Protein-coding genes in eukaryotes are transcribed by RNA polymerase II (Pol II) and introns are removed from pre-mRNA by the spliceosome. Understanding the time lag between Pol II progression and splicing could provide mechanistic insights into the regulation of gene expression. Here, we present two single-molecule nascent RNA sequencing methods that directly determine the progress of splicing...

The endosomal sorting complexes required for transport (ESCRT) machinery functions in HIV-1 budding, cytokinesis, multivesicular body biogenesis, and other pathways, in the course of which it interacts with concave membrane necks and bud rims. To test the role of membrane shape in regulating ESCRT assembly, we nanofabricated templates for invaginated supported lipid bilayers. The assembly of th...

Protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus is mediated by coat complex II (COPII) vesicles. It has been believed that COPII vesicles containing cargo are released from the ER exit sites (ERES) into the cytosol and then reach and fuse with the first post-ER compartment, cis-Golgi or ER-to-Golgi intermediate compartment (ERGIC). However, it still remains elusive ...

We performed a large-scale cDNA analysis to explore the transcriptome of the budding yeast Saccharomyces cerevisiae. We sequenced two cDNA libraries, one from the cells exponentially growing in a minimal medium and the other from meiotic cells. Both libraries were generated by using a vector-capping method that allows the accurate mapping of transcription start sites (TSSs). Consequently, we id...

Cyclin-dependent kinases (CDKs) function as central regulators of both the cell cycle and transcription. CDK activation depends on phosphorylation by a CDK-activating kinase (CAK). Different CAKs have been identified in budding yeast, fission yeast, and metazoans. All known CAKs belong to the extended CDK family. The sole budding yeast CAK, CAK1, and one of the two CAKs in fission yeast, csk1, ...

The sorting of transmembrane cargo proteins into the lumenal vesicles of multivesicular bodies (MVBs) depends on the recruitment of endosomal sorting complexes required for transport (ESCRTs) to the cytosolic face of endosomal membranes. The subsequent dissociation of ESCRT complexes from endosomes requires Vps4, a member of the AAA family of adenosine triphosphatases. We show that Did2 directs...

We have analysed the influence of size, intracellular localisation, and sorting of various human immunodeficiency virus type 1 (HIV-1)-derived Gag and Env polypeptides containing well defined H2(d)-restricted cytotoxic T lymphocyte (CTL) epitopes on the induction of a humoral and cellular immune response after DNA vaccination. Thus, expression vectors were generated based on RNA- and codon-opti...

The matrix protein VP40 is essential for Ebola virus (EBOV) and Marburg virus assembly and budding at the plasma membrane. In this study we have investigated the effect of single amino acid substitutions in a conserved proline-rich region of the EBOV VP40 located in the carboxy-terminal part of the protein. We demonstrate that substitutions within this region result in an alteration of intracel...

Cellular responses to environmental stimuli require conserved signal transduction pathways. In budding yeast (Saccharomyces cerevisiae), nutrient limitation induces morphological changes that depend on the protein kinase A (PKA) pathway and the Kss1 mitogen-activated protein kinase (MAPK) pathway. It was unclear to what extent and at what level there is synergy between these two distinct signal...