CDC5, a DNA binding protein, positively regulates posttranscriptional processing and/or transcription of primary microRNA transcripts

Abstract

CDC5 is a MYB-related protein that exists in plants, animals, and fungi. In Arabidopsis, CDC5 regulates both growth and immunity through unknown mechanisms. Here, we show that CDC5 from Arabidopsis positively regulates the accumulation of microRNAs (miRNAs), which control many biological processes including development and adaptations to environments in plants. CDC5 interacts with both the promoters of genes encoding miRNAs (MIR) and the DNA-dependent RNA polymerase II. As a consequence, lack of CDC5 reduces the occupancy of polymerase II at MIR promoters, as well as MIR promoter activities. In addition, CDC5 is associated with the DICER-LIKE1 complex, which generates miRNAs from their primary transcripts and is required for efficient miRNA production. These results suggest that CDC5 may have dual roles in miRNA biogenesis: functioning as a positive transcription factor of MIR and/or acting as a component of the DICER-LIKE1 complex to enhance primary miRNA processing.M icroRNAs (miRNAs) and small interfering RNAs (siRNAs) are ∼22-nucleotide (nt) noncoding RNAs that regulate various biological processes including development, metabolism, and immunity in plants and animals (1-3). miRNAs are generated from primary miRNA transcripts (pri-miRNAs) containing stem-loop structure, whereas siRNAs are derived from long, perfect, doublestranded RNAs (dsRNAs) (1-3). They are associated with members of the Argonaute protein family to repress gene expression at posttranscriptional and/or transcriptional levels (1-3). In addition to miRNAs, plants encode two major classes of siRNAs: siRNAs derived from repeated DNAs (ra-siRNAs) and transacting siRNAs (ta-siRNAs) (4-6).Studies in Arabidopsis have established the framework of miRNA biogenesis in plants (1-3). In Arabidopsis, pri-miRNAs are primarily transcribed by DNA-dependent RNA polymerase II (Pol II), with assistance from the mediator complex and the transcription factor Negative on TATA less2 (NOT2) (7,8). After transcription, pri-miRNAs are processed by an RNase III enzyme called DICER-LIKE1 (DCL1) to miRNA precursors and then to mature miRNAs (9, 10). The efficient processing of pri-miRNA requires SERRATE (SE; a zinc finger protein), TOUGH (an RNA-binding protein), and a dephosphorylated HYPONASTIC LEAVES1 (HYL1; a double-stranded RNA binding protein) that form a complex with DCL1 (11-18). SE and HYL1 also promote the processing accuracy of pri-miRNAs (19). Four other proteins, DAWDLE (DDL; an RNA binding protein), Cap-binding protein 20, Cap-binding protein 80, and NOT2, which are associated with the DCL1 complex (8,(20)(21)(22), also function in miRNA biogenesis. Recent studies also reveal that the correct localization of DCL1 requires NOT2 and MODIFIER OF SNC1, 2 (an RNA binding protein) (8,23). In addition, the accumulation of a subset of miRNAs requires a proline-rich protein named SICKLE (24).The cell division cycle 5 (CDC5) protein is a conserved protein in animals, plants, and fungi (25). It was first isolated from Schizosaccharomyces pombe as a cell cycle regulat...

DELAY OF GERMINATION1 ( DOG1 ) regulates both seed dormancy and flowering time through microRNA pathways

“…Loss of function or gain of function of DOG1 decreases or increases, respectively, expression of DICER-related genes (Fig. 4 E-G), and previous studies found that miR156 and miR172 levels are altered in mutants of DCL1, HYL1, SE, TGH, and CDC5 (42,56,58,62,63). However, multiple feedback loops are associated with transcription and processing of miRNAs (41), so further study is needed to clarify the specific mechanism by which DOG1 affects miRNA levels.…”

“…4 A and B), we tested whether transcripts of genes encoding proteins associated with miRNA transcription or processing were altered. After preliminary screening, we focused on five genes: DICER-LIKE 1 (DCL1), HYPONASTIC LEAVES1 (HYL1), the G-patch domain protein TOUGH (TGH), the zinc finger protein SERRATE (SE), and CELL DIVISION CYCLE 5 (CDC5) that binds RNA polymerase II and MIR promoters to positively regulate transcription of MIR-encoding genes (41,42). Although the specific effects varied somewhat among the Arabidopsis and lettuce genotypes and mutants tested, transcript levels of all of these genes differed from their respective WTs in at least some cases (Fig.…”

STV1, a ribosomal protein, binds primary microRNA transcripts to promote their interaction with the processing complex in Arabidopsis

“…In Arabidopsis, the transcription factors, including the mediator complex, the elongator complex, Negative on TATA less 2 (NOT2), and CELL DIVISION CYCLE 5 (CDC5) have been shown to positively regulate MIR transcription through recruiting Pol II to MIR promoters (15)(16)(17). The transcription of some MIRs, such as MIR172 and MIR156, is also subject to physiological, temporal, or spatial regulation by specific transcription factors (18,19).…”

Regulation of Plant miRNA Biogenesis

“…General transcription factors like Mediator complex, elongator complex, Negative on TATA less 2 (NOT2), Cell Division Cycle 5 (CDC5) are shown to increase the Pol II occupancy at the MIR promoters and activate their transcription (Kim et al, 2011;Fang et al, 2015;Zhang et al, 2013) similar to protein coding genes. In line with this, mutants of these genes have reduced expression of miRNAs.…”

“…A forkhead domain containing protein named Dawdle (DDL) is another RNA binding protein that affects miRNA accumulation by interacting with phosphorylated DCL1 and pri-miRNA, helping in the recruitment to miRNA processing complex (Yu et al, 2008;Machida and Adam Yuan, 2013). The NOT2 and MOS4-associated complex (MAC) complex members -CDC5 and PRL1 interact with components of miRNA biogenesis such as DCL1, SE, HYL1 and CBP20/80 Zhang et al, 2014Zhang et al, , 2013Jia et al, 2017). Since these components couple transcription and processing, probably these are involved in assembly of dicing bodies and recruiting pri-miRNAs to the site of primiRNA processing.…”