Ubiquitination-dependent degradation of MEL1 is critical for microsporogenesis

Grass anthers are enriched for 21-nucleotide (nt), phased, small interfering RNAs (phasiRNAs) derived from hundreds to thousands oflong noncoding loci. Mutations in a few such loci in rice (Oryza sativa) are known to confer temperature/photoperiod-sensitive male sterility, and are the basis for hybrid rice production (reviewed in Liu et al., 2020). The rice MEIOSIS ARRESTED AT LEPTOTENE1 (MEL1) gene encodes an Argonaute family protein that can load 21-nt phasiRNAs and mediate cleavage of their target mRNAs in male germinal cells (Jiang et al., 2020; Zhang et al., 2020). A prior study had shown that the accumulation of the MEL1 protein is spatiotemporally regulated; it accumulates to high abundance in pollen mother cells and during early meiosis, but is downregulated after meiosis (Nonomura et al., 2007).

Lian et al. (2021) now investigate whether the stage-specific accumulation pattern of MEL1 is critical for its function in male germinal development. In transgenic rice lines (OXMEL1) overexpressing MEL1, they show that microspogenesis is perturbed at anaphase II; about half of the OXMEL1 microspore tetrads underwent abnormal division. Consequently, about half of the pollen grains aborted at the mature pollen stage, and the transgenic plants were semi-sterile (see Figure). These results suggest that temporal downregulation of MEL1 in the wild type is critical for microsporogenesis.

Figure.

Overexpression of MEL1 causes partial male sterility. Panicles (left) and I₂-KI-stained pollen grains (right) of a MEL1 overexpression line (OXMEL1) and wild type (WT). Adapted from Lian et al. (2021), Figure 1D.

To characterize MEL1 protein turnover, the authors performed a series of biochemical assays and showed that MEL1 is ubiquitinated and degraded by the 26S proteasome pathway. Co-immunoprecipitation followed by mass spectrometry analyses identified three putative E3 ubiquitin ligases that interact with MEL1 in vivo. Only one of them, XBOS36, was found to ubiquitinate MEL1 to facilitate its degradation.

To understand whether the degradation of MEL1 mediated by XBOS36 is essential for normal microsporogenesis, the authors knocked out the XBOS36 gene using CRISPR-Cas9. MEL1 protein abundance significantly increased in the xbos36 mutant compared to wild type during microsporogenesis, whereas no significant change in MEL1 mRNA abundance was detected, supporting the notion that temporal downregulation of MEL1 in the wild type is mediated by XBOS36 at the post-translational level. The xbos36 mutant exhibited a semi-sterile phenotype similar to that of the OXMEL1 transgenics, suggesting that the timely degradation of the MEL1 protein is essential for successful microsporogenesis.

The authors next examined which lysine residues of the MEL1 are ubiquitinated. Incubation of N- and C-terminal MEL1 protein fragments with protein extracts from rice panicles at the early microspore stage showed that the N-terminus is degraded more rapidly than the C-terminus, suggesting that XBOS36 preferentially interacts with the N-terminal region of MEL1. Five lysine residues within the MEL1 protein were predicted to be potential ubiquitination sites. The authors generated MEL1 variants with one or more lysine residues substituted by arginine. Only the variant with all the four N-terminal lysine residues substituted showed reduced ubiquitination, suggesting that ubiquitination of the N-terminal lysines targets the MEL1 protein for degradation by the 26S proteasome pathway.

Finally, the authors asked whether perturbation of MEL1 degradation in microspores causes off-target cleavage of mRNAs. A transcriptome analysis of spikelets from wild type, OXMEL1, and xbos36 plants identified 289 protein-coding genes that are commonly downregulated in OXMEL1 and xbos36. Interrogation of previously published degradome data showed that the mRNAs of 30% of these genes are cleaved by 21-nt phasiRNAs in the wild type, suggesting that the other downregulated mRNAs are possibly off-targets of phasiRNAs as consequences of prolonged accumulation of MEL1 protein, although direct evidence of cleavage is yet to be obtained.

The work of Lian et al. (2021) highlights the importance of the post-translational regulation of MEL1 for successful microsporogenesis. The identification of XBOS36, which may modulate the targeting activity of the 21-nt reproductive phasiRNAs through MEL1, further advances our understanding of the regulation of male fertility and may facilitate future development of hybrid crops.