Intrinsically disordered proteins SAID1/2 condensate on SERRATE for dual inhibition of miRNA biogenesis in Arabidopsis.

Intrinsically disordered proteins (IDPs) SAID1/2 are hypothetic dentin sialophosphoprotein-like proteins, but their true functions are unknown. Here, we identified SAID1/2 as negative regulators of SERRATE (SE), a core factor in miRNA biogenesis complex (microprocessor). Loss-of-function double mutants of said1; said2 caused pleiotropic developmental defects and thousands of differentially expressed genes that partially overlapped with those in se. said1; said2 also displayed increased assembly of microprocessor and elevated accumulation of microRNAs (miRNAs). Mechanistically, SAID1/2 promote pre-mRNA processing 4 kinase A-mediated phosphorylation of SE, causing its degradation in vivo. Unexpectedly, SAID1/2 have strong binding affinity to hairpin-structured pri-miRNAs and can sequester them from SE. Moreover, SAID1/2 directly inhibit pri-miRNA processing by microprocessor in vitro. Whereas SAID1/2 did not impact SE subcellular compartmentation, the proteins themselves exhibited liquid-liquid phase condensation that is nucleated on SE. Thus, we propose that SAID1/2 reduce miRNA production through hijacking pri-miRNAs to prevent microprocessor activity while promoting SE phosphorylation and its destabilization in Arabidopsis.

Reciprocal regulation of m 6 A modification and miRNA production machineries via phase separation-dependent and -independent mechanisms.

Methyltransferase complex (MTC) deposits N 6-adenosine (m 6 A) onto RNA, whereas microprocessor produces miRNA. Whether and how these two distinct complexes cross-regulate each other has been poorly studied. Here we report that the MTC subunit B (MTB) tends to form insoluble condensates with poor activity, with its level monitored by 20S proteasome. Conversely, the microprocessor component SERRATE (SE) forms liquid-like condensates, which in turn promotes solubility and stability of MTB, leading to increased MTC activity. Consistently, the hypomorphic lines expressing SE variants, defective in MTC interaction or liquid-like phase behavior, exhibit reduced m 6 A level. Reciprocally, MTC can recruit microprocessor to MIRNA loci, prompting co-transcriptional cleavage of primary miRNA (pri-miRNAs) substrates. Additionally, pri-miRNAs carrying m 6 A modifications at their single-stranded basal regions are enriched by m 6 A readers, which retain microprocessor in the nucleoplasm for continuing processing. This reveals an unappreciated mechanism of phase separation in RNA modification and processing through MTC and microprocessor coordination.

SERRATE drives phase separation behaviours to regulate m6A modification and miRNA biogenesis.

The methyltransferase complex (MTC) deposits N6-adenosine (m6A) onto RNA, whereas the microprocessor produces microRNA. Whether and how these two distinct complexes cross-regulate each other has been poorly studied. Here we report that the MTC subunit B tends to form insoluble condensates with poor activity, with its level monitored by the 20S proteasome. Conversely, the microprocessor component SERRATE (SE) forms liquid-like condensates, which in turn promote the solubility and stability of the MTC subunit B, leading to increased MTC activity. Consistently, the hypomorphic lines expressing SE variants, defective in MTC interaction or liquid-like phase behaviour, exhibit reduced m6A levels. Reciprocally, MTC can recruit the microprocessor to the MIRNA loci, prompting co-transcriptional cleavage of primary miRNA substrates. Additionally, primary miRNA substrates carrying m6A modifications at their single-stranded basal regions are enriched by m6A readers, which retain the microprocessor in the nucleoplasm for continuing processing. This reveals an unappreciated mechanism of phase separation in RNA modification and processing through MTC and microprocessor coordination.