HYL1-CLEAVAGE SUBTILASE 1 (HCS1) suppresses miRNA biogenesis in response to light-to-dark transition

Abstract

The core plant microprocessor consists of DICER-LIKE 1 (DCL1), SERRATE (SE), and HYPONASTIC LEAVES 1 (HYL1) and plays a pivotal role in microRNA (miRNA) biogenesis. However, the proteolytic regulation of each component remains elusive. Here, we show that HYL1-CLEAVAGE SUBTILASE 1 (HCS1) is a cytoplasmic protease for HYL1-destabilization. HCS1-excessiveness reduces HYL1 that disrupts miRNA biogenesis, while HCS1-deficiency accumulates HYL1. Consistently, we identified the HYL1K154A mutant that is insensitive to the proteolytic activity of HCS1, confirming the importance of HCS1 in HYL1 proteostasis. Moreover, HCS1-activity is regulated by light/dark transition. Under light, cytoplasmic CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) E3 ligase suppresses HCS1-activity. COP1 sterically inhibits HCS1 by obstructing HYL1 access into the catalytic sites of HCS1. In contrast, darkness unshackles HCS1-activity for HYL1-destabilization due to nuclear COP1 relocation. Overall, the COP1-HYL1-HCS1 network may integrate two essential cellular pathways: the miRNA-biogenetic pathway and light signaling pathway.

Dynamic Phosphorylation of miRNA Biogenesis Factor HYL1 by MPK3 Involving Nuclear–Cytoplasmic Shuttling and Protein Stability in Arabidopsis

“…Interestingly, the N-terminal of HYL1 was unaffected by the proteolytic cleavage as compared to the full length AtHYL1 ( Figure 5 B). This analysis supported the recent report where HCS1 protease is confirmed as the protease for HYL1-destabilization [ 20 ] that might have an action like that of trypsin proteases. The incubation of bacterially purified full length AtHYL1 and AtHYL1N with crude protein extracted as a source of proteases from Col-0 further proves the tryptic nature of these proteases.…”

“…Another recent study proposed that cytoplasmic HYL1 regulates miRNA-mediated mRNA translation repression in association with argonaute 1 (AGO1) and altered meristem program 1 (AMP1) [ 19 ]. Studies have also found that an unknown cytoplasmic protease has been involved in regulating HYL1 proteolysis [ 16 , 20 ]. A recent report suggested that HYL1-cleavage subtilase 1 (HCS1) is a cytoplasmic protease for HYL1 destabilization [ 21 ].…”

“…They show that the HYL1 K154A mutant is insensitive to the proteolytic activity of HCS1, which is also regulated by the light/dark transition [ 22 ]. Recently, in plants, the role of reversible phosphorylation is reported with respect to HYL1 reactivation by light to a prolonged dark/shade transition [ 20 , 23 ]. It is also known that CPL1/2 (C-terminal phosphatase-like 1/2) dephosphorylates HYL1 and regulates its functions in miRNA biogenesis [ 14 ].…”

In Arabidopsis thaliana, RNA-Induced Silencing Complex-Loading of MicroRNAs Plays a Minor Regulatory Role During Photomorphogenesis Except for miR163

“…Moreover, the dcl1-9 and hyl1-2 miRNA biogenesis mutants were hypersensitive to red light in the hypocotyl elongation assay ( Sun et al, 2018 ). Furthermore, if dark-grown Arabidopsis seedlings are de-etiolated (shifted to light), the DCL1, HYL1, and SE microprocessor proteins accumulate to high levels and the pri-miRNA levels of most miRNAs are enhanced ( Choi et al, 2020 ; Bhagat et al, 2022 ; Jung et al, 2022 ). The expression of microprocessor components is under complex regulation, for instance, altered transcription, phosphorylation, and protein stability all contribute to the increased HYL1 level during de-etiolation ( Choi et al, 2020 ; Sacnun et al, 2020 ; Bhagat et al, 2022 ; Jung et al, 2022 ).…”

“…Furthermore, if dark-grown Arabidopsis seedlings are de-etiolated (shifted to light), the DCL1, HYL1, and SE microprocessor proteins accumulate to high levels and the pri-miRNA levels of most miRNAs are enhanced ( Choi et al, 2020 ; Bhagat et al, 2022 ; Jung et al, 2022 ). The expression of microprocessor components is under complex regulation, for instance, altered transcription, phosphorylation, and protein stability all contribute to the increased HYL1 level during de-etiolation ( Choi et al, 2020 ; Sacnun et al, 2020 ; Bhagat et al, 2022 ; Jung et al, 2022 ). However, high throughput miRNA sequencing (miRNA-seq) revealed that although pri-miRNA levels and microprocessor concentrations are increased during photomorphogenesis, the total miRNAome is only moderately altered ( Choi et al, 2020 ).…”