Assessing RNA Interactions with Proteins by DRaCALA

Patel, Darshan; Gebbie, Margo; Lee, Vincent T.

doi:10.1016/b978-0-12-801122-5.00021-0

Cited by 9 publications

(8 citation statements)

References 28 publications

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“… a : RNAs were labeled with 32 P at their 5’ end. b : Affinities were measured using an established binding assay for radiolabeled RNAs (Roelofs et al, 2011; Patel et al, 2014). …”

Section: Resultsmentioning

confidence: 99%

“…To measure K d , serial dilutions of purified His 10 -MBP-Orn, His 10 -MBP Orn mutants, or untagged Orn in binding buffer (10 mM Tris, pH 8, 100 mM NaCl, and 5 mM CaCl 2 ) were mixed with radiolabeled nucleotides, applied to nitrocellulose sheets, dried, imaged and K d values were calculated as described previously (Roelofs et al, 2011; Patel et al, 2014).…”

Section: Methodsmentioning

confidence: 99%

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A dedicated diribonuclease resolves a key bottleneck for the terminal step of RNA degradation

Kim

Lormand

Weiss

et al. 2019

eLife

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Degradation of RNA polymers, an ubiquitous process in all cells, is catalyzed by specific subsets of endo- and exoribonucleases that together recycle RNA fragments into nucleotide monophosphate. In γ-proteobacteria, 3-‘5’ exoribonucleases comprise up to eight distinct enzymes. Among them, Oligoribonuclease (Orn) is unique as its activity is required for clearing short RNA fragments, which is important for cellular fitness. However, the molecular basis of Orn’s unique cellular function remained unclear. Here, we show that Orn exhibits exquisite substrate preference for diribonucleotides. Crystal structures of substrate-bound Orn reveal an active site optimized for diribonucleotides. While other cellular RNases process oligoribonucleotides down to diribonucleotide entities, Orn is the one and only diribonucleotidase that completes the terminal step of RNA degradation. Together, our studies indicate RNA degradation as a step-wise process with a dedicated enzyme for the clearance of a specific intermediate pool, diribonucleotides, that affects cellular physiology and viability.

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“… a : RNAs were labeled with 32 P at their 5’ end. b : Affinities were measured using an established binding assay for radiolabeled RNAs (Roelofs et al, 2011; Patel et al, 2014). …”

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

A dedicated diribonuclease resolves a key bottleneck for the terminal step of RNA degradation

Kim

Lormand

Weiss

et al. 2019

eLife

View full text Add to dashboard Cite

show abstract

“…To measure K D , serial dilutions of purified His-MBP-Orn or His-MBP Orn mutants in binding buffer (10 mM Tris, pH 8, 100 mM NaCl, and 5 mM CaCl 2 ) were mixed with radiolabeled nucleotides, applied to nitrocellulose sheets, dried, imaged and K D values were calculated as described previously 28,50 .…”

Section: Methodsmentioning

confidence: 99%

Oligoribonuclease functions as a diribonucleotidase to bypass a key bottleneck in RNA degradation

et al. 2019

Preprint

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23Degradation of RNA polymers is a multistep process catalyzed by specific subsets of RNases. In 24 all cases, degradation is completed by exoribonucleases that recycle RNA fragments into 25 nucleotide monophosphate. In γ-proteobacteria, a group of up to eight 3'-5' exoribonucleases 26 have been implicated in RNA degradation. Oligoribonuclease (Orn) is unique among them as its 27 activity is required for clearing short RNA fragments, a function important for cellular fitness. 28 However, the mechanistic basis for this substrate selectivity remained unclear. Here we show 29 that Orn's activity as a general exoribonuclease has been vastly overestimated by demonstrating 30 that the enzyme exhibits a much narrower substrate preference for diribonucleotides. Co-crystal 31 structures of Orn with substrates reveal an active site optimized for diribonucleotides that does 32 not accommodate longer substrates. While other cellular RNases process oligoribonucleotides 33 down to diribonucleotide entities, our functional studies demonstrate that Orn is the one and 34 only diribonucleotidase that completes the final stage of the RNA degradation pathway. 35Together, these results indicate that Orn is a dedicated diribonucleotidase that clears the 36 diribonucleotide pool that otherwise affects cellular physiology and viability. 37 38 14 . Since the discovery of c-di-GMP over 30 years ago, it has been known that the signal is degraded 56 by a two-step process with a linear pGpG diribonucleotide intermediate 15 . While the enzyme for 57 linearizing c-di-GMP to pGpG was discovered early on 16,17 , the identity of the enzyme that degrades 58 pGpG remained elusive. Two recent studies showed that Orn is the primary enzyme that degrades 59 pGpG in Pseudomonas aeruginosa 18,19 . In an orn deletion strain, the accrual of linear pGpG has a 60 profound effect on cells. Specifically, the increase in pGpG inhibits upstream phosphodiesterases that 61 degrade c-di-GMP, thereby triggering phenotypes associated with high cellular c-di-GMP levels. 62 However, the molecular basis of Orn's unique cellular functions in γ-proteobacteria that distinguishes 63 it from all other exoribonucleases remains unexplained. 64Since its discovery over 50 years ago 20 , Orn has been presumed to degrade 65 oligoribonucleotides 21-23 . This notion largely derived from assays utilizing two types of substrates. In 66 one series of experiments, 3 H polyuridine (poly(U)) was incubated with Orn, other enzymes, or lysates 67 and analyzed by paper chromatography, which offers limited resolution overall [21][22][23] . In a second set of 68 experiments, Orn was incubated with oligoribonucleotides that had been tagged at their 5' terminus by 69

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“…DRaCALAs were performed as described previously [ 26 ]. Briefly, RNAs were dephosphorylated and 5’ radiolabeled with 30μCi ATP.…”

Section: Methodsmentioning

confidence: 99%

Regulation of coronavirus nsp15 cleavage specificity by RNA structure

Salukhe,

Choi,

Van Voorhis

et al. 2023

PLoS ONE

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SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, has had an enduring impact on global public health. However, SARS-CoV-2 is only one of multiple pathogenic human coronaviruses (CoVs) to have emerged since the turn of the century. CoVs encode for several nonstructural proteins (nsps) that are essential for viral replication and pathogenesis. Among them is nsp15, a uridine-specific viral endonuclease that is important in evading the host immune response and promoting viral replication. Despite the established endonuclease function of nsp15, little is known about other determinants of its cleavage specificity. In this study we investigate the role of RNA secondary structure in SARS-CoV-2 nsp15 endonuclease activity. Using a series of in vitro endonuclease assays, we observed that thermodynamically stable RNA structures were protected from nsp15 cleavage relative to RNAs lacking stable structure. We leveraged the s2m RNA from the SARS-CoV-1 3’UTR as a model for our structural studies as it adopts a well-defined structure with several uridines, two of which are unpaired and thus highly probable targets for nsp15 cleavage. We found that SARS-CoV-2 nsp15 specifically cleaves s2m at the unpaired uridine within the GNRNA pentaloop of the RNA. Further investigation revealed that the position of uridine within the pentaloop also impacted nsp15 cleavage efficiency suggesting that positioning within the pentaloop is necessary for optimal presentation of the scissile uridine and alignment within the nsp15 catalytic pocket. Our findings indicate that RNA secondary structure is an important determinant of nsp15 cleavage and provides insight into the molecular mechanisms of RNA recognition by nsp15.

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Assessing RNA Interactions with Proteins by DRaCALA

Cited by 9 publications

References 28 publications

A dedicated diribonuclease resolves a key bottleneck for the terminal step of RNA degradation

A dedicated diribonuclease resolves a key bottleneck for the terminal step of RNA degradation

Oligoribonuclease functions as a diribonucleotidase to bypass a key bottleneck in RNA degradation

Regulation of coronavirus nsp15 cleavage specificity by RNA structure

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