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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 positively influences pri-miRNA levels.(A) The abundance of pri-miRNAs in inflorescences of prl1-2 and Col. The transcript levels of pri-miRNAs in prl1-2 were determined by quantitative RT-PCR (qRT-PCR), normalized to that of UBQUITIN5 (UBQ5) and compared with those in Col. Value of Col was set to 1. Error bars indicate standard deviation of three technical replications. **: P<0.01. (B) GUS expression in PRL1+ and prl1-2 harboring pMIR167a::GUS. PRL1+: PRL1/PRL1 or PRL1/prl1-2. Twenty plants containing GUS were stained for each genotype and an image was shown. (C) Relative GUS mRNA levels in PRL1+ and prl1-2 harboring pMIR167a::GUS. GUS transcript levels were determined by qRT-PCR and normalized to UBQ5. Value of PRL1+ was set to 1. Standard deviation of three technical replications was shown as error bars. P = 0.11 (t-test).
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pgen-1004841-g003: PRL1 positively influences pri-miRNA levels.(A) The abundance of pri-miRNAs in inflorescences of prl1-2 and Col. The transcript levels of pri-miRNAs in prl1-2 were determined by quantitative RT-PCR (qRT-PCR), normalized to that of UBQUITIN5 (UBQ5) and compared with those in Col. Value of Col was set to 1. Error bars indicate standard deviation of three technical replications. **: P<0.01. (B) GUS expression in PRL1+ and prl1-2 harboring pMIR167a::GUS. PRL1+: PRL1/PRL1 or PRL1/prl1-2. Twenty plants containing GUS were stained for each genotype and an image was shown. (C) Relative GUS mRNA levels in PRL1+ and prl1-2 harboring pMIR167a::GUS. GUS transcript levels were determined by qRT-PCR and normalized to UBQ5. Value of PRL1+ was set to 1. Standard deviation of three technical replications was shown as error bars. P = 0.11 (t-test).

Mentions: The interaction of PRL1 with Pol II suggests that PRL1 may positively regulate MIR transcription. If so, lack of PRL1 will impair MIR transcription, resulting in reduced levels of pri-miRNAs. To test this, we compared the pri-miRNA levels in prl1-2 with those in Col using qRT-PCR. In fact, the levels of all seven examined pri-miRNAs in prl1-2 were less than those in Col (Fig. 3A), which were recovered in the complementation line of prl1-2 (Fig. 3A). To test whether the reduction of pri-miRNA levels is due to impaired MIR promoter activity, we introduced the prl1-2 mutation into a Col transgenic line containing a single cope of GUS transgene driven by MIR167a promoter (pMIR167a::GUS), which was previously used to test the function of the mediator complex in regulating MIR transcription [6]. However, GUS staining and qRT-PCR analysis showed a similar GUS expression level in PRL1+ (PRL1/PRL1 or PRL1/prl1 genotype) and prl1-2 containing the pMIR167a::GUS transgene (Fig. 3B and 3C). This result demonstrated that PRL1 does not affect MIR promoter activity. Consistent with this notion, prl1 did not show obvious effect on MIR172b promoter activity (Figure S2A).


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 positively influences pri-miRNA levels.(A) The abundance of pri-miRNAs in inflorescences of prl1-2 and Col. The transcript levels of pri-miRNAs in prl1-2 were determined by quantitative RT-PCR (qRT-PCR), normalized to that of UBQUITIN5 (UBQ5) and compared with those in Col. Value of Col was set to 1. Error bars indicate standard deviation of three technical replications. **: P<0.01. (B) GUS expression in PRL1+ and prl1-2 harboring pMIR167a::GUS. PRL1+: PRL1/PRL1 or PRL1/prl1-2. Twenty plants containing GUS were stained for each genotype and an image was shown. (C) Relative GUS mRNA levels in PRL1+ and prl1-2 harboring pMIR167a::GUS. GUS transcript levels were determined by qRT-PCR and normalized to UBQ5. Value of PRL1+ was set to 1. Standard deviation of three technical replications was shown as error bars. P = 0.11 (t-test).
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pgen-1004841-g003: PRL1 positively influences pri-miRNA levels.(A) The abundance of pri-miRNAs in inflorescences of prl1-2 and Col. The transcript levels of pri-miRNAs in prl1-2 were determined by quantitative RT-PCR (qRT-PCR), normalized to that of UBQUITIN5 (UBQ5) and compared with those in Col. Value of Col was set to 1. Error bars indicate standard deviation of three technical replications. **: P<0.01. (B) GUS expression in PRL1+ and prl1-2 harboring pMIR167a::GUS. PRL1+: PRL1/PRL1 or PRL1/prl1-2. Twenty plants containing GUS were stained for each genotype and an image was shown. (C) Relative GUS mRNA levels in PRL1+ and prl1-2 harboring pMIR167a::GUS. GUS transcript levels were determined by qRT-PCR and normalized to UBQ5. Value of PRL1+ was set to 1. Standard deviation of three technical replications was shown as error bars. P = 0.11 (t-test).
Mentions: The interaction of PRL1 with Pol II suggests that PRL1 may positively regulate MIR transcription. If so, lack of PRL1 will impair MIR transcription, resulting in reduced levels of pri-miRNAs. To test this, we compared the pri-miRNA levels in prl1-2 with those in Col using qRT-PCR. In fact, the levels of all seven examined pri-miRNAs in prl1-2 were less than those in Col (Fig. 3A), which were recovered in the complementation line of prl1-2 (Fig. 3A). To test whether the reduction of pri-miRNA levels is due to impaired MIR promoter activity, we introduced the prl1-2 mutation into a Col transgenic line containing a single cope of GUS transgene driven by MIR167a promoter (pMIR167a::GUS), which was previously used to test the function of the mediator complex in regulating MIR transcription [6]. However, GUS staining and qRT-PCR analysis showed a similar GUS expression level in PRL1+ (PRL1/PRL1 or PRL1/prl1 genotype) and prl1-2 containing the pMIR167a::GUS transgene (Fig. 3B and 3C). This result demonstrated that PRL1 does not affect MIR promoter activity. Consistent with this notion, prl1 did not show obvious effect on MIR172b promoter activity (Figure S2A).

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.