Crystal Structure of Arabidopsis thaliana Dawdle Forkhead-Associated Domain Reveals a Conserved Phospho-Threonine Recognition Cleft for Dicer-Like 1 Binding

Dawdle （DDL） is a microRNA processing protein essential for the development of Arabidopsis. DDL contains a putative nuclear localization signal at its amino-terminus and forkhead-associated （FHA） domain at the carboxyl-termi- nus. Here, we report the crystal structure of the FHA domain of Arabidopsis Dawdle, determined by multiple-wavelength anomalous dispersion method at 1.7-A resolution. DDL FHA structure displays a seven-stranded 13-sandwich architec- ture that contains a unique structural motif comprising two long anti-parallel strands. Strikingly, crystal packing of the DDL FHA domain reveals that a glutamate residue from the symmetry-related DDL FHA domain, a structural mimic of the phospho-threonine, is specifically recognized by the structurally conserved phospho-threonine binding cleft. Consistently with the structural observations, co-immuno-precipitation experiments performed in Nicotiana benthami- ana show that the DDL FHA domain co-immuno-precipitates with DCL1 fragments containing the predicted pThr＋3（lle/ Val/Leu/Asp） motif. Taken together, we count the recognition of the target residue by the canonical binding cleft of the DDL FHA domain as the key molecular event to instate FHA domain-mediated protein-protein interaction in plant miRNA processing.

LHP1 Interacts with ATRX through Plant-Specific Domains at Specific Loci Targeted by PRC2

Heterochromatin Protein 1 （HP1） is a major regulator of chromatin structure and function. In animals, the network of proteins interacting with HP1 is mainly associated with constitutive heterochromatin marked by H3K9me3. HP1 physically interacts with the putative ortholog of the SNF2 chromatin remodeler ATRX, which controls deposition of histone variant H3.3 in mammals. In this study, we show that the Arabidopsis thaliana ortholog of ATRX participates in H3.3 deposition and possesses specific conserved domains in plants. We found that plant Like HP1 （LHP1） protein interacts with ATRX through domains that evolved specifically in land plant ancestors. Loss of ATRX function in Arabidopsis affects the expression of a limited subset of genes controlled by PRC2 （POLYCOMB REPRESSIVE COMPLEX 2）, including the flowering time regulator FLC. The function of ATRX in regulation of flowering time requires novel LHPl-interacting domain and ATPase activity of the ATRX SNF2 helicase domain. Taken together, these results suggest that distinct evolutionary pathways led to the interaction between ATRX and HP1 in mammals and its counterpart LHP1 in plants, resulting in distinct modes of transcriptional regulation.

Reverse Genetic Analysis of Adaptive Mutations within the Capsid Proteins of Enterovirus 71(EV-A71) Strains Necessary for Infection of CHO-K1 Cells

Dear Editor,Previous studies had described the adaptation of enterovirus 71(EV-A71)strains that enabled entry and viral replication in Chinese Hamster Ovary(CHO)cell line(Zaini and McMinn 2012;Zaini et al.2012).These adapted strains derived from serial passage of a clinical isolate in CHO cells exhibited an amino acid substitution at VP2149,which enhanced viral replication by 100*1000-fold compared to the clinical isolate.The VP2149 mutation was claimed responsible for adaptation to CHO-K1 cells without performing detailed molecular analyses to support these claims.In this study,we evaluate various VP1 and VP2 mutations in two CHO-adapted EV-A71 strains derived in our lab to assess their contribution to the phenotype of CHO cell adaptation.