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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 is required for the accumulation of miRNAs.(A) The effect of various MAC components on the abundance of miRNAs (B) The levels of miRNAs are reduced in prl1-2. (C) The levels of siRNAs are reduced in prl1-2. Col: wild-type control. For miR159/319: upper band, miR159; lower band, miR319. Northern blot was used to detect small RNAs and the radioactive signals were quantified with ImageQuant (V5.2). To determine the amount of miRNAs/siRNAs in various mutants relative to that in Col, the radioactive signals of miRNAs/siRNAs were normalized to U6 RNA and compared with that in Col (set as 1). The numbers indicate the average value of three repeats (P<0.05).
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pgen-1004841-g001: PRL1 is required for the accumulation of miRNAs.(A) The effect of various MAC components on the abundance of miRNAs (B) The levels of miRNAs are reduced in prl1-2. (C) The levels of siRNAs are reduced in prl1-2. Col: wild-type control. For miR159/319: upper band, miR159; lower band, miR319. Northern blot was used to detect small RNAs and the radioactive signals were quantified with ImageQuant (V5.2). To determine the amount of miRNAs/siRNAs in various mutants relative to that in Col, the radioactive signals of miRNAs/siRNAs were normalized to U6 RNA and compared with that in Col (set as 1). The numbers indicate the average value of three repeats (P<0.05).

Mentions: Given the role of CDC5 in miRNA biogenesis, it is possible that other components of the MAC complex may also be required for miRNA accumulation. Therefore, we examined the effect of the mutants mac3b (SALK_050811), mos4 (SALK_0090851C) and prl1-2 on miRNA abundance using Northern blot. We also included snc1 (SALK_047058C) in the analysis since SNC1 is related to the MAC complex and snc1 causes development defects. These mutants are likely since the transcripts of corresponding genes could not be detected by RT-PCR (Figure S1A). Like in cdc5-1, the abundance of all four tested miRNAs (miR167, miR171, miR172 and miR173) was decreased in prl1-2 compared to Col (wild-type control). In contrast, miRNA levels in mos4, mac3b and snc1 were comparable with those in Col (Fig. 1A). We examined the accumulation of additional miRNAs in prl1-2 and found that all these miRNAs were reduced in abundance in prl1-2 relative to Col (Fig. 1B). In addition, expression a wild-type copy of PRL1 fused with a YFP tag under the control of its native promoter (pPRL1::PRL1-YFP) fully recovered miRNA levels in prl1-2 (Fig. 1B). These results demonstrated that PRL1 but not MOS4 and MAC3b is required for miRNA accumulation. We next analyzed the transcript levels of several miRNA targets (ARF3, CUC1, MYB33, MYB65 and PHV) in prl1-2 and Col by quantitative RT-PCR (qRT-PCR) in order to test the effect of prl1-2 on miRNA function. The transcription levels of these targets were slightly increased in prl1-2 relative to Col (Figure. S1B). The PRL1 transgene fully recovered miRNA function in prl1 (Figure S1B). We also asked if PRL1 has a role in siRNA biogenesis. The levels of nine examined siRNAs (three ta-siRNAs and six ra-siRNAs) were reduced compared to those in Col (Fig. 1B and 1C), which was complemented by the expression of pPRL1::PRL1-YFP. These results revealed that like CDC5, PRL1 positively regulates the levels of miRNAs and siRNAs in Arabidopsis.


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 is required for the accumulation of miRNAs.(A) The effect of various MAC components on the abundance of miRNAs (B) The levels of miRNAs are reduced in prl1-2. (C) The levels of siRNAs are reduced in prl1-2. Col: wild-type control. For miR159/319: upper band, miR159; lower band, miR319. Northern blot was used to detect small RNAs and the radioactive signals were quantified with ImageQuant (V5.2). To determine the amount of miRNAs/siRNAs in various mutants relative to that in Col, the radioactive signals of miRNAs/siRNAs were normalized to U6 RNA and compared with that in Col (set as 1). The numbers indicate the average value of three repeats (P<0.05).
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pgen-1004841-g001: PRL1 is required for the accumulation of miRNAs.(A) The effect of various MAC components on the abundance of miRNAs (B) The levels of miRNAs are reduced in prl1-2. (C) The levels of siRNAs are reduced in prl1-2. Col: wild-type control. For miR159/319: upper band, miR159; lower band, miR319. Northern blot was used to detect small RNAs and the radioactive signals were quantified with ImageQuant (V5.2). To determine the amount of miRNAs/siRNAs in various mutants relative to that in Col, the radioactive signals of miRNAs/siRNAs were normalized to U6 RNA and compared with that in Col (set as 1). The numbers indicate the average value of three repeats (P<0.05).
Mentions: Given the role of CDC5 in miRNA biogenesis, it is possible that other components of the MAC complex may also be required for miRNA accumulation. Therefore, we examined the effect of the mutants mac3b (SALK_050811), mos4 (SALK_0090851C) and prl1-2 on miRNA abundance using Northern blot. We also included snc1 (SALK_047058C) in the analysis since SNC1 is related to the MAC complex and snc1 causes development defects. These mutants are likely since the transcripts of corresponding genes could not be detected by RT-PCR (Figure S1A). Like in cdc5-1, the abundance of all four tested miRNAs (miR167, miR171, miR172 and miR173) was decreased in prl1-2 compared to Col (wild-type control). In contrast, miRNA levels in mos4, mac3b and snc1 were comparable with those in Col (Fig. 1A). We examined the accumulation of additional miRNAs in prl1-2 and found that all these miRNAs were reduced in abundance in prl1-2 relative to Col (Fig. 1B). In addition, expression a wild-type copy of PRL1 fused with a YFP tag under the control of its native promoter (pPRL1::PRL1-YFP) fully recovered miRNA levels in prl1-2 (Fig. 1B). These results demonstrated that PRL1 but not MOS4 and MAC3b is required for miRNA accumulation. We next analyzed the transcript levels of several miRNA targets (ARF3, CUC1, MYB33, MYB65 and PHV) in prl1-2 and Col by quantitative RT-PCR (qRT-PCR) in order to test the effect of prl1-2 on miRNA function. The transcription levels of these targets were slightly increased in prl1-2 relative to Col (Figure. S1B). The PRL1 transgene fully recovered miRNA function in prl1 (Figure S1B). We also asked if PRL1 has a role in siRNA biogenesis. The levels of nine examined siRNAs (three ta-siRNAs and six ra-siRNAs) were reduced compared to those in Col (Fig. 1B and 1C), which was complemented by the expression of pPRL1::PRL1-YFP. These results revealed that like CDC5, PRL1 positively regulates the levels of miRNAs and siRNAs in Arabidopsis.

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.