Anti-Myc Tag Antibody [MYC.A7] (A85281)

Cajal bodies (CBs) are major sub-nuclear structures in most eucaryotic cells. In human adenovirus 5 (Ad5) infection, CBs are reorganized into microfoci in the late phase of infection. Here we show that many CB protein components (p80-coilin, SMN-1, and WRAP53) remained stable throughout most of the infectious cycle of Ad type 5 (Ad5) in human A549 epithelial cells, even when CBs were reorganized into microfoci. Reduction of p80-coilin expression by RNA interference resulted in significant reductions in the levels of early (E1A, E2A-DBP), intermediate (pIX and IVa2), and late (L1-IIIa, L2-penton base, L3-hexon, L4-100K, and L5-fiber) proteins in Ad5-infected A549 cells. Depletion of p80-coilin did not significantly alter the total cellular levels of the corresponding Ad cytoplasmic mRNAs (with the exception of E1A 12S and 13S and pIX mRNA) or the production of the Ad5 pre-mRNAs tested (E1A, E2A-DBP, IVa2, or late pre-mRNAs containing the tripartite leader). However, the ratio of viral cytoplasmic to nuclear-spliced Ad RNAs was reduced in p80-coilin-depleted, Ad5-infected cells compared to control-infected cells. Immunofluorescent staining of Ad5-infected cells revealed co-localization of p80-coilin with areas of immunoreactivity defined by a polyclonal antibody that recognized the L4-22K and L4-33K proteins in a fraction of microfoci. Immunoprecipitation analysis showed that only the L4-22K protein formed a stable complex with p80-coilin in Ad5-infected cells and in cells co-transfected with plasmids encoding p80-coilin and either the L4-22K or L4-33K protein. p80-coilin therefore plays an important role in Ad replication by complex formation with L4-22K and facilitating nuclear export of Ad mRNAs. IMPORTANCE The architecture of sub-nuclear structures of eucaryotic cells is often changed during the infectious cycle of many animal and plant viruses. Cajal bodies (CBs) form a major sub-nuclear structure whose functions may include the regulation of cellular RNA metabolism. During the lifecycle of human adenovirus 5 (Ad5), CBs are reorganized from their spherical-like structure into smaller clusters termed microfoci. The mechanism of this reorganization and its significance for virus replication has yet to be established. Here we show that the major CB protein, p80-coilin, facilitates the nuclear export of Ad5 transcripts. Depletion of p80-coilin by RNA interference led to lowered levels of viral proteins and infectious virus. p80-coilin was found to form a complex with the viral L4-22K protein in Ad5-infected cells and in some reorganized microfoci. These findings assign a new role for p80-coilin as a potential regulator of infection by a human DNA virus.

The Acetylation-dependent (Ac/) N-degron pathway degrades proteins through recognition of their acetylated N-termini (Nt) by E3-ligases called Ac/N-recognins. To date, specific Ac/N-recognins have not been defined in plants. Here we used molecular, genetic, and multi-omics approaches to characterise potential roles for Arabidopsis (Arabidopsis thaliana) DEGRADATION OF ALPHA2 10 (DOA10)-like E3-ligases in the Nt-acetylation-(NTA-) dependent turnover of proteins at global and protein-specific scales. Arabidopsis has two ER-localised DOA10-like proteins. AtDOA10A, but not the Brassicaceae-specific AtDOA10B, can compensate for loss of yeast (Saccharomyces cerevisiae) ScDOA10 function. Transcriptome and Nt-acetylome profiling of an Atdoa10a/b RNAi mutant revealed no obvious differences in the global NTA profile compared to wildtype, suggesting that AtDOA10s do not regulate the bulk turnover of NTA substrates. Using protein steady-state and cycloheximide-chase degradation assays in yeast and Arabidopsis, we showed that turnover of ER-localised SQUALENE EPOXIDASE 1 (AtSQE1), a critical sterol biosynthesis enzyme, is mediated by AtDOA10s. Degradation of AtSQE1 in planta did not depend on NTA, but Nt-acetyltransferases indirectly impacted its turnover in yeast, indicating kingdom-specific differences in NTA and cellular proteostasis. Our work suggests that, in contrast to yeast and mammals, targeting of Nt-acetylated proteins is not a major function of DOA10-like E3 ligases in Arabidopsis and provides further insight into plant ERAD and the conservation of regulatory mechanisms controlling sterol biosynthesis in eukaryotes.