RRP41L,a Putative Core Subunit of the Exosome,Plays an Important Role in Seed Germination and Early Seedling Growth in Arabidopsis

In prokaryotic and eukaryotic cells, the 3′-5′-exonucleolytic decay and processing of RNAs are essential for RNA metabolism. However, the understanding of the mechanism of 3′-5′-exonucleolytic decay in plants is very limited. Here, we report the characterization of an Arabidopsis (Arabidopsis thaliana) transfer DNA insertional mutant that shows severe growth defects in early seedling growth, including delayed germination and cotyledon expansion, thinner yellow/pale-green leaves, and a slower growth rate. High-efficiency thermal asymmetric interlaced polymerase chain reaction analysis showed that the insertional locus was in the sixth exon of AT4G27490, encoding a predicted 3′-5′-exonuclease, that contained a conserved RNase phosphorolytic domain with high similarity to RRP41, designated RRP41L. Interestingly, we detected highly accumulated messenger RNAs (mRNAs) that encode seed storage protein and abscisic acid (ABA) biosynthesis and signaling pathway-related protein during the early growth stage in rrp41l mutants. The mRNA decay kinetics analysis for seed storage proteins, 9-cis-epoxycarotenoid dioxygenases, and ABA INSENSITIVEs revealed that RRP41L catalyzed the decay of these mRNAs in the cytoplasm. Consistent with these results, the rrp41l mutant was more sensitive to ABA in germination and root growth than wild-type plants, whereas overexpression lines of RRP41L were more resistant to ABA in germination and root growth than wild-type plants. RRP41L was localized to both the cytoplasm and nucleus, and RRP41L was preferentially expressed in seedlings. Altogether, our results showed that RRP41L plays an important role in seed germination and early seedling growth by mediating specific cytoplasmic mRNA decay in Arabidopsis.RNA decay is an essential step in gene expression regulation that influences many aspects of development and growth. In eukaryotes, mRNA decay is normally initiated by the removal of the poly(A) tail (Couttet et al., 1997; Parker and Song, 2004) and then enters one of two decay pathways: (1) the decapping complex cleaves the 5′ cap, after which the 5′-3′-exoribonuclease, such as XRN1 in animals and yeast (Saccharomyces cerevisiae) and XRN4 in plants, hydrolyzes the mRNA from the 5′ end (Hsu and Stevens, 1993; Kastenmayer and Green, 2000; Garneau et al., 2007; Rymarquis et al., 2011), and (2) the mRNA decays from the 3′ end by the 3′-5′-exonuclease.In eukaryotic cells, the 3′-5′-exonuclease can act alone to process the substrate in some cases, but the vast majority of 3′-5′-exonuclease activity is attributed to the exosome, which is an evolutionarily conserved macromolecular complex that mediates numerous reactions of 3′-5′ RNA processing/degradation and is essential for viability (Mitchell et al., 1997; Estévez et al., 2003). The structure of the exosome has been determined in archaea and eukaryotes, with the core forming a ring-shaped structure (Büttner et al., 2005; Lorentzen et al., 2005; Liu et al., 2006). In eukaryotes, the salient feature of the ring is defined by three distinct heterodimers of six RNase phosphorolytic (PH) domain-type proteins, MTR3-RRP42, RRP41-RRP45, and RRP43-RRP46 (Lehner and Sanderson, 2004; Hernández et al., 2006; Liu et al., 2006). However, the six-protein ring is not stable on its own in vitro and requires three subunits that contain S1 and KH domains (RRP4 links RRP41 and RRP42, RRP40 links RRP45 and RRP46, and CSL4 contacts MTR3 and RRP43) to form a stable core complex (Liu et al., 2006). In yeast, the loss of any individual subunit of the nine-component conserved core is lethal, resulting in similar ribosomal RNA (rRNA) processing defect profiles (Allmang et al., 1999a, 1999b). Moreover, x-ray crystallographic analysis of the human exosome revealed that all of its core subunits are required for its integrity (Liu et al., 2006). Using tandem affinity purification tagging in Arabidopsis (Arabidopsis thaliana) transgenic lines that expressed tagged versions of RRP4 and RRP41, Chekanova et al. (2007) first purified and characterized the exosome complex and revealed that the plant exosome complex contains six RNase PH domain-containing proteins and three S1 and/or KH domain proteins. Although the composition and structure of the plant exosome is similar to other eukaryotes, the function of individual subunits of the exosome appears to be different in Arabidopsis. Down-regulation of distinct subunits of the core complex results in different defects in plant development and RNA-processing profiles. For example, csl4 null mutant plants did not manifest any obvious phenotype, and the null mutation affected only a subset of exosome targets (Chekanova et al., 2007). Therefore, the CSL4 subunit appears to be nonessential for exosome function in Arabidopsis. However, the CSL4 subunit is essential for viability in yeast (Baker et al., 1998; Allmang et al., 1999b). In contrast, the rrp4 mutant shows seed arrest during early stages of embryonic development. RRP41 was shown to be essential for the development of female gametophytes, and homozygous rrp41 is lethal (Chekanova et al., 2007). Additionally, RRP45 is encoded by duplicate genes: RRP45A and RRP45B. Arabidopsis single mutants that lack either RRP45A or RRP45B have no phenotype or only a mild one, respectively, whereas simultaneous down-regulation of both proteins is lethal (Hooker et al., 2007). These data indicate that subunits of the Arabidopsis exosome core complex have specialized roles in plant growth and development and make unequal contributions to the activity of the exosome in vivo. However, the functions of other predicted core subunits of the exosome, with the exception of those mentioned above, are still unclear in Arabidopsis.Here, we report the characterization of an Arabidopsis transfer DNA (T-DNA) insertional mutant that displays severe defects in early seedling growth. High-efficiency thermal asymmetric interlaced (hiTAIL)-PCR analysis revealed that the insertional locus was in the sixth exon of AT4G27490, encoding a predicted 3′-5′-exonuclease that contained a conserved RNase PH domain. A previous study presumed that AT4G27490 was one subunit of the core exosome in Arabidopsis, a homolog of yeast Mtr3 (Chekanova et al., 2007), but another study suggested that it was a homolog of yeast Rrp41 (Zimmer et al., 2008). Here, we refer to AT4G27490 as RRP41L. Interestingly, we detected highly accumulated mRNAs that encode seed storage protein (SSP) and abscisic acid (ABA) biosynthesis and signaling pathway-related protein during the early growth stage in the rrp41l mutant. The mRNA decay kinetics analysis for SSPs, 9-cis-epoxycarotenoid dioxygenases (NCEDs), and ABA INSENSITIVEs (ABIs) revealed that RRP41L catalyzed the decay of these mRNAs in the cytoplasm. Consistent with these results, the rrp41l mutant was more sensitive in seed germination and root growth than wild-type plants, whereas the overexpression (OE) lines of RRP41L were more resistant to ABA in seed germination and root growth than wild-type plants. RRP41L is localized to both the cytoplasm and nucleus, and RRP41L is preferentially expressed in seedlings. Collectively, our results showed that RRP41L plays an important role in seed germination and early seedling growth by mediating specific cytoplasmic mRNA decay in Arabidopsis.     

Humoral immunogenicity and reactogenicity of CoronaVac or ZF2001 booster after two doses of inactivated vaccine

Dear Editor, COVID-19 vaccination campaigns are being conducted in countries worldwide,and 47.4％ of the world population has received at least one dose of a COV...     

Humoral immune response to circulating SARS-CoV-2 variants elicited by inactivated and RBD-subunit vaccines

SARS-CoV-2 variants could induce immune escape by mutations on the receptor-binding domain (RBD) and N-terminal domain (NTD). Here we report the humoral immune response to circulating SARS-CoV-2 variants, such as 501Y.V2 (B.1.351), of the plasma and neutralizing antibodies (NAbs) elicited by CoronaVac (inactivated vaccine), ZF2001 (RBD-subunit vaccine) and natural infection. Among 86 potent NAbs identified by high-throughput single-cell VDJ sequencing of peripheral blood mononuclear cells from vaccinees and convalescents, near half anti-RBD NAbs showed major neutralization reductions against the K417N/E484K/N501Y mutation combination, with E484K being the dominant cause. VH3-53/VH3-66 recurrent antibodies respond differently to RBD variants, and K417N compromises the majority of neutralizing activity through reduced polar contacts with complementarity determining regions. In contrast, the 242–244 deletion (242–244Δ) would abolish most neutralization activity of anti-NTD NAbs by interrupting the conformation of NTD antigenic supersite, indicating a much less diversity of anti-NTD NAbs than anti-RBD NAbs. Plasma of convalescents and CoronaVac vaccinees displayed comparable neutralization reductions against pseudo- and authentic 501Y.V2 variants, mainly caused by E484K/N501Y and 242–244Δ, with the effects being additive. Importantly, RBD-subunit vaccinees exhibit markedly higher tolerance to 501Y.V2 than convalescents, since the elicited anti-RBD NAbs display a high diversity and are unaffected by NTD mutations. Moreover, an extended gap between the third and second doses of ZF2001 leads to better neutralizing activity and tolerance to 501Y.V2 than the standard three-dose administration. Together, these results suggest that the deployment of RBD-vaccines, through a third-dose boost, may be ideal for combating SARS-CoV-2 variants when necessary, especially for those carrying mutations that disrupt the NTD supersite.Subject terms: Immunology, Cryoelectron microscopy, X-ray crystallography