A Sequence-Indexed Mutator Insertional Library for Maize Functional Genomics Study.

Sequence-indexed insertional libraries are important resources for functional gene study in model plants. However, the maize (Zea mays) UniformMu library covers only 36% of the annotated maize genes. Here, we generated a new sequence-indexed maize Mutator insertional library named ChinaMu through high-throughput sequencing of enriched Mu-tagged sequences. A total of 2,581 Mu F2 lines were analyzed, and 311,924 nonredundant Mu insertion sites were obtained. Based on experimental validation, ChinaMu contains about 97,000 germinal Mu insertions, about twice as many as UniformMu. About two-thirds (66,565) of the insertions are high-quality germinal insertions (positive rate > 90%), 89.6% of which are located in genic regions. Furthermore, 45.7% (20,244) of the 44,300 annotated maize genes are effectively tagged and about two-thirds (13,425) of these genes harbor multiple insertions. We tested the utility of ChinaMu using pentatricopeptide repeat (PPR) genes. For published PPR genes with defective kernel phenotypes, 17 out of 20 were tagged, 11 of which had the previously reported mutant phenotype. For 16 unstudied PPR genes with both Mu insertions and defective kernel phenotypes, 6 contained insertions that cosegregated with the mutant phenotype. Our sequence-indexed Mu insertional library provides an important resource for functional genomics study in maize.

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.

The epigenetic factor FVE orchestrates cytoplasmic SGS3-DRB4-DCL4 activities to promote transgene silencing in Arabidopsis.

Posttranscriptional gene silencing (PTGS) is a regulatory mechanism to suppress undesired transcripts. Here, we identified Flowering locus VE (FVE), a well-known epigenetic component, as a new player in cytoplasmic PTGS. Loss-of-function fve mutations substantially reduced the accumulation of transgene-derived small interfering RNAs (siRNAs). FVE interacts with suppressor of gene silencing 3 (SGS3), a master component in PTGS. FVE promotes SGS3 homodimerization that is essential for its function. FVE can bind to single-stranded RNA and double-stranded RNA (dsRNA) with moderate affinities, while its truncated form FVE-8 has a significantly increased binding affinity to dsRNA. These affinities affect the association and channeling of SGS3-RNA to downstream dsRNA binding protein 4 (DRB4)/Dicer-like protein 2/4 (DCL2/4) complexes. Hence, FVE, but not FVE-8, biochemically enhances the DRB4/DCL2/4 activity in vitro. We surmise that FVE promotes production of transgene-derived siRNAs through concertedly tuning SGS3-DRB4/DCL2/4 functions. Thus, this study revealed a noncanonical role of FVE in PTGS.