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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.


The role of PRL1 in siRNA biogenesis.(A) PRL1 interacts with DCL3 and DCL4. Co-IP was performed to detect the interaction of PRL1 with DCL3 or DCL4. MBP and MBP-PRL1 fused protein were expressed in E.coli. YFP, DCL3-YFP and DCL4-YFP were expressed in N. benthamiana leaves. Anti-YFP was used for IP. For loading, ten percent and one percent of input proteins were used for IP and Co-IP, respectively. (B) prl1-2 impairs siRNA production from double-stranded RNAs (dsRNAs). Protein extracts isolated from inflorescences of Col, prl1-2 and prl1-2 containing a PRL1-YFP transgene were incubated dsRNAs for 120 min. dsRNAs were synthesized through in vitro transcription of a DNA fragment (5′ portion of UBQ5 gene, ∼460 bp) under the presence of [α-32P] UTP.
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pgen-1004841-g005: The role of PRL1 in siRNA biogenesis.(A) PRL1 interacts with DCL3 and DCL4. Co-IP was performed to detect the interaction of PRL1 with DCL3 or DCL4. MBP and MBP-PRL1 fused protein were expressed in E.coli. YFP, DCL3-YFP and DCL4-YFP were expressed in N. benthamiana leaves. Anti-YFP was used for IP. For loading, ten percent and one percent of input proteins were used for IP and Co-IP, respectively. (B) prl1-2 impairs siRNA production from double-stranded RNAs (dsRNAs). Protein extracts isolated from inflorescences of Col, prl1-2 and prl1-2 containing a PRL1-YFP transgene were incubated dsRNAs for 120 min. dsRNAs were synthesized through in vitro transcription of a DNA fragment (5′ portion of UBQ5 gene, ∼460 bp) under the presence of [α-32P] UTP.

Mentions: We next asked the role of PRL1 in siRNA biogenesis, as prl1-2 reduces the accumulation of siRNAs. By analog, we examined the interaction of PRL1 with DCL3 and DCL4 and the effect of prl1-2 on dsRNA processing. To test the PRL1-DCL3/DCL4 interaction, we expressed a recombined PRL1 fused with a maltose-binding protein at its N-terminus (MBP-PRL1) and MBP in E.coli. The protein extracts containing MBP-PRL1 or MBP were mixed with protein extracts containing DCL3-YFP or DCL4-YFP, which were transiently expressed in N. benthamiana. Then the DCL3-YFP or DCL4-YFP complex was IPed with anti-YFP antibodies. MBP-PRL1, but not MBP, was co-IPed with DCL3-YFP and DCL4-YFP (Fig. 5A). In addition, YFP did not interact with MBP or MBP-PRL1. These results demonstrated that PRL1 interacts with DCL3 and DCL4 (Fig. 5A).


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

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

The role of PRL1 in siRNA biogenesis.(A) PRL1 interacts with DCL3 and DCL4. Co-IP was performed to detect the interaction of PRL1 with DCL3 or DCL4. MBP and MBP-PRL1 fused protein were expressed in E.coli. YFP, DCL3-YFP and DCL4-YFP were expressed in N. benthamiana leaves. Anti-YFP was used for IP. For loading, ten percent and one percent of input proteins were used for IP and Co-IP, respectively. (B) prl1-2 impairs siRNA production from double-stranded RNAs (dsRNAs). Protein extracts isolated from inflorescences of Col, prl1-2 and prl1-2 containing a PRL1-YFP transgene were incubated dsRNAs for 120 min. dsRNAs were synthesized through in vitro transcription of a DNA fragment (5′ portion of UBQ5 gene, ∼460 bp) under the presence of [α-32P] UTP.
© Copyright Policy
Related In: Results  -  Collection

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pgen-1004841-g005: The role of PRL1 in siRNA biogenesis.(A) PRL1 interacts with DCL3 and DCL4. Co-IP was performed to detect the interaction of PRL1 with DCL3 or DCL4. MBP and MBP-PRL1 fused protein were expressed in E.coli. YFP, DCL3-YFP and DCL4-YFP were expressed in N. benthamiana leaves. Anti-YFP was used for IP. For loading, ten percent and one percent of input proteins were used for IP and Co-IP, respectively. (B) prl1-2 impairs siRNA production from double-stranded RNAs (dsRNAs). Protein extracts isolated from inflorescences of Col, prl1-2 and prl1-2 containing a PRL1-YFP transgene were incubated dsRNAs for 120 min. dsRNAs were synthesized through in vitro transcription of a DNA fragment (5′ portion of UBQ5 gene, ∼460 bp) under the presence of [α-32P] UTP.
Mentions: We next asked the role of PRL1 in siRNA biogenesis, as prl1-2 reduces the accumulation of siRNAs. By analog, we examined the interaction of PRL1 with DCL3 and DCL4 and the effect of prl1-2 on dsRNA processing. To test the PRL1-DCL3/DCL4 interaction, we expressed a recombined PRL1 fused with a maltose-binding protein at its N-terminus (MBP-PRL1) and MBP in E.coli. The protein extracts containing MBP-PRL1 or MBP were mixed with protein extracts containing DCL3-YFP or DCL4-YFP, which were transiently expressed in N. benthamiana. Then the DCL3-YFP or DCL4-YFP complex was IPed with anti-YFP antibodies. MBP-PRL1, but not MBP, was co-IPed with DCL3-YFP and DCL4-YFP (Fig. 5A). In addition, YFP did not interact with MBP or MBP-PRL1. These results demonstrated that PRL1 interacts with DCL3 and DCL4 (Fig. 5A).

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.