Functional Analysis Of Arabidopsis Spliceosome Components AtBud13 And JANUS

In eukaryotic cells,pre-mRNA splicing is an important step for gene expression that requires the precise removal of introns.Pre-mRNA splicing is mediated by large RNA-protein complexes known as spliceosomes.Although spliceosomes have been extensively investigated in yeast or animals,its function in plants is still rather elusive.Embryogenesis involves precise cell fate determination based on the position of cells along the embryo axes.In Arabidopsis,the crucial cell types are established extremely early during embryogenesis as reflected by the stereotypic sequence of oriented cell divisions.In the past decades,mechanisms underlying embryogenesis have been well documented.However,there are still unresolved issues to be addresses.In this study,we demonstrated the key role of spliceosome components in early embryogenesis as detailed blow.(1)At1g31870(AtBud13)is the Arabidopsis homolog of yeast Bud13,a component of spliceosome.AtBud13 is expressed throughout plant development and the protein is localized in the nucleus.Mutations of AtBud13 resulted in embryo lethality and seed abortion.RNA-seq analyses revealed that functional loss of AtBud13 predominantly resulted in the retention of introns,especially shorter introns(?100 bases).Among genes whose pre-mRNA splicing was altered due to functional loss of AtBud13,52 were involved in embryogenesis.Our results demonstrated that AtBud13 plays critical roles in early embryo development by affecting pre-mRNA splicing.(2)Arabidopsis JANUS encodes a putative component of spliceosome,homologous to mammalian SF4 b.Functional loss of JANUS is essential for embryonic pattern formation.Intriguingly,functional loss of JANUS resulted in the reduced transcription but not pre-mRNA processing of WOX2 and PIN7,two critical genes for embryogenesis.We demonstrated that JANUS interacts with and likelyrecruits RNA polymerase ?(Pol ?)through it RRM2 domain.These findings suggested that JANUS recruits Pol ? for the activation of two parallel pathways to ensure proper pattern formation during embryogenesis.(3)Embryo lethality of the janus mutant prevented it being used to study sporophytic roles of JANUS.To encounter the problem,we generate JANUS-RNAi plants.The significantly reduced root length,root meristem length,and cell number of the JANUS-RNAi plants indicated that JANUS is a positive regulator of root meristem development.Indeed,we showed that JANUS positively regulates PLT expression in root meristem by recruiting Pol ? on PLT and by interacting with PLT1.JANUS-dependent recruitment of Pol ? is inhibited through the competitive binding of JANUS by GRF-INTERACTING FACTOR1/ANGUSTIFOLIA3(GIF1/AN3),a transcriptional co-factor negatively regulating PLT expression.Finally,GIF1 and JANUS,the opposing regulators of PLT,both depend on Arabidopsis IMPORTIN ?4(IMB4)for their nuclear accumulation.The combination of an importin and its two opposing cargos in PLT transcription may have logistic benefits in fine-tuning the concentration gradient of PLT in root meristem.(4)In summary,this study demonstrated key roles of spliceosome components in plant development.In addition to the regulation of pre-mRNA splicing,components of the spliceosome may mediate gene transcription.