An ATP-independent role for Prp16 in promoting aberrant splicing.

The spliceosome is assembled through a step-wise process of binding and release of its components to and from the pre-mRNA. The remodeling process is facilitated by eight DExD/H-box RNA helicases, some of which have also been implicated in splicing fidelity control. In this study, we unveil a contrasting role for the prototypic splicing proofreader, Prp16, in promoting the utilization of aberrant 5' splice sites and mutated branchpoints.

Dynamic protein-RNA interactions in mediating splicing catalysis.

The spliceosome is assembled via sequential interactions of pre-mRNA with five small nuclear RNAs and many proteins. Recent determination of cryo-EM structures for several spliceosomal complexes has provided deep insights into interactions between spliceosomal components and structural changes of the spliceosome between steps, but information on how the proteins interact with pre-mRNA to mediate the reaction is scarce. By systematic analysis of proteins interacting with the splice sites (SSs), we have identified many previously unknown interactions of spliceosomal components with the pre-mRNA.

A central role of Cwc25 in spliceosome dynamics during the catalytic phase of pre-mRNA splicing.

Splicing of precursor mRNA occurs via two consecutive steps of transesterification reaction; both require ATP and several proteins. Despite the energy requirement in the catalytic phase, incubation of the purified spliceosome under proper ionic conditions can elicit competitive reversible transesterification, debranching, and spliced-exon-reopening reactions without the necessity for ATP or other factors, suggesting that small changes in the conformational state of the spliceosome can lead to disparate chemical consequences for the substrate. We show here that Cwc25 plays a central role in modulating the conformational state of the catalytic spliceosome during normal splicing reactions.

Role of Cwc24 in the First Catalytic Step of Splicing and Fidelity of 5' Splice Site Selection.

Cwc24 is an essential splicing factor but only transiently associates with the spliceosome, with an unknown function. The protein contains a RING finger and a zinc finger domain in the carboxyl terminus. The human ortholog of Cwc24, RNF113A, has been associated with the disorder trichothiodystrophy.

Sad1 counteracts Brr2-mediated dissociation of U4/U6.U5 in tri-snRNP homeostasis.

The yeast Sad1 protein was previously identified in a screen for factors involved in the assembly of the U4/U6 di-snRNP particle. Sad1 is required for pre-mRNA splicing both in vivo and in vitro, and its human orthologue has been shown to associate with U4/U6.U5 tri-snRNP.

Link of NTR-mediated spliceosome disassembly with DEAH-box ATPases Prp2, Prp16, and Prp22.

The DEAH-box ATPase Prp43 is required for disassembly of the spliceosome after the completion of splicing or after the discard of the spliceosome due to a splicing defect. Prp43 associates with Ntr1 and Ntr2 to form the NTR complex and is recruited to the spliceosome via the interaction of Ntr2 and U5 component Brr2. Ntr2 alone can bind to U5 and to the spliceosome.

Integrin β1 mediates vaccinia virus entry through activation of PI3K/Akt signaling.

Vaccinia virus has a broad range of infectivity in many cell lines and animals. Although it is known that the vaccinia mature virus binds to cell surface glycosaminoglycans and extracellular matrix proteins, whether additional cellular receptors are required for virus entry remains unclear. Our previous studies showed that the vaccinia mature virus enters through lipid rafts, suggesting the involvement of raft-associated cellular proteins.

The lipid raft-associated protein CD98 is required for vaccinia virus endocytosis.

Mature vaccinia virus (vaccinia MV) infects a broad range of animals in vivo and cell cultures in vitro; however, the cellular receptors that determine vaccinia MV tropism and entry pathways are poorly characterized. Here, we performed quantitative proteomic analyses of lipid raft-associated proteins upon vaccinia MV entry into HeLa cells. We found that a type II membrane glycoprotein, CD98, is enriched in lipid rafts upon vaccinia MV infection compared to mock-infected HeLa cells.

Splicing factor Cwc22 is required for the function of Prp2 and for the spliceosome to escape from a futile pathway.

Cwc22 was previously identified to associate with the pre-mRNA splicing factor Cef1/Ntc85, a component of the Prp19-associated complex (nineteen complex [NTC]) involved in spliceosome activation. We show here that Cwc22 is required for pre-mRNA splicing both in vivo and in vitro but is neither tightly associated with the NTC nor required for spliceosome activation. Cwc22 is associated with the spliceosome prior to catalytic steps and remains associated throughout the reaction.

Disulfide bond formation at the C termini of vaccinia virus A26 and A27 proteins does not require viral redox enzymes and suppresses glycosaminoglycan-mediated cell fusion.

Vaccinia virus A26 protein is an envelope protein of the intracellular mature virus (IMV) of vaccinia virus. A mutant A26 protein with a truncation of the 74 C-terminal amino acids was expressed in infected cells but failed to be incorporated into IMV (W. L.

Vaccinia virus proteome: identification of proteins in vaccinia virus intracellular mature virion particles.

Vaccinia virus is a large enveloped poxvirus with more than 200 genes in its genome. Although many poxvirus genomes have been sequenced, knowledge of the host and viral protein components of the virions remains incomplete. In this study, we used gel-free liquid chromatography and tandem mass spectroscopy to identify the viral and host proteins in purified vaccinia intracellular mature virions (IMV).

The oligomeric structure of vaccinia viral envelope protein A27L is essential for binding to heparin and heparan sulfates on cell surfaces: a structural and functional approach using site-specific mutagenesis.

The soluble domain of the self-assembly vaccinia virus envelope protein A27L, sA27L-aa, consists of a flexible extended coil at the N terminus and a rigid hydrophobic coiled-coil region at the C terminus. In the former, a basic strip of 12 residues is responsible for binding to cell-surface heparan sulfates. Although the latter is believed to mediate self-assembly, its biological role is unclear.

Vaccinia virus penetration requires cholesterol and results in specific viral envelope proteins associated with lipid rafts.

Vaccinia virus infects a wide variety of mammalian cells from different hosts, but the mechanism of virus entry is not clearly defined. The mature intracellular vaccinia virus contains several envelope proteins mediating virion adsorption to cell surface glycosaminoglycans; however, it is not known how the bound virions initiate virion penetration into cells. For this study, we investigated the importance of plasma membrane lipid rafts in the mature intracellular vaccinia virus infection process by using biochemical and fluorescence imaging techniques.

The cowpox virus host range gene, CP77, affects phosphorylation of eIF2 alpha and vaccinia viral translation in apoptotic HeLa cells.

Host restriction of vaccinia virus has been previously described in CHO and RK13 cells in which a cowpox virus CP77 gene rescues vaccinia virus growth at the viral protein translation level. Here we investigate the restrictive stage of vaccinia virus in HeLa cells using a vaccinia mutant virus (VV-hr) that contains a deletion of 18-kb genome sequences resulting in no growth in HeLa cells. Insertion of CP77 gene into VV-hr generated a recombinant virus (VV-36hr) that multiplied well in HeLa cells.

Role of the serine-threonine kinase PAK-1 in myxoma virus replication.

Subversion or appropriation of cellular signal transduction pathways is a common strategy employed by viruses to promote an environment within infected cells that supports the viral replicative cycle. Using subsets of 3T3 murine fibroblasts previously shown to differ in their ability to support myxoma virus (MV) replication, we investigated the role of host serine-threonine kinases (STKs) as potential mediators of the permissive phenotype. Both permissive and nonpermissive 3T3 cells supported equivalent levels of virion binding, entry, and early virus gene expression, indicating that MV tropism in 3T3 cells was not determined by receptor-mediated entry.

Molecular chaperone Hsp90 is important for vaccinia virus growth in cells.

Molecular chaperones assist protein folding, and some chaperones are induced by heat, nutrient depletion, or pathogen invasion. This study investigates the role played by Hsp90 in the life cycle of vaccinia virus. The titer of vaccinia intracellular mature virions (IMV) was reduced by 2 orders of magnitude in RK13 cells treated with geldanamycin (GA), which blocks the ATPase activity of Hsp90.