A ribonuclease activity linked to DYW1 in vitro is inhibited by RIP/MORF proteins

Abstract

Organellar C-to-U RNA editing in plants occurs in complexes composed of various classes of nuclear-encoded proteins. DYW-deaminases are zinc metalloenzymes that catalyze hydrolytic deamination required for C-to-U modification editing. Solved crystal structures for DYW-deaminase domains display all structural features consistent with a canonical cytidine deamination mechanism. However, some recombinant DYW-deaminases from plants have been associated with ribonuclease activity in vitro. Direct ribonuclease activity by an editing factor is confounding since it is not required for deamination of cytosine, theoretically would be inimical for mRNA editing, and does not have a clear physiological function in vivo. His-tagged recombinant DYW1 from Arabidopsis thaliana (rAtDYW1) was expressed and purified using immobilized metal affinity chromatography (IMAC). Fluorescently labeled RNA oligonucleotides were incubated with recombinant AtDYW1 under different conditions. Percent relative cleavage of RNA probes was recorded at multiple time points from triplicate reactions. The effects of treatment with zinc chelators EDTA and 1, 10-phenanthroline were examined for rAtDYW1. Recombinant His-tagged RNA editing factors AtRIP2, ZmRIP9, AtRIP9, AtOZ1, AtCRR4, and AtORRM1 were expressed in E. coli and purified. Ribonuclease activity was assayed for rAtDYW1 in the presence of different editing factors. Lastly, the effects on nuclease activity in the presence of nucleotides and modified nucleosides were investigated. RNA cleavage observed in this study was linked to the recombinant editing factor rAtDYW1 in vitro. The cleavage reaction is sensitive to high concentrations of zinc chelators, indicating a role for zinc ions for activity. The addition of equal molar concentrations of recombinant RIP/MORF proteins reduced cleavage activity associated with rAtDYW1. However, addition of equal molar concentrations of purified recombinant editing complex proteins AtCRR4, AtORRM1, and AtOZ1 did not strongly inhibit ribonuclease activity on RNAs lacking an AtCRR4 cis-element. Though AtCRR4 inhibited AtDYW1 activity for oligonucleotides with a cognate cis-element. The observation that editing factors limit ribonuclease activity of rAtDYW1 in vitro, suggests that nuclease activities are limited to RNAs in absence of native editing complex partners. Purified rAtDYW1 was associated with the hydrolysis of RNA in vitro, and activity was specifically inhibited by RNA editing factors.

Figure 1

Recombinant AtDYW1 has nuclease activity in vitro. (A) An image of a Coomassie-stained SDS-PAGE to assess purity of the IMAC purified rAtDYW1 with 1, 2, and 5 μg loaded per lane. (B) RNA oligonucleotides labeled with a 5′ Cy-5 fluorophore visualized after separation on a 6 M Urea 20% Polyacrylamide gel from assay of nuclease activity. From left to right lanes represent rAtDYW1 and BSA were incubated from < 1 to 60 min followed by a 2.5 μg/mL RNaseA control at 60 min. (C) Relative cleavage $\left[\frac{(\text{Intensity at}0\text{min}-\text{Intesity at timepoint})}{\text{Intensity at}0\text{min}},\times ,100\right]$ was calculated and plotted in an X-ray scatterplot for timepoints 20, 40, and 60 min for rAtDYW1 and an equivalent reaction with addition of 40 U/mL of Proteinase K. Error bars represent 1 standard deviation of the mean for three separate reaction replicates. (D) Ribonuclease activity was assayed in reactions with a titration of rAtDYW1 from 225 to 28:1 molar ratio compared to labeled RNA probe at 15 nM. Dotted trendline is a best fit line using a logarithmic fit. For (B–D) the oligo Zmrps14 was used.

Figure 2

AtDYW1 was incubated with 5 different RNA oligonucleotides (AtndhD, M13 FAM, ZmndhB, Zmrps14, and PpccmFC) at 28 °C for 20 min in triplicate reactions (lanes A–C) and compared to the oligonucleotide without incubation with DYW1 (lanes O). RNA species from reactions were separated on a 20% polyacrylamide gel in 6 M urea and imaged using an Azure c400 imager using channels for each specific fluorophore. At left of each gel image, letters represent the likely 3′ nucleotide identify. Bands were ranked as highly (***), moderately (**), and slightly (*) increased species compared to the oligonucleotide control.

Figure 3

Ribonuclease activity for rAtDYW1 is sensitive to treatment with zinc ion chelators. Fractions of rAtDYW1 were dialyzed for 3 days with and without an inhibitor cocktail of 25 mM EDTA and 0.65 mM 1,10-phenanthroline. Ribonuclease activity was assayed for dialyzed rAtDYW1 fractions. At top, a picture of RNA oligos with the Zmrps14 sequence separated on a 6 M urea 20% PAGE. Lanes from left to right represent an RNA oligo control, triplicate reactions of the RNA oligo subjected to reaction conditions without enzyme, RNA oligonucleotides treated with rAtDYW1 dialyzed 3 days at 4 °C without chelator, and RNA treated with rAtDYW1 dialyzed in a buffer containing EDTA and 1,10-phenanthroline.

Figure 4

Recombinant AtRIP2 inhibits ribonuclease activity of rAtDYW1. (A) The Coomassie stained SDS-PAGE image indicates the purity of rAtRIP2 fractions after one round of Ni-NTA affinity chromatography and one round of size exclusion chromatography. (B) Ribonuclease activity on RNA oligonucleotides was assayed at 0 min, 20 min, 40 min and 60 min in triplicate reactions (representative image from one time course shown) in the presence of rAtDYW1, rAtRIP2, and rAtDYW1 mixed with rAtRIP2 in an equimolar ratio (1:1). At top a representative image is shown of the set of the 6 M urea 20% PAGE used to quantify percent relative cleavage. Below a graph displays % relative cleavage of all triplicate reactions across 4 timepoints. (C) Ribonuclease activity was assayed for triplicate reactions containing 2.5 μg/mL RNaseA, 2.5 ng/μL RNaseA + 5 μM rAtRIP2, and 2.5 μg/mL RNaseA + 4 U/μL Ribonuclease inhibitor (RNaseIN). At top a representative image of one time-course reaction is shown and below a X–Y scatterplot displays % relative cleavage as a function of time for triplicate reactions. (D) Activity was measured in triplicate reactions with various stoichiometric ratios of rAtRIP2 versus rAtDYW1 from (1:1 to 1:200). Representative gel images are shown at left and % relative cleavage for triplicate reactions are represented in a X–Y scatterplot at right. (B–D) Error bars represent 1 standard deviation from the mean for triplicate reaction replicates. (B–D) used the oligo Zmrps14.

Figure 5

Recombinant ZmRIP9 progressively inhibits ribonuclease activity of rAtDYW1 at increasing relative molar ratios. (A) Recombinant ZmRIP9 was purified using Ni-NTA affinity followed by size exclusion chromatography steps. An image of a representative Coomassie SDS-PAGE indicates purity. (B) At top, ribonuclease activity was assayed for triplicate reactions at 0, 20, 40, and 60 min timepoints containing rZmRIP9 mixed with rAtDYW1 with stoichiometric ratios of 5:1, 2:1, 1:1, and 1:2. Below, a X–Y scatterplot displays % relative cleavage at 4 timepoints. Error bars represent 1 standard deviation from the mean for triplicate reactions. (B) Nuclease reaction use the Zmrps14 oligo.

Figure 6

The editing factor rAtOZ1 does not strongly inhibit rAtDYW1 at an equimolar ratio. (A) Recombinant AtOZ1 was purified using a single round of IMAC and an SDS-PAGE image is shown to evaluate protein purity. (B) Ribonuclease activity was assayed for triplicate reactions containing rAtDYW1, rAtOZ1, and rAtDYW1 + rAtOZ1 mixed in an equimolar ratio on the oligonucleotide Zmrps14. A representative image of a 6 M urea 20% PAGE is shown at top and the data from the triplicate reactions is represented in the X–Y scatterplot below. (B) Error bars represent 1 standard deviation from the mean.

Figure 7

Recombinant AtDYW1 ribonuclease activity was reduced on oligonucleotides with AtndhD sequences when incubated with rAtCRR4 but not affected by not affected by addition of nucleotides or tetrahydrouridine. (A) Recombinant AtCRR4 was purified by IMAC followed by gel filtration and the SDS-PAGE image relates purity. (B) Hydrolysis of RNA Zmrps14 oligonucleotides was assayed in triplicate reactions with rAtDYW1, rAtDYW1 + rAtCRR4 mixed in an equimolar ratio, and rAtCRR4. At top a representative image of RNA oligonucleotides separated on a 6 M urea 20% PAGE. Below a scatterplot represents % relative cleavage at 4 time points for the triplicate reactions. (C) Images of 20% PAGE in 6 M urea show RNA species created through out a 0, 20, 40, 60 min time course from incubation of AtndhD oligonucleotides with rAtDYW1 alone (top), rAtDYW1 with equimolar rAtCRR4 (middle), and rAtDYW1 with equimolar rAtCRR4 in the presence of THU. Reactions were run in triplicate and lanes are labeled for each reaction with (A–C). (D) % relative cleavage was plotted versus time calculated from the intensity of bands from the gel using ImageJ. Error bars represent one standard deviation from the mean for triplicate reactions. (B,D) Error bars represent 1 standard deviation from the mean.