A rapid method for assessing the RNA-binding potential of a protein

Bendak, Katerina; Loughlin, Fionna E.; Cheung, Vivian G.; O’Connell, Mitchell R.; Crossley, Merlin; Mackay, Joel P.

doi:10.1093/nar/gks285

Cited by 30 publications

(42 citation statements)

References 37 publications

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“…To assess more broadly the ability of YY1(F1–4) to bind to RNA, we tested this construct in EMSAs with Pentaprobes (PPs), a set of twelve 100-bp, single-stranded RNA sequences that collectively represent all possible 5-mers (33,34). The diverse set of binding sites available on these probes makes them a good screen for nucleic acid binding activity, and it is likely that they contain both single- and double-stranded elements.…”

Section: Resultsmentioning

confidence: 99%

The zinc fingers of YY1 bind single-stranded RNA with low sequence specificity

et al. 2016

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Classical zinc fingers (ZFs) are traditionally considered to act as sequence-specific DNA-binding domains. More recently, classical ZFs have been recognised as potential RNA-binding modules, raising the intriguing possibility that classical-ZF transcription factors are involved in post-transcriptional gene regulation via direct RNA binding. To date, however, only one classical ZF-RNA complex, that involving TFIIIA, has been structurally characterised. Yin Yang-1 (YY1) is a multi-functional transcription factor involved in many regulatory processes, and binds DNA via four classical ZFs. Recent evidence suggests that YY1 also interacts with RNA, but the molecular nature of the interaction remains unknown. In the present work, we directly assess the ability of YY1 to bind RNA using in vitro assays. Systematic Evolution of Ligands by EXponential enrichment (SELEX) was used to identify preferred RNA sequences bound by the YY1 ZFs from a randomised library over multiple rounds of selection. However, a strong motif was not consistently recovered, suggesting that the RNA sequence selectivity of these domains is modest. YY1 ZF residues involved in binding to single-stranded RNA were identified by NMR spectroscopy and found to be largely distinct from the set of residues involved in DNA binding, suggesting that interactions between YY1 and ssRNA constitute a separate mode of nucleic acid binding. Our data are consistent with recent reports that YY1 can bind to RNA in a low-specificity, yet physiologically relevant manner.

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Section: Resultsmentioning

confidence: 99%

The zinc fingers of YY1 bind single-stranded RNA with low sequence specificity

et al. 2016

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“…40 The Pentaprobes have been used to determine the sequence specificity of RNases with recognition sequences of five or fewer nucleotides. Cleavage of each Pentaprobe by the RNase of interest is assessed by PAGE.…”

Section: Resultsmentioning

confidence: 99%

Light Activation of Staphylococcus aureus Toxin YoeB_Sa1 Reveals Guanosine-Specific Endoribonuclease Activity

Larson

Hergenrother

2013

Biochemistry

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The Staphylococcus aureus chromosome harbors two homologues of the YefM-YoeB toxin-antitoxin (TA) system. The toxins YoeBSa1 and YoeBSa2 possess ribosome-dependent ribonuclease (RNase) activity in Escherichia coli. This activity is similar to that of the E. coli toxin YoeBEc, an enzyme that, in addition to ribosome-dependent RNase activity, possesses ribosome-independent RNase activity in vitro. To investigate whether YoeBSa1 is also a ribosome-independent RNase, YoeBSa1 was expressed using a novel strategy, and its in vitro RNase activity, sequence specificity, and kinetics were characterized. Y88 of YoeBSa1 was critical for in vitro activity and cell culture toxicity. This residue was mutated to ortho-nitrobenzyl tyrosine (ONBY) via unnatural amino acid mutagenesis. YoeBSa1-Y88ONBY could be expressed in the absence of the antitoxin YefMSa1 in E. coli. Photocaged YoeBSa1-Y88ONBY displayed UV light-dependent RNase activity toward free mRNA in vitro. The in vitro ribosome-independent RNase activity of YoeBSa1-Y88ONBY, YoeBSa1-Y88F, and YoeBSa1-Y88TAG was significantly reduced or abolished. In contrast to YoeBEc, which cleaves RNA at both adenosine and guanosine with a preference for adenosine, YoeBSa1 cleaved mRNA specifically at guanosine. Using this information, a fluorometric assay was developed and used to determine the kinetic parameters for ribosome-independent RNA cleavage by YoeBSa1.

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“…The 12 Pentaprobe vectors can be used to make ss and ds RNA and DNA substrates that encode every combination of five bases (Bendak et al 2012). We have demonstrated ribonuclease activity for VapC Rv0065 and VapC Rv0617 using one of the ss Pentaprobe RNA substrates (Ahidjo et al 2011).…”

Section: Screening For Vapc Sequence-specificity Using Pentaprobesmentioning

confidence: 98%

“…This sequence was then encoded in six overlapping dsDNA molecules from which RNA can be transcribed, resulting in 12 ssRNA segments (six in the forward direction and six complementary sequences in the reverse direction) covering every combination of five bases (Bendak et al 2012). The reverse Pentaprobe sequences also allow dsRNA to be synthesized.…”

Section: Introductionmentioning

confidence: 99%

Determination of ribonuclease sequence-specificity using Pentaprobes and mass spectrometry

McKenzie

Duyvestyn²,

Smith³

et al. 2012

RNA

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The VapBC toxin-antitoxin (TA) family is the largest of nine identified TA families. The toxin, VapC, is a metal-dependent ribonuclease that is inhibited by its cognate antitoxin, VapB. Although the VapBCs are the largest TA family, little is known about their biological roles. Here we describe a new general method for the overexpression and purification of toxic VapC proteins and subsequent determination of their RNase sequence-specificity. Functional VapC was isolated by expression of the nontoxic VapBC complex, followed by removal of the labile antitoxin (VapB) using limited trypsin digestion. We have then developed a sensitive and robust method for determining VapC ribonuclease sequence-specificity. This technique employs the use of Pentaprobes as substrates for VapC. These are RNA sequences encoding every combination of five bases. We combine the RNase reaction with MALDI-TOF MS to detect and analyze the cleavage products and thus determine the RNA cut sites. Successful MALDI-TOF MS analysis of RNA fragments is acutely dependent on sample preparation methods. The sequencespecificity of four VapC proteins from two different organisms (VapC PAE0151 and VapC PAE2754 from Pyrobaculum aerophilum, and VapC Rv0065 and VapC Rv0617 from Mycobacterium tuberculosis) was successfully determined using the described strategy. This rapid and sensitive method can be applied to determine the sequence-specificity of VapC ribonucleases along with other RNA interferases (such as MazF) from a range of organisms.

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A rapid method for assessing the RNA-binding potential of a protein

Cited by 30 publications

References 37 publications

The zinc fingers of YY1 bind single-stranded RNA with low sequence specificity

The zinc fingers of YY1 bind single-stranded RNA with low sequence specificity

Light Activation of Staphylococcus aureus Toxin YoeB_Sa1 Reveals Guanosine-Specific Endoribonuclease Activity

Determination of ribonuclease sequence-specificity using Pentaprobes and mass spectrometry

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A rapid method for assessing the RNA-binding potential of a protein

Cited by 30 publications

References 37 publications

The zinc fingers of YY1 bind single-stranded RNA with low sequence specificity

The zinc fingers of YY1 bind single-stranded RNA with low sequence specificity

Light Activation of Staphylococcus aureus Toxin YoeBSa1 Reveals Guanosine-Specific Endoribonuclease Activity

Determination of ribonuclease sequence-specificity using Pentaprobes and mass spectrometry

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Light Activation of Staphylococcus aureus Toxin YoeB_Sa1 Reveals Guanosine-Specific Endoribonuclease Activity