HASTY modulates miRNA biogenesis by linking pri-miRNA transcription and processing

Abstract

Post-transcriptional gene silencing mediated by microRNAs (miRNAs) modulates numerous developmental and stress response pathways. For the last two decades, HASTY (HST), the ortholog of human EX-PORTIN 5, was considered to be a candidate protein that exports plant miRNAs from the nucleus to the cytoplasm. Here, we report that HST functions in the miRNA pathway independent of its cargo-exporting activity in Arabidopsis. We found that Arabidopsis mutants with impaired HST shuttling exhibit normal subcellular distribution of miRNAs. Interestingly, protein-protein interaction and microscopy assays showed that HST directly interacts with the microprocessor core component DCL1 through its N-terminal domain. Moreover, mass spectrometry analysis revealed that HST also interacts independently of its N-terminal domain with the mediator complex subunit MED37. Further experiments revealed that HST could act as a scaffold to facilitate the recruitment of DCL1 to genomic MIRNA loci by stabilizing the DCL1-MED37 complex, which in turn promotes the transcription and proper processing of primary miRNA transcripts (pri-miRNAs). Taken together, these results suggest that HST is likely associated with the formation of the miRNA biogenesis complex at MIRNA genes, promoting the transcription and processing of pri-miRNAs rather than the direct export of processed miRNAs from the nucleus.

HASTY , the Arabidopsis EXPORTIN5 ortholog, regulates cell‐to‐cell and vascular microRNA movement

“…Potential candidates were back‐crossed once to the parental reporter pSUC2::amiRSUL . One hundred F2 segregating mutant plants (2 weeks old) were then harvested and pooled for genomic DNA extraction following a CTAB DNA extraction (Clarke, 2009 ), in parallel with the pSUC2::amiRSUL parental line. Whole‐genome resequencing of parental and pooled mutant DNA was performed using a TruSeq Nano DNA Library Prep kit and a HiSeq4000 sequencing system (Illumina) to get an average genome coverage of 50X.…”

Protective host-dependent antagonism amongPseudomonasin theArabidopsisphyllosphere

“…Combined with 96% EtOH, the solution was loaded onto a plasmid DNA extraction column (QIAprep Spin Miniprep Kit; Qiagen), and went through several washes before elution of the RNA. mRNA enrichment and sequencing libraries were prepared as previously described [45]. Briefly, mRNA enrichment was done using NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, USA), followed by heat fragmentation.…”

Nucleocytoplasmic Communication in Healthy and Diseased Plant Tissues

“…Mutants of the Arabidopsis exportins PAUSED ( PSD )/ EXPORTIN-T and HASTY ( HST )/ EXPORTIN5 display a delay or acceleration in a range of developmental processes, respectively ( Bollman et al, 2003 ; Hunter et al, 2003 ; Li and Chen, 2003 ). While PSD exports tRNAs, hst mutants show reduced miRNA levels without affecting the subcellular distribution of miRNAs ( Park et al, 2005 ; Bologna et al, 2018 ; Cambiagno et al, 2020 ). Since HST interacts with proteins of the mediator (MED) complex that is part of the transcriptional machinery, HST might have a function as an export-independent scaffold for transcription and processing of primary miRNA transcripts ( Cambiagno et al, 2020 ).…”

“…While PSD exports tRNAs, hst mutants show reduced miRNA levels without affecting the subcellular distribution of miRNAs ( Park et al, 2005 ; Bologna et al, 2018 ; Cambiagno et al, 2020 ). Since HST interacts with proteins of the mediator (MED) complex that is part of the transcriptional machinery, HST might have a function as an export-independent scaffold for transcription and processing of primary miRNA transcripts ( Cambiagno et al, 2020 ).…”