Tandem Affinity Purification and Mass-Spectrometric Analysis of FACT and Associated Proteins.

Isolation of a protein/complex is important for its biochemical and structural characterization with mechanistic insights. TAP (tandem affinity purification) strategy allows rapid isolation of cellular proteins/complexes with a high level of purity. This methodology involves an immuno-affinity-based purification followed by a conformation-based isolation to obtain a highly homogeneous protein/complex.

A novel ubiquitin-proteasome system regulation of Sgf73/ataxin-7 that maintains the integrity of the coactivator SAGA in orchestrating transcription.

Ataxin-7 maintains the integrity of Spt-Ada-Gcn5-Acetyltransferase (SAGA), an evolutionarily conserved coactivator in stimulating preinitiation complex (PIC) formation for transcription initiation, and thus, its upregulation or downregulation is associated with various diseases. However, it remains unknown how ataxin-7 is regulated that could provide new insights into disease pathogenesis and therapeutic interventions. Here, we show that ataxin-7's yeast homologue, Sgf73, undergoes ubiquitylation and proteasomal degradation.

Transcription-coupled DNA double-strand break repair.

The genomic DNA is constantly under attack by cellular and/or environmental factors. Fortunately, the cell is armed to safeguard its genome by various mechanisms such as nucleotide excision, base excision, mismatch and DNA double-strand break repairs. While these processes maintain the integrity of the genome throughout, DNA repair occurs preferentially faster at the transcriptionally active genes.

Genome-Wide Regulations of the Preinitiation Complex Formation and Elongating RNA Polymerase II by an E3 Ubiquitin Ligase, San1.

San1 ubiquitin ligase is involved in nuclear protein quality control via its interaction with intrinsically disordered proteins for ubiquitylation and proteasomal degradation. Since several transcription/chromatin regulatory factors contain intrinsically disordered domains and can be inhibitory to transcription when in excess, San1 might be involved in transcription regulation. To address this, we analyzed the role of San1 in the genome-wide association of TATA box binding protein (TBP; which nucleates preinitiation complex [PIC] formation for transcription initiation) and RNA polymerase II (Pol II).

Viral regulation of mRNA export with potentials for targeted therapy.

Eukaryotic gene expression begins with transcription in the nucleus to synthesize mRNA (messenger RNA), which is subsequently exported to the cytoplasm for translation to protein. Like transcription and translation, mRNA export is an important regulatory step of eukaryotic gene expression. Various factors are involved in regulating mRNA export, and thus gene expression.

An F-Box Protein, Mdm30, Interacts with TREX Subunit Sub2 To Regulate Cellular Abundance Cotranscriptionally in Orchestrating mRNA Export Independently of Splicing and Mitochondrial Function.

Although an F-box protein, Mdm30, is found to regulate ubiquitylation of the Sub2 component of TREX (anscription-port) complex for proteasomal degradation in stimulation of mRNA export, it remains unknown whether such ubiquitin-proteasome system (UPS) regulation of Sub2 occurs cotranscriptionally via its interaction with Mdm30. Further, it is unclear whether impaired UPS regulation of Sub2 in the absence of Mdm30 alters mRNA export via splicing defects of export factors and/or mitochondrial dynamics/function, since Sub2 controls mRNA splicing and Mdm30 regulates mitochondrial aggregation. Here, we show that Mdm30 interacts with Sub2, and temporary shutdown of Mdm30 enhances Sub2's abundance and impairs mRNA export.