Mitosis in metazoa requires nuclear envelope (NE) disassembly and reassembly. NE disassembly is driven by multiple phosphorylation events. Mitotic phosphorylation of the protein BAF reduces its affinity for chromatin and the LEM family of inner nuclear membrane proteins; loss of this BAF-mediated chromatin-NE link contributes to NE disassembly. BAF must reassociate with chromatin and LEM proteins at mitotic exit to reform the NE; however, how its dephosphorylation is regulated is unknown. Here, we show that the C. elegans protein LEM-4L and its human ortholog Lem4 (also called ANKLE2) are both required for BAF dephosphorylation. They act in part by inhibiting BAF's mitotic kinase, VRK-1, in vivo and in vitro. In addition, Lem4/LEM-4L interacts with PP2A and is required for it to dephosphorylate BAF during mitotic exit. By coordinating VRK-1- and PP2A-mediated signaling on BAF, Lem4/LEM-4L controls postmitotic NE formation in a function conserved from worms to humans.

Proteins interact with nucleic acids to regulate cellular functions. The study of these regulatory interactions is often hampered by the limited efficiency of current protocols to isolate the relevant nucleic acid–protein complexes. In this report, we describe a rapid and simple procedure to highly enrich cross-linked nucleic acid–bound proteins, referred to as “2C” for “complex capture.” This method is based on the observation that silica matrix–based columns used for nucleic acid purification also effectively retain UV cross-linked nucleic acid–protein complexes. As a proof of principle, 2C was used to isolate RNA-bound proteins from yeast and mammalian Huh7 cells. The 2C method makes RNA labelling redundant, and specific RNA–protein interactions can be observed and validated by Western blotting. RNA–protein complexes isolated by 2C can subsequently be immunoprecipitated, showing that 2C is in principle compatible with sensitive downstream applications. We suggest that 2C can dramatically simplify the study of nucleic acid–protein interactions and benefit researchers in the fields of DNA and RNA biology.

Ubiquinone (coenzyme Q; Q) is a key factor in the mitochondria electron transport chain, but it also functions as an antioxidant and as a cofactor of mitochondrial uncoupling proteins. Furthermore, Q isoforms balance in Caenorhabditis elegans is determined by both dietary intake and endogenous biosynthesis. In the absence of synthesis, withdrawal of dietary Q8 in adulthood extends life span. Thus, Q plays an important role in the aging process and understanding its synthesis acquires a new impetus. We have identified by RNA interference (RNAi) eight genes, including clk-1, involved in ubiquinone biosynthesis in C. elegans feeding animals with dsRNA-containing Escherichia coli HT115 strains. Silenced C. elegans showed lower levels of both endogenous Q9 and Q8 provided by diet, produced less superoxide without a significant modification of mitochondrial electron chain, and extended life span compared with non-interfered animals. E. coli strains harboring dsRNA also interfered with their own Q8 biosynthesis. These findings suggest that more efficient electron transport between a lower amount of Q and electron transport capacity of the mitochondrial complexes leads to less production of reactive oxygen species that contributes to extension of life span in the nematode C. elegans.

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