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PRL1, an RNA-binding protein, positively regulates the accumulation of miRNAs and siRNAs in Arabidopsis.

Zhang S, Liu Y, Yu B - PLoS Genet. (2014)

Bottom Line: In addition, prl1 reduces pri-miRNA levels without affecting pri-miRNA transcription.These results suggest that PRL1 may stabilize pri-miRNAs and function as a co-factor to enhance DCL1 activity.Based on these results, we propose that CDC5 and PRL1 cooperatively regulate pri-miRNA levels, which results in their synergistic effects on miRNA accumulation, while they function together as a complex to enhance DCL1 activity.

View Article: PubMed Central - PubMed

Affiliation: Center for Plant Science Innovation & School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska, United States of America.

ABSTRACT
The evolutionary conserved WD-40 protein PRL1 plays important roles in immunity and development. Here we show that PRL1 is required for the accumulation of microRNAs (miRNAs) and small interfering RNAs (siRNAs). PRL1 positively influences the processing of miRNA primary transcripts (pri-miRNAs) and double-stranded RNAs (dsRNAs). Furthermore, PRL1 interacts with the pri-miRNA processor, DCL1, and the dsRNA processors (DCL3 and DCL4). These results suggest that PRL1 may function as a general factor to promote the production of miRNAs and siRNAs. We also show that PRL1 is an RNA-binding protein and associates with pri-miRNAs in vivo. In addition, prl1 reduces pri-miRNA levels without affecting pri-miRNA transcription. These results suggest that PRL1 may stabilize pri-miRNAs and function as a co-factor to enhance DCL1 activity. We further reveal the genetic interaction of PRL1 with CDC5, which interacts with PRL1 and regulates transcription and processing of pri-miRNAs. Both miRNA and pri-miRNA levels are lower in cdc5 prl1 than those in either cdc5 or prl1. However, the processing efficiency of pri-miRNAs in cdc5 prl1 is similar to that in cdc5 and slightly lower than that in prl1. Based on these results, we propose that CDC5 and PRL1 cooperatively regulate pri-miRNA levels, which results in their synergistic effects on miRNA accumulation, while they function together as a complex to enhance DCL1 activity.

No MeSH data available.


PRL1 binds pri-miRNAs in vitro and in vivo.(A) Proteins used for in vitro RNA binding assay. Purified MBP and MBP-PRL1 proteins were resolved on SDS-page gel and stained by Coomassie Blue. The lower bands in MBP-PRL1 line are degraded MBP-PRL1 since they can be detected by anti-MBP antibody. M: marker. (B) PRL1 binds MIR162b, pre-miR162b and ssRNA in vitro. MIR162b, pre-miR162b and ssRNA were produced by in vitro transcription. SsRNA: single stranded-RNA; DsRNA: double stranded-RNA. (C) PRL1 binds pri-miRNAs in vivo. NoAb: No antibody control. Transgenic plants harboring pPRL1::PRL1-YFP, pTGH: TGH-YFP or YFP transgene were used for RIP assay with anti-YFP anti-bodies. Ten percent immunoprecipitates and two percent input proteins were analyzed by western blot. Five percent RNAs were used as input RNA.
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pgen-1004841-g007: PRL1 binds pri-miRNAs in vitro and in vivo.(A) Proteins used for in vitro RNA binding assay. Purified MBP and MBP-PRL1 proteins were resolved on SDS-page gel and stained by Coomassie Blue. The lower bands in MBP-PRL1 line are degraded MBP-PRL1 since they can be detected by anti-MBP antibody. M: marker. (B) PRL1 binds MIR162b, pre-miR162b and ssRNA in vitro. MIR162b, pre-miR162b and ssRNA were produced by in vitro transcription. SsRNA: single stranded-RNA; DsRNA: double stranded-RNA. (C) PRL1 binds pri-miRNAs in vivo. NoAb: No antibody control. Transgenic plants harboring pPRL1::PRL1-YFP, pTGH: TGH-YFP or YFP transgene were used for RIP assay with anti-YFP anti-bodies. Ten percent immunoprecipitates and two percent input proteins were analyzed by western blot. Five percent RNAs were used as input RNA.

Mentions: Given the role of PRL1 in RNA metabolism, it is reasonable to speculate that PRL1 might have an RNA-binding activity. We therefore performed an in vitro RNA pull-down assay to test this possibility. In this assay, recombinant PRL1 fused with a maltose-binding protein at its N-terminus (MBP-PRL1) and MBP were expressed in E.coli and purified with amylose resin (Fig. 7A). MBP-PRL1 and MBP were then incubated with [32P]-labeled MIR162b or pre-miR162b, respectively. MBP-PRL1 but not MBP bound MIR162b and pre-miR162b after incubation. In addition, when excess amount of unlabeled MIR162b or pre-miR162b was added in the reaction, radioactive labeled MIR162b or pre-miR162b could not be retained in the MBP-PRL1 complex. These results suggested that PRL1 binds RNAs in vitro. We next tested the binding ability of MBP-PRL1 to dsRNA, ssRNA and DNA using the in vitro RNA pull-down assay described above. MBP-PRL1 was able to bind a ∼100-nt RNA corresponding to a portion of the 5′ end of the UBIQUITIN 5 (UBQ5) [13], but not an in vitro synthesized ∼50 bp DNA fragment [32] and a dsRNA generated through in vitro transcription vitro transcription of a DNA fragment (5′ portion of UBQ5, ∼460 bp) containing the T7 promoter at end of each strand [13] (Fig. 7B).


PRL1, an RNA-binding protein, positively regulates the accumulation of miRNAs and siRNAs in Arabidopsis.

Zhang S, Liu Y, Yu B - PLoS Genet. (2014)

PRL1 binds pri-miRNAs in vitro and in vivo.(A) Proteins used for in vitro RNA binding assay. Purified MBP and MBP-PRL1 proteins were resolved on SDS-page gel and stained by Coomassie Blue. The lower bands in MBP-PRL1 line are degraded MBP-PRL1 since they can be detected by anti-MBP antibody. M: marker. (B) PRL1 binds MIR162b, pre-miR162b and ssRNA in vitro. MIR162b, pre-miR162b and ssRNA were produced by in vitro transcription. SsRNA: single stranded-RNA; DsRNA: double stranded-RNA. (C) PRL1 binds pri-miRNAs in vivo. NoAb: No antibody control. Transgenic plants harboring pPRL1::PRL1-YFP, pTGH: TGH-YFP or YFP transgene were used for RIP assay with anti-YFP anti-bodies. Ten percent immunoprecipitates and two percent input proteins were analyzed by western blot. Five percent RNAs were used as input RNA.
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pgen-1004841-g007: PRL1 binds pri-miRNAs in vitro and in vivo.(A) Proteins used for in vitro RNA binding assay. Purified MBP and MBP-PRL1 proteins were resolved on SDS-page gel and stained by Coomassie Blue. The lower bands in MBP-PRL1 line are degraded MBP-PRL1 since they can be detected by anti-MBP antibody. M: marker. (B) PRL1 binds MIR162b, pre-miR162b and ssRNA in vitro. MIR162b, pre-miR162b and ssRNA were produced by in vitro transcription. SsRNA: single stranded-RNA; DsRNA: double stranded-RNA. (C) PRL1 binds pri-miRNAs in vivo. NoAb: No antibody control. Transgenic plants harboring pPRL1::PRL1-YFP, pTGH: TGH-YFP or YFP transgene were used for RIP assay with anti-YFP anti-bodies. Ten percent immunoprecipitates and two percent input proteins were analyzed by western blot. Five percent RNAs were used as input RNA.
Mentions: Given the role of PRL1 in RNA metabolism, it is reasonable to speculate that PRL1 might have an RNA-binding activity. We therefore performed an in vitro RNA pull-down assay to test this possibility. In this assay, recombinant PRL1 fused with a maltose-binding protein at its N-terminus (MBP-PRL1) and MBP were expressed in E.coli and purified with amylose resin (Fig. 7A). MBP-PRL1 and MBP were then incubated with [32P]-labeled MIR162b or pre-miR162b, respectively. MBP-PRL1 but not MBP bound MIR162b and pre-miR162b after incubation. In addition, when excess amount of unlabeled MIR162b or pre-miR162b was added in the reaction, radioactive labeled MIR162b or pre-miR162b could not be retained in the MBP-PRL1 complex. These results suggested that PRL1 binds RNAs in vitro. We next tested the binding ability of MBP-PRL1 to dsRNA, ssRNA and DNA using the in vitro RNA pull-down assay described above. MBP-PRL1 was able to bind a ∼100-nt RNA corresponding to a portion of the 5′ end of the UBIQUITIN 5 (UBQ5) [13], but not an in vitro synthesized ∼50 bp DNA fragment [32] and a dsRNA generated through in vitro transcription vitro transcription of a DNA fragment (5′ portion of UBQ5, ∼460 bp) containing the T7 promoter at end of each strand [13] (Fig. 7B).

Bottom Line: In addition, prl1 reduces pri-miRNA levels without affecting pri-miRNA transcription.These results suggest that PRL1 may stabilize pri-miRNAs and function as a co-factor to enhance DCL1 activity.Based on these results, we propose that CDC5 and PRL1 cooperatively regulate pri-miRNA levels, which results in their synergistic effects on miRNA accumulation, while they function together as a complex to enhance DCL1 activity.

View Article: PubMed Central - PubMed

Affiliation: Center for Plant Science Innovation & School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska, United States of America.

ABSTRACT
The evolutionary conserved WD-40 protein PRL1 plays important roles in immunity and development. Here we show that PRL1 is required for the accumulation of microRNAs (miRNAs) and small interfering RNAs (siRNAs). PRL1 positively influences the processing of miRNA primary transcripts (pri-miRNAs) and double-stranded RNAs (dsRNAs). Furthermore, PRL1 interacts with the pri-miRNA processor, DCL1, and the dsRNA processors (DCL3 and DCL4). These results suggest that PRL1 may function as a general factor to promote the production of miRNAs and siRNAs. We also show that PRL1 is an RNA-binding protein and associates with pri-miRNAs in vivo. In addition, prl1 reduces pri-miRNA levels without affecting pri-miRNA transcription. These results suggest that PRL1 may stabilize pri-miRNAs and function as a co-factor to enhance DCL1 activity. We further reveal the genetic interaction of PRL1 with CDC5, which interacts with PRL1 and regulates transcription and processing of pri-miRNAs. Both miRNA and pri-miRNA levels are lower in cdc5 prl1 than those in either cdc5 or prl1. However, the processing efficiency of pri-miRNAs in cdc5 prl1 is similar to that in cdc5 and slightly lower than that in prl1. Based on these results, we propose that CDC5 and PRL1 cooperatively regulate pri-miRNA levels, which results in their synergistic effects on miRNA accumulation, while they function together as a complex to enhance DCL1 activity.

No MeSH data available.