Limits...
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.


Related in: MedlinePlus

PRL1 associates with the Pol II and DCL1 complexes.(A) and (B) Co-immunoprecipitation (Co-IP) between PRL1 and Pol II. Protein extracts from transgenic plants containing PRL1-YFP were incubated with Anti-YFP or anti-RPB2 antibodies to precipitate PRL1-YFP or Pol II. PRL1-YFP and RBP2 were detected with western blot and labeled on the left side of the picture. Ten percent of input proteins were used for IP and one percent of input proteins were used for Co-IP. (C) BiFC analysis of PRL1 with DCL1, HYL1, SE, AGO1 and CDC5. Paired cCFP- and nVenus-fusion proteins were co-infiltrated into N. benthamiana leaves. The BiFC signal (Yellow fluorescence) was detected at 48 h after infiltration by confocal microscopy, assigned as green color and marked with arrow. 30 nuclei were examined for each pair and an image is shown. Red: auto fluorescence of chlorophyll. (D) and (E) Co-immunoprecipitation between PRL1 and DCL1. (F) and (G) Co-immunoprecipitation between PRL1 and SE. PRL1-YFP or YFP were co-expressed with DCL1-MYC and SE-MYC in N. benthamiana, respectively. Anti-YFP and anti-MYC (MBL) antibodies were used to detect YFP- and MYC-fused proteins, respectively. The protein pairs in the protein extracts were indicated on the on tope of the picture and proteins detected by western blot were indicated on the left side of the picture. Ten percent of input proteins were used for IP and one percent of inputs proteins were used for Co-IP.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4256206&req=5

pgen-1004841-g002: PRL1 associates with the Pol II and DCL1 complexes.(A) and (B) Co-immunoprecipitation (Co-IP) between PRL1 and Pol II. Protein extracts from transgenic plants containing PRL1-YFP were incubated with Anti-YFP or anti-RPB2 antibodies to precipitate PRL1-YFP or Pol II. PRL1-YFP and RBP2 were detected with western blot and labeled on the left side of the picture. Ten percent of input proteins were used for IP and one percent of input proteins were used for Co-IP. (C) BiFC analysis of PRL1 with DCL1, HYL1, SE, AGO1 and CDC5. Paired cCFP- and nVenus-fusion proteins were co-infiltrated into N. benthamiana leaves. The BiFC signal (Yellow fluorescence) was detected at 48 h after infiltration by confocal microscopy, assigned as green color and marked with arrow. 30 nuclei were examined for each pair and an image is shown. Red: auto fluorescence of chlorophyll. (D) and (E) Co-immunoprecipitation between PRL1 and DCL1. (F) and (G) Co-immunoprecipitation between PRL1 and SE. PRL1-YFP or YFP were co-expressed with DCL1-MYC and SE-MYC in N. benthamiana, respectively. Anti-YFP and anti-MYC (MBL) antibodies were used to detect YFP- and MYC-fused proteins, respectively. The protein pairs in the protein extracts were indicated on the on tope of the picture and proteins detected by western blot were indicated on the left side of the picture. Ten percent of input proteins were used for IP and one percent of inputs proteins were used for Co-IP.

Mentions: The PRL1-CDC5 interaction suggests that similar to CDC5, PRL1 may act as a co-factor of Pol II and DCL1 to regulate miRNA accumulation. To test these two possibilities, we first examined the PRL1-Pol II association using co-immunoprecipitation (co-IP) assay. In this experiment, anti-YFP and anti-RPB2 that detects the second largest subunit of Pol II (RPB2) [6] were used to capture the PRL1-YFP and Pol II complex, respectively, from the protein extracts of prl1-2 complementation line expressing the pPRL1::PRL1-YFP transgene. After IP, PRL1-YFP was detected in the Pol II precipitates whereas RPB2 existed in the PRL1-YFP complex (Fig. 2A and 2B). In contrast, no interaction was detected in the control reactions (Fig. 2A and 2B), demonstrating the PRL1-Pol II association.


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 associates with the Pol II and DCL1 complexes.(A) and (B) Co-immunoprecipitation (Co-IP) between PRL1 and Pol II. Protein extracts from transgenic plants containing PRL1-YFP were incubated with Anti-YFP or anti-RPB2 antibodies to precipitate PRL1-YFP or Pol II. PRL1-YFP and RBP2 were detected with western blot and labeled on the left side of the picture. Ten percent of input proteins were used for IP and one percent of input proteins were used for Co-IP. (C) BiFC analysis of PRL1 with DCL1, HYL1, SE, AGO1 and CDC5. Paired cCFP- and nVenus-fusion proteins were co-infiltrated into N. benthamiana leaves. The BiFC signal (Yellow fluorescence) was detected at 48 h after infiltration by confocal microscopy, assigned as green color and marked with arrow. 30 nuclei were examined for each pair and an image is shown. Red: auto fluorescence of chlorophyll. (D) and (E) Co-immunoprecipitation between PRL1 and DCL1. (F) and (G) Co-immunoprecipitation between PRL1 and SE. PRL1-YFP or YFP were co-expressed with DCL1-MYC and SE-MYC in N. benthamiana, respectively. Anti-YFP and anti-MYC (MBL) antibodies were used to detect YFP- and MYC-fused proteins, respectively. The protein pairs in the protein extracts were indicated on the on tope of the picture and proteins detected by western blot were indicated on the left side of the picture. Ten percent of input proteins were used for IP and one percent of inputs proteins were used for Co-IP.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4256206&req=5

pgen-1004841-g002: PRL1 associates with the Pol II and DCL1 complexes.(A) and (B) Co-immunoprecipitation (Co-IP) between PRL1 and Pol II. Protein extracts from transgenic plants containing PRL1-YFP were incubated with Anti-YFP or anti-RPB2 antibodies to precipitate PRL1-YFP or Pol II. PRL1-YFP and RBP2 were detected with western blot and labeled on the left side of the picture. Ten percent of input proteins were used for IP and one percent of input proteins were used for Co-IP. (C) BiFC analysis of PRL1 with DCL1, HYL1, SE, AGO1 and CDC5. Paired cCFP- and nVenus-fusion proteins were co-infiltrated into N. benthamiana leaves. The BiFC signal (Yellow fluorescence) was detected at 48 h after infiltration by confocal microscopy, assigned as green color and marked with arrow. 30 nuclei were examined for each pair and an image is shown. Red: auto fluorescence of chlorophyll. (D) and (E) Co-immunoprecipitation between PRL1 and DCL1. (F) and (G) Co-immunoprecipitation between PRL1 and SE. PRL1-YFP or YFP were co-expressed with DCL1-MYC and SE-MYC in N. benthamiana, respectively. Anti-YFP and anti-MYC (MBL) antibodies were used to detect YFP- and MYC-fused proteins, respectively. The protein pairs in the protein extracts were indicated on the on tope of the picture and proteins detected by western blot were indicated on the left side of the picture. Ten percent of input proteins were used for IP and one percent of inputs proteins were used for Co-IP.
Mentions: The PRL1-CDC5 interaction suggests that similar to CDC5, PRL1 may act as a co-factor of Pol II and DCL1 to regulate miRNA accumulation. To test these two possibilities, we first examined the PRL1-Pol II association using co-immunoprecipitation (co-IP) assay. In this experiment, anti-YFP and anti-RPB2 that detects the second largest subunit of Pol II (RPB2) [6] were used to capture the PRL1-YFP and Pol II complex, respectively, from the protein extracts of prl1-2 complementation line expressing the pPRL1::PRL1-YFP transgene. After IP, PRL1-YFP was detected in the Pol II precipitates whereas RPB2 existed in the PRL1-YFP complex (Fig. 2A and 2B). In contrast, no interaction was detected in the control reactions (Fig. 2A and 2B), demonstrating the PRL1-Pol II association.

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.


Related in: MedlinePlus