we report that, in addition to Pum, Brat also directly interacts with the hb mRNA. We identify Brat-binding sites distinct from the Pum consensus motif and show that RNA binding and translational repression by Brat do not require Pum, suggesting so far unrecognized Pum-independent Brat functions. Using various biochemical and biophysical methods, we also demonstrate that the NHL (NCL-1, HT2A, and LIN-41) domain of Brat, a domain previously believed to mediate protein-protein interactions, is a novel, sequence-specific ssRNA-binding domain. The Brat-NHL domain folds into a six-bladed b propeller, and we identify its positively charged top surface as the RNA-binding site. Brat belongs to the functional diverse TRIM (tripartite motif)-NHL protein family. Using structural homology modeling, we predict that the NHL domains of all TRIM-NHL proteins have the potential to bind RNA, indicating that Brat is part of a conserved family of RNA-binding proteins.

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“…In addition, we also took an unbiased approach using UV-cross-linking followed by MS to identify protein-RNA contact sites (Luo et al 2008;Kramer et al 2011). …”

Section: Genes and Development 755mentioning

confidence: 99%

The NHL domain of BRAT is an RNA-binding domain that directly contacts the hunchback mRNA for regulation

et al. 2014

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“…After digestion with trypsin and RNases A and T1, the crosslinked sites were identified by Nano-LC-ESI-MS as previously described (Kramer et al 2011;Schmitzová et al 2012). …”

Section: Protein Expression and Purificationmentioning

confidence: 99%

The Prp8 RNase H-like domain inhibits Brr2-mediated U4/U6 snRNA unwinding by blocking Brr2 loading onto the U4 snRNA

et al. 2012

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The spliceosomal RNA helicase Brr2 catalyzes unwinding of the U4/U6 snRNA duplex, an essential step for spliceosome catalytic activation. Brr2 is regulated in part by the spliceosomal Prp8 protein by an unknown mechanism. We demonstrate that the RNase H (RH) domain of yeast Prp8 binds U4/U6 small nuclear RNA (snRNA) with the single-stranded regions of U4 and U6 preceding U4/U6 stem I, contributing to its binding. Via cross-linking coupled with mass spectrometry, we identify RH domain residues that contact the U4/U6 snRNA. We further demonstrate that the same single-stranded region of U4 preceding U4/U6 stem I is recognized by Brr2, indicating that it translocates along U4 and first unwinds stem I of the U4/U6 duplex. Finally, we show that the RH domain of Prp8 interferes with U4/U6 unwinding by blocking Brr2's interaction with the U4 snRNA. Our data reveal a novel mechanism whereby Prp8 negatively regulates Brr2 and potentially prevents premature U4/U6 unwinding during splicing. They also support the idea that the RH domain acts as a platform for the exchange of U6 snRNA for U1 at the 59 splice site. Our results provide insights into the mechanism whereby Brr2 unwinds U4/U6 and show how this activity is potentially regulated prior to spliceosome activation.[Keywords: pre-mRNA splicing; RNA helicase; RNA-protein complex; RNA-protein cross-linking; spliceosome catalytic activation] Supplemental material is available for this article. Received July 11, 2012; revised version accepted September 18, 2012. Pre-mRNA splicing is catalyzed by a multisubunit RNAprotein enzyme, the spliceosome, which carries out two successive trans-esterification reactions that lead to removal of an intron and the ligation of its flanking exons. Spliceosomes are formed via the stepwise recruitment of small nuclear ribonucleoprotein particles (snRNPs) and numerous non-snRNP proteins to the pre-mRNA substrate (for review, see Wahl et al. 2009). Initially, U1 and U2 snRNPs bind the 59 splice site (59 SS) and the branch site (BS) of the pre-mRNA's intron, respectively. This is followed by the recruitment of the U4/U6.U5 tri-snRNP to the spliceosome, yielding the B complex, which does not yet have an active center. The U4 and U6 snRNAs are extensively base-paired in the tri-snRNP, thereby keeping U6 small nuclear RNA (snRNA) catalytically inert (Staley and Guthrie 1998). For catalytic activation of the spliceosome, U4 snRNA must be displaced from U6 snRNA, which allows the formation of new U2/U6 base-pairing interactions and a catalytically important U6 internal stem-loop (ISL) ). Concomitant with or prior to this, the base-pairing interaction between the U1 snRNA and the 59 SS must be disrupted to allow the highly conserved ACAGAGA sequence of U6 snRNA to base-pair with the 59 end of the intron. This newly formed U2-U6-pre-mRNA RNA interaction network is thought to comprise the heart of the catalytic center of the spliceosome (Madhani and Guthrie 1992;Villa et al. 2002).Brr2 catalyzes the dissociation of the U4/U6 duplex and thus plays a ke...

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“…The first step in the workflow is a conventional database search to identify residual non-cross-linked peptides and also non-cross-linked oligonucleotides. The latter can be deduced from the fractional masses which differ from those of peptides and cross-linked peptides [40]. Once these precursors have been discarded from the MS data, the precursor masses of putative cross-links between the non-UV-irradiated control and the UV-irradiated sample are compared.…”

Section: Discussionmentioning

confidence: 99%

“…Precursors with identical masses and the same retention times in LC-MS/MS are deleted, as these cannot represent UV-induced peptide-RNA oligonucleotide cross-links (i.e., because they are present in both the irradiated and the non-irradiated sample). In the remaining set of spectra, a so-called precursor variant approach is applied [40]. Basically, as the composition and hence the mass of the cross-linked RNA are not known, several combinations of theoretical masses of putative RNA oligonucleotides are subtracted from each precursor mass.…”

Section: Discussionmentioning

confidence: 99%

“…UV-induced cross-linking between proteins and RNA is a radical-induced reaction with relatively low yields [40]. Therefore, one essential step is enrichment of cross-linked species, i.e., cross-linked peptide-RNA oligonucleotides from the complex mixture obtained after digestion of protein-RNA complexes; this mixture consists mainly of non-cross-linked peptides and RNA oligonucleotides.…”

Section: Enrichment Of Cross-linked Peptide-rna Oligonucleotidesmentioning

confidence: 99%

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Analysis of protein–RNA interactions in CRISPR proteins and effector complexes by UV-induced cross-linking and mass spectrometry

Sharma

Hrle

Kramer

et al. 2015

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a b s t r a c tRibonucleoprotein (RNP) complexes play important roles in the cell by mediating basic cellular processes, including gene expression and its regulation. Understanding the molecular details of these processes requires the identification and characterization of protein-RNA interactions. Over the years various approaches have been used to investigate these interactions, including computational analyses to look for RNA binding domains, gel-shift mobility assays on recombinant and mutant proteins as well as co-crystallization and NMR studies for structure elucidation. Here we report a more specialized and direct approach using UV-induced cross-linking coupled with mass spectrometry. This approach permits the identification of cross-linked peptides and RNA moieties and can also pin-point exact RNA contact sites within the protein. The power of this method is illustrated by the application to different singleand multi-subunit RNP complexes belonging to the prokaryotic adaptive immune system, CRISPR-Cas (CRISPR: clustered regularly interspaced short palindromic repeats; Cas: CRISPR associated). In particular, we identified the RNA-binding sites within three Cas7 protein homologs and mapped the cross-linking results to reveal structurally conserved Cas7 -RNA binding interfaces. These results demonstrate the strong potential of UV-induced cross-linking coupled with mass spectrometry analysis to identify RNA interaction sites on the RNA binding proteins.

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Mass-spectrometric analysis of proteins cross-linked to 4-thio-uracil- and 5-bromo-uracil-substituted RNA

Cited by 32 publications

References 34 publications

The NHL domain of BRAT is an RNA-binding domain that directly contacts the hunchback mRNA for regulation

The NHL domain of BRAT is an RNA-binding domain that directly contacts the hunchback mRNA for regulation

The Prp8 RNase H-like domain inhibits Brr2-mediated U4/U6 snRNA unwinding by blocking Brr2 loading onto the U4 snRNA

Analysis of protein–RNA interactions in CRISPR proteins and effector complexes by UV-induced cross-linking and mass spectrometry

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