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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 miRNA maturation in vitro.(A) and (B) A schematic diagram of the MIR162b (A) and pre-miR162b (B) used in vitro processing assay. (C) and (D) The amount of miR162b produced from MIR162b and pre-miR162b were reduced in prl1-2. Proteins were isolated from inflorescences of prl1-2 and Col and incubated with MIR162b or pre-miR162b. The reactions were stopped at various time points as indicated in the picture. (E) and (F) Quantification of miR162b production in prl1-2 compared to that in Col. Quantification analysis was performed at 80 min. The radioactive signal of miR162 were normalized to input and compared with that of Col. The amount of miR162 produced in Col was set as 1. The value represents mean of three repeats (*** P<0.001; t-test).
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pgen-1004841-g004: PRL1 is required for miRNA maturation in vitro.(A) and (B) A schematic diagram of the MIR162b (A) and pre-miR162b (B) used in vitro processing assay. (C) and (D) The amount of miR162b produced from MIR162b and pre-miR162b were reduced in prl1-2. Proteins were isolated from inflorescences of prl1-2 and Col and incubated with MIR162b or pre-miR162b. The reactions were stopped at various time points as indicated in the picture. (E) and (F) Quantification of miR162b production in prl1-2 compared to that in Col. Quantification analysis was performed at 80 min. The radioactive signal of miR162 were normalized to input and compared with that of Col. The amount of miR162 produced in Col was set as 1. The value represents mean of three repeats (*** P<0.001; t-test).

Mentions: We next asked whether PRL1 has a role in processing of miRNA precursors through an in vitro processing assay [13], [31] since it is associated with DCL1 and SE. In this experiment, a portion of pri-miR162b that contains the stem-loop of miR162b with 6-nt arms at each end (MIR162b; Fig. 4A) and pre-miR162b (Fig. 4B) were first produced through in vitro transcription in the presence of [α-32P] UTP. [32P]-labeled MIR162b and pre-miR162b were then processed in the protein extracts of young flower buds of prl1-2 or Col. The production of miR162b from both MIR162b and pre-miR162b in prl1-2 at various time points was less than that in Col (Fig. 4C and 4D). The processing of MIR162b and pre-miRNA162b was recovered in the PRL1 complementation line (Figure S3) The levels of miR162 produced from MIR162b and pre-miRNA162b in prl1 at 80 min were ∼40% of those produced in Col (Fig. 4E and 4F). These results suggested that PRL1 might have a role in promoting miRNA maturation.


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 miRNA maturation in vitro.(A) and (B) A schematic diagram of the MIR162b (A) and pre-miR162b (B) used in vitro processing assay. (C) and (D) The amount of miR162b produced from MIR162b and pre-miR162b were reduced in prl1-2. Proteins were isolated from inflorescences of prl1-2 and Col and incubated with MIR162b or pre-miR162b. The reactions were stopped at various time points as indicated in the picture. (E) and (F) Quantification of miR162b production in prl1-2 compared to that in Col. Quantification analysis was performed at 80 min. The radioactive signal of miR162 were normalized to input and compared with that of Col. The amount of miR162 produced in Col was set as 1. The value represents mean of three repeats (*** P<0.001; t-test).
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004841-g004: PRL1 is required for miRNA maturation in vitro.(A) and (B) A schematic diagram of the MIR162b (A) and pre-miR162b (B) used in vitro processing assay. (C) and (D) The amount of miR162b produced from MIR162b and pre-miR162b were reduced in prl1-2. Proteins were isolated from inflorescences of prl1-2 and Col and incubated with MIR162b or pre-miR162b. The reactions were stopped at various time points as indicated in the picture. (E) and (F) Quantification of miR162b production in prl1-2 compared to that in Col. Quantification analysis was performed at 80 min. The radioactive signal of miR162 were normalized to input and compared with that of Col. The amount of miR162 produced in Col was set as 1. The value represents mean of three repeats (*** P<0.001; t-test).
Mentions: We next asked whether PRL1 has a role in processing of miRNA precursors through an in vitro processing assay [13], [31] since it is associated with DCL1 and SE. In this experiment, a portion of pri-miR162b that contains the stem-loop of miR162b with 6-nt arms at each end (MIR162b; Fig. 4A) and pre-miR162b (Fig. 4B) were first produced through in vitro transcription in the presence of [α-32P] UTP. [32P]-labeled MIR162b and pre-miR162b were then processed in the protein extracts of young flower buds of prl1-2 or Col. The production of miR162b from both MIR162b and pre-miR162b in prl1-2 at various time points was less than that in Col (Fig. 4C and 4D). The processing of MIR162b and pre-miRNA162b was recovered in the PRL1 complementation line (Figure S3) The levels of miR162 produced from MIR162b and pre-miRNA162b in prl1 at 80 min were ∼40% of those produced in Col (Fig. 4E and 4F). These results suggested that PRL1 might have a role in promoting miRNA maturation.

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.