Functional Analysis Of The Exosome Subunit RRP42 In Arabidopsis

The exosome is an evolutionarily conserved macromolecular complex. It can mediate numerous reactions of 3'-5J RNA processing and degradation. The salient feature of the exosome core is the hexameric ring defined by three distinct heterodimers of six RNase PH domain-type proteins:RRP41-RRP45, MTR3-RRP42 and RRP43-RRP46. These heterodimers are bridged on one side by three subunits (RRP40, RRP4 and CSL4) containing S1 and KH domains to form a stable nine subunite cyclic complex. In yeast and mammals, there is a deeper study of the structure and function of exosome.However, current research on functions of exosome subunit in plants is limited. In this study, we mainly analyzed the function of the exosome core subunit RRP42, and also preliminaryly analyzed another core subunit RRP46.Firstly, we used the CRISPR/Cas9 system to knock out RRP42. Three rrp42/RRP42 lines were obtained. The mutation types in each rrp42/RRP42 line was different with each other, and the mutant female gametophytes arrested at the stage of FG2. These results show that the null mutation of rrp42 is lethal and RRP42 has an essential role for development of the female gametophyte in Arabidopsis. To address the functions of RRP42 during vegetative growth, we designed three different amiRNAs targeting RRP42 using the artificial microRNA system and obtained numbers of T1 lines with similar phenotypes. The rrp42 knock-down mutants mainly displayed wrinkled and smaller cotyledon in seedling. During later growth stages, the rrp42 knock-down mutants displayed primarily variegated,wrinkled and serrated leaves, especially in cauline leaves. The results of internal leaf anatomy by means of cross sections showed that the palisade cell arrangement and morphology in rrp42 knock-down mutants leaves were abnormal and the density of mesophyll cells were reduced. We tested the expression levels of RRP42 in these rrp42 knock-down mutants, the expression levels of RRP42 were consistent with the severity of rrp42 knock-down mutant defects. These results indicated that the defects of rrp42 knock-down mutants were induced by the repressive activity of RRP42. The spatial and temporal expression patterns of RRP42 in plant tissue also showed that RRP42 was preferentially expressed in ovule and leaves. These results indicated that the RRP42 may play an important role in female gametophytes and leaf development in Arabidopsis.The phenotypic defects of rrp42 knock-down mutants prompted us to use qRT-PCR to measure the level of transcripts that encode proteins participating in photosynthesis, cell division, cell wall assembly and leaf morphogenesis. We found that expression of the XTHs and expansins, both involved in cell wall assembly, were highly accumulated in rrp42 knock-down mutant. Moreover, the mRNA decay kinetics analysis for XTH19, EXPA10 and EXPA11 revealed that the decay of these mRNAs in rrp42 knock-down mutant leaves was clearly slower than that in wild type leaves. The intracellular distribution of RRP42 showed that it was localized to both the cytoplasm and nucleus. Our results indicated that RRP42 has functions in cytoplasmic mRNA decay in Arabidopsis. In addition, we compared the target mRNA of RRP42 with other subunit target mRNAs. The results showed that the expression of RRP42 target mRNA did not change significantly in other subunit mutants (rrp41iRNAi,rrp4iRVAi and rrp41l). We also randomly chose a number of mRNAs up-regulated in the rrp41iRVAi,rrp4iRNAi and rrp41l mutants, and examined their transcript levels in rrp42 knock-down mutant by qRT-PCR. The results showed that the accumulation of these mRNAs was consistent with that of wild type. These results indicate that exosome subunit seems to have specificity in the degradation of cytoplasmic mRNA.In addition, we also obtained several heterozygotes of rrp46/RRP46 with mutations in different ways, and they all were embryogenic lethal. The analysis of RRP46 function required more research in later work.