SummaryPolyadenylation regulation and efficient nuclear export of mature mRNPs both require the polyadenosine-RNA-binding protein, Nab2, which contains seven CCCH Zn fingers. We describe here the solution structure of fingers 5-7, which are necessary and sufficient for high-affinity polyadenosine-RNA binding, and identify key residues involved. These Zn fingers form a single structural unit. Structural coherence is lost in the RNA-binding compromised Nab2-C437S mutant, which also suppresses the rat8-2 allele of RNA helicase Dbp5. Structure-guided Nab2 variants indicate that dbp5(rat8-2) suppression is more closely linked to hyperadenylation and suppression of mutant alleles of the nuclear RNA export adaptor, Yra1, than to affinity for polyadenosine-RNA. These results indicate that, in addition to modulating polyA tail length, Nab2 has an unanticipated function associated with generating export-competent mRNPs, and that changes within fingers 5-7 lead to suboptimal assembly of mRNP export complexes that are more easily disassembled by Dbp5 upon reaching the cytoplasm.

Pontocerebellar hypoplasia type 1b (PCH1b) is an autosomal recessive disorder that causes cerebellar hypoplasia and spinal motor neuron degeneration, leading to mortality in early childhood. PCH1b is caused by mutations in the RNA exosome subunit gene, EXOSC3. The RNA exosome is an evolutionarily conserved complex, consisting of nine different core subunits, and one or two 39-59 exoribonuclease subunits, that mediates several RNA degradation and processing steps. The goal of this study is to assess the functional consequences of the amino acid substitutions that have been identified in EXOSC3 in PCH1b patients. To analyze these EXOSC3 substitutions, we generated the corresponding amino acid substitutions in the Saccharomyces cerevisiae ortholog of EXOSC3, Rrp40. We find that the rrp40 variants corresponding to EXOSC3-G31A and -D132A do not affect yeast function when expressed as the sole copy of the essential Rrp40 protein. In contrast, the rrp40-W195R variant, corresponding to EXOSC3-W238R in PCH1b patients, impacts cell growth and RNA exosome function when expressed as the sole copy of Rrp40. The rrp40-W195R protein is unstable, and does not associate efficiently with the RNA exosome in cells that also express wild-type Rrp40. Consistent with these findings in yeast, the levels of mouse EXOSC3 variants are reduced compared to wild-type EXOSC3 in a neuronal cell line. These data suggest that cells possess a mechanism for optimal assembly of functional RNA exosome complex that can discriminate between wildtype and variant exosome subunits. Budding yeast can therefore serve as a useful tool to understand the molecular defects in the RNA exosome caused by PCH1b-associated amino acid substitutions in EXOSC3, and potentially extending to disease-associated substitutions in other exosome subunits.KEYWORDS pontocerebellar hypoplasia type 1b; RNA exosome; EXOSC3; Rrp40; EXOSC2; RNA processing/degradation T HE RNA exosome is an evolutionarily conserved ribonuclease complex that is responsible for several essential RNA processing and degradation steps (Mitchell et al. 1997;Allmang et al. 1999;Schneider and Tollervey 2013). Major exosome substrates include mRNA, rRNA, and small RNAs. In addition to degrading aberrant and unneeded transcripts, the RNA exosome also trims precursor RNAs, including 5.8S rRNA (Mitchell et al. 1996). The RNA exosome thus plays critical roles in both RNA degradation and maturation.The subunits of the RNA exosome complex are evolutionarily conserved and many were first identified in Saccharomyces cerevisiae in a screen for ribosomal RNA processing (rrp) mutants (Mitchell et al. 1996(Mitchell et al. , 1997Allmang et al. 1999). S. cerevisiae Rrp subunits correspond to human EXOSC subunits. The functions of the RNA exosome have been extensively characterized in budding yeast (Sloan et al. 2012), and many are conserved in humans (Schilders et al. 2005;Staals et al. 2010;Lubas et al. 2011Lubas et al. , 2015 the RNA exosome, six subunits comprise a core ring, and three putative RNA-binding sub...

The C4 protein of beet curly top virus [BCTV-B (US:Log:76)] induces hyperplasia in infected phloem tissue and tumorigenic growths in transgenic plants. The protein offers an excellent model for studying cell cycle control, cell differentiation, and plant development. To investigate the role of the C4 protein in plant development, transgenic Arabidopsis thaliana plants were generated in which the C4 transgene was expressed under the control of an inducible promoter. A detailed analysis of the developmental changes that occur in cotyledons and hypocotyls of seedlings expressing the C4 transgene showed extensive cell division in all tissues types examined, radically altered tissue layer organization, and the absence of a clearly defined vascular system. Induced seedlings failed to develop true leaves, lateral roots, and shoot and root apical meristems, as well as vascular tissue. Specialized epidermis structures, such as stomata and root hairs, were either absent or developmentally impaired in seedlings that expressed C4 protein. Exogenous application of brassinosteroid and abscisic acid weakly rescued the C4-induced phenotype, while induced seedlings were hypersensitive to gibberellic acid and kinetin. These results indicate that ectopic expression of the BCTV C4 protein in A. thaliana drastically alters plant development, possibly through the disruption of multiple hormonal pathways.

Even though plant cells are highly plastic, plants only develop hyperplasia under very specific abiotic and biotic stresses, such as when exposed to pathogens like Beet curly top virus (BCTV). The C4 protein of BCTV is sufficient to induce hyperplasia and alter Arabidopsis development. It was previously shown that C4 interacts with two Arabidopsis Shaggy-like protein kinases, AtSK21 and 23, which are negative regulators of brassinosteroid (BR) hormone signaling. Here we show that the C4 protein interacts with five additional AtSK family members. Bikinin, a competitive inhibitor of the seven AtSK family members that interact with C4, induced hyperplasia similar to that induced by the C4 protein. The Ser49 residue of C4 was found to be critical for C4 function, since: 1) mutagenesis of Ser49 to Ala abolished the C4-induced phenotype, abolished C4/AtSK interactions, and resulted in a mutant protein that failed to induce changes in the BR signaling pathway; 2) Ser49 is phosphorylated in planta; and 3) plant-encoded AtSKs must be catalytically active to interact with C4. A C4 N-myristoylation site mutant that does not localize to the plasma membrane and does not induce a phenotype, retained the ability to bind AtSKs. Taken together, these results suggest that plasma membrane associated C4 interacts with and co-opts multiple AtSKs to promote its own phosphorylation and activation to subsequently compromise cell cycle control.