PRL1, an RNA-Binding Protein, Positively Regulates the Accumulation of miRNAs and siRNAs in Arabidopsis

PLoS Genetics, Dec 2014

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.

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PRL1, an RNA-Binding Protein, Positively Regulates the Accumulation of miRNAs and siRNAs in Arabidopsis

Positively Regulates the Accumulation of miRNAs and siRNAs in Arabidopsis. PLoS Genet 10(12): e1004841. doi:10.1371/journal.pgen.1004841 PRL1, an RNA-Binding Protein, Positively Regulates the Accumulation of miRNAs and siRNAs in Arabidopsis Shuxin Zhang 0 Yuhui Liu 0 Bin Yu 0 Gregory P. Copenhaver, The University of North Carolina at Chapel Hill, United States of America 0 1 Center for Plant Science Innovation & School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska, United States of America, 2 Biotechnology Research Institute, Chinese Academy of Agricultural Sciences & Key Laboratory of Agricultural Genomics, Ministry of Agriculture , Beijing , China 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. - Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by National Science Foundation Grants MCB-1121193 (to BY). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. In plants and animals, microRNAs (miRNAs), ,2025 nucleotides (nt) in size, regulate gene expression in various biological processes such as development and metabolism [13]. They are produced as duplexes through precise excision from imperfect fold-back primary transcripts (pri-miRNAs) [13]. In the miRNA duplex, the miRNA strand is loaded into ARGONAUTE (AGO) proteins to repress the expression of target genes containing its complementary sequences while the other strand (passenger strand; miRNA*) is degraded [13]. Plants and animals also use small interfering RNAs (siRNAs) to repress gene expression. siRNAs are chemically identical to miRNAs [2]. However they are produced from long double stranded RNAs. The two major classes of plant siRNAs are siRNAs derived from repeated DNAs (rasiRNAs) and trans-acting siRNAs (ta-siRNAs) [4,5]. In plants, most pri-miRNAs are transcribed by DNA-dependent RNA polymerase II (Pol II) from endogenous miRNA encoding genes (MIR) [1,2]. The mediator complex and Negative on TATA less2 (NOT2; a transcription factor) regulate the transcription of MIR [6,7]. After generation, pri-miRNAs are proposed to be stabilized by DAWDLE (DDL), an RNA binding protein [8]. PrimiRNAs are then processed to stem-loop precursors (pre-miRNAs) and finally to the miRNA/miRNA* duplex by Dicer-LIKE 1 (DCL1; an RNAse III enzyme) in the nucleus in plants [9,10]. The C2H2 zinc-finger protein SERRATE (SE) and the RNA binding proteins HYPONASTIC LEAVES 1 (HYL1) and TOUGH (TGH) form a complex with DCL1 to ensure efficient and accurate process of pri-miRNAs [9,1117]. To ensure its proper function, HYL1 needs to be dephosphorylated during pri-miRNA processing [18]. Several other proteins including DDL, CapBinding Protein 20 (CBP20), CBP80, RACK1 and NOT2 are associated with the DCL1 complex to facilitate miRNA maturation [7,8,1921]. NOT2 and MODIFIER OF SNC1, 2 (MOS2; an RNA binding protein) have been shown to guide the correct localization of the DCL1 complex [7,22]. SICKLE (SIC; a proline rich protein) is shown to regulate the accumulation of some miRNAs [23]. Besides protein factors, the structure of pri-miRNAs plays essential roles in regulating DCL1 activity [2427]. For instance, an imperfectly paired lower stem of ,15 bp below the miRNA:miRNA* duplex is crucial for the initial pri-miRNA cleavage [2527]. We previously showed that Cell Div (...truncated)


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Shuxin Zhang, Yuhui Liu, Bin Yu. PRL1, an RNA-Binding Protein, Positively Regulates the Accumulation of miRNAs and siRNAs in Arabidopsis, PLoS Genetics, 2014, 12, DOI: 10.1371/journal.pgen.1004841