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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 and CDC5 synergistically regulate miRNA accumulation.(A) Morphological phenotypes of Col, cdc5-1, prl1-2 and cdc5-1 prl1-2. (B) The abundance of miRNAs is lower in cdc5-1 prl1-2 than that in cdc5-1 or prl1-2. Small RNAs were detected by Northern Blot. To determine the amount of miRNAs, radioactive signals of miRNAs were normalized to U6 RNA. The number represents the relative abundance compared to Col (set as 1) quantified by three repeats (P<0.05). (C) The abundance of pri-miRNAs is reduced in cdc5-1 prl1-2. The levels of pri-miRNAs in various mutants were determined by qRT-PCR, normalized to UBQUITIN5 (UBQ5) and compared with those of Col (set as 1). Standard deviation of three technical replications was shown as error bars. **: P<0.01. (D) miR162b production from pre-miR162b in Col, cdc5-1 prl1-2, cdc5-1 and prl1-2. The reaction was stopped at 120 min. The radioactive signals of miR162b were normalized to input. The number represents the relative production in various genotypes compared to Col (set as 1) quantified by three repeats (P<0.05).
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pgen-1004841-g006: PRL1 and CDC5 synergistically regulate miRNA accumulation.(A) Morphological phenotypes of Col, cdc5-1, prl1-2 and cdc5-1 prl1-2. (B) The abundance of miRNAs is lower in cdc5-1 prl1-2 than that in cdc5-1 or prl1-2. Small RNAs were detected by Northern Blot. To determine the amount of miRNAs, radioactive signals of miRNAs were normalized to U6 RNA. The number represents the relative abundance compared to Col (set as 1) quantified by three repeats (P<0.05). (C) The abundance of pri-miRNAs is reduced in cdc5-1 prl1-2. The levels of pri-miRNAs in various mutants were determined by qRT-PCR, normalized to UBQUITIN5 (UBQ5) and compared with those of Col (set as 1). Standard deviation of three technical replications was shown as error bars. **: P<0.01. (D) miR162b production from pre-miR162b in Col, cdc5-1 prl1-2, cdc5-1 and prl1-2. The reaction was stopped at 120 min. The radioactive signals of miR162b were normalized to input. The number represents the relative production in various genotypes compared to Col (set as 1) quantified by three repeats (P<0.05).

Mentions: CDC5 and PRL1 have been shown to directly interact with each other. Both CDC5 and PRL1 interact with DCL1 and positively regulate miRNA processing. These results raise a possibility that CDC5 and PRL1 may act as a complex to regulate DCL1 activity. In addition, CDC5 regulates MIR promoter activity while PRL1 does not. These suggest that PRL1 and CDC5 might act additionally in miRNA pathway. To test these two possibilities, we constructed a cdc5-1 prl1-2 double mutant by crossing prl1-2 into cdc5-1 and compared miRNA levels in cdc5-1 prl1-2 with those in cdc5-1 and prl1-2, respectively. The cdc5-1 prl1-2 double mutant displayed more severe developmental defects than either cdc5-1 or prl1-2, suggesting that PRL1 and CDC5 function additionally in regulating development (Fig. 6A). Northern blot analyses showed that the levels of several examined miRNAs in cdc5-1 prl1-2 were lower than those in either prl1-2 or cdc5-1 (Fig. 6B), indicating that PRL1 and CDC5 function synergistically in miRNA pathway.


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 and CDC5 synergistically regulate miRNA accumulation.(A) Morphological phenotypes of Col, cdc5-1, prl1-2 and cdc5-1 prl1-2. (B) The abundance of miRNAs is lower in cdc5-1 prl1-2 than that in cdc5-1 or prl1-2. Small RNAs were detected by Northern Blot. To determine the amount of miRNAs, radioactive signals of miRNAs were normalized to U6 RNA. The number represents the relative abundance compared to Col (set as 1) quantified by three repeats (P<0.05). (C) The abundance of pri-miRNAs is reduced in cdc5-1 prl1-2. The levels of pri-miRNAs in various mutants were determined by qRT-PCR, normalized to UBQUITIN5 (UBQ5) and compared with those of Col (set as 1). Standard deviation of three technical replications was shown as error bars. **: P<0.01. (D) miR162b production from pre-miR162b in Col, cdc5-1 prl1-2, cdc5-1 and prl1-2. The reaction was stopped at 120 min. The radioactive signals of miR162b were normalized to input. The number represents the relative production in various genotypes compared to Col (set as 1) quantified by three repeats (P<0.05).
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pgen-1004841-g006: PRL1 and CDC5 synergistically regulate miRNA accumulation.(A) Morphological phenotypes of Col, cdc5-1, prl1-2 and cdc5-1 prl1-2. (B) The abundance of miRNAs is lower in cdc5-1 prl1-2 than that in cdc5-1 or prl1-2. Small RNAs were detected by Northern Blot. To determine the amount of miRNAs, radioactive signals of miRNAs were normalized to U6 RNA. The number represents the relative abundance compared to Col (set as 1) quantified by three repeats (P<0.05). (C) The abundance of pri-miRNAs is reduced in cdc5-1 prl1-2. The levels of pri-miRNAs in various mutants were determined by qRT-PCR, normalized to UBQUITIN5 (UBQ5) and compared with those of Col (set as 1). Standard deviation of three technical replications was shown as error bars. **: P<0.01. (D) miR162b production from pre-miR162b in Col, cdc5-1 prl1-2, cdc5-1 and prl1-2. The reaction was stopped at 120 min. The radioactive signals of miR162b were normalized to input. The number represents the relative production in various genotypes compared to Col (set as 1) quantified by three repeats (P<0.05).
Mentions: CDC5 and PRL1 have been shown to directly interact with each other. Both CDC5 and PRL1 interact with DCL1 and positively regulate miRNA processing. These results raise a possibility that CDC5 and PRL1 may act as a complex to regulate DCL1 activity. In addition, CDC5 regulates MIR promoter activity while PRL1 does not. These suggest that PRL1 and CDC5 might act additionally in miRNA pathway. To test these two possibilities, we constructed a cdc5-1 prl1-2 double mutant by crossing prl1-2 into cdc5-1 and compared miRNA levels in cdc5-1 prl1-2 with those in cdc5-1 and prl1-2, respectively. The cdc5-1 prl1-2 double mutant displayed more severe developmental defects than either cdc5-1 or prl1-2, suggesting that PRL1 and CDC5 function additionally in regulating development (Fig. 6A). Northern blot analyses showed that the levels of several examined miRNAs in cdc5-1 prl1-2 were lower than those in either prl1-2 or cdc5-1 (Fig. 6B), indicating that PRL1 and CDC5 function synergistically in miRNA pathway.

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.