Insight into the RNA Exosome Complex Through Modeling Pontocerebellar Hypoplasia Type 1b Disease Mutations in Yeast.

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 3'-5' 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 wild-type 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.

Toward understanding the molecular mechanism of a geminivirus C4 protein.

Geminiviruses are ssDNA plant viruses that cause significant agricultural losses worldwide. The viruses do not encode a polymerase protein and must reprogram differentiated host cells to re-enter the S-phase of the cell cycle for the virus to gain access to the host-replication machinery for propagation. To date, 3 Beet curly top virus (BCTV) encoded proteins have been shown to restore DNA replication competency: the replication-initiator protein (Rep), the C2 protein, and the C4 protein. Ectopic expression of the BCTV C4 protein leads to a severe developmental phenotype characterized by extensive hyperplasia. We recently demonstrated that C4 interacts with 7 of the 10 members of the Arabidopsis thaliana SHAGGY-like protein kinase gene family and characterized the interactions of C4 and C4 mutants with AtSKs. Herein, we propose a model of how C4 functions.

The roles of phosphorylation and SHAGGY-like protein kinases in geminivirus C4 protein induced hyperplasia.

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.

Structural basis for polyadenosine-RNA binding by Nab2 Zn fingers and its function in mRNA nuclear export.

Polyadenylation 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.

Geminivirus C4 protein alters Arabidopsis development.

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.