3 research outputs found
Identification of protein partners in Mycobacteria using a single step affinity purification method
Tuberculosis is a leading cause of death in developing countries. Efforts are being made
to both prevent its spreading and improve curability rates. Understanding the biology of
the bacteria causing the disease, Mycobacterium tuberculosis (M. tuberculosis), is thus
vital. We have implemented improved screening methods for protein-protein
interactions based on affinity purification followed by high-resolution mass
spectrometry. This method is attractively applicable to both medium- and highthroughput
studies aiming to characterize protein-protein interaction networks of
tubercle bacilli. From four tested epitopes, FLAG, eGFP, Protein A, and hemagglutinin,
the eGFP tag was found most useful based on easily monitored expression and as a
simultaneous tool for sub-cellular localization studies. It presents a relatively low
background with cost effective purification.
RNA polymerase subunit A (RpoA) was used as a model for investigation of a large
protein complex. When used as a bait, it co-purified with all remaining RNA polymerase
core subunits as well as many accessory proteins. The amount of RpoA strongly
correlated with the amount of quantification peptide used as part of the tagging system
in this study (SH), making it applicable for semi-quantification studies. Interactions
between the components of the RpoA-eGFP protein complex were further confirmed
using protein cross-linking.
Dynamic changes in the composition of protein complexes under induction of UV
damage were observed when UvrA-eGFP expressing cells, treated with UV light were
used to co-purify UvrA interaction partners
The RNA exosome complex central channel controls both exonuclease and endonuclease Dis3 activities in vivo and in vitro.
The RNA exosome is an essential ribonuclease complex involved in RNA processing and decay. It consists of a 9-subunit catalytically inert ring composed of six RNase PH-like proteins forming a central channel and three cap subunits with KH/S1 domains located at the top. The yeast exosome catalytic activity is supplied by the Dis3 (also known as Rrp44) protein, which has both endo- and exoribonucleolytic activities and the nucleus-specific exonuclease Rrp6. In vitro studies suggest that substrates reach the Dis3 exonucleolytic active site following passage through the ring channel, but in vivo support is lacking. Here, we constructed an Rrp41 ring subunit mutant with a partially blocked channel that led to thermosensitivity and synthetic lethality with Rrp6 deletion. Rrp41 mutation caused accumulation of nuclear and cytoplasmic exosome substrates including the non-stop decay reporter, for which degradation is dependent on either endonucleolytic or exonucleolytic Dis3 activities. This suggests that the central channel also controls endonucleolytic activity. In vitro experiments performed using Chaetomium thermophilum exosomes reconstituted from recombinant subunits confirmed this notion. Finally, we analysed the impact of a lethal mutation of conserved basic residues in Rrp4 cap subunit and found that it inhibits digestion of single-stranded and structured RNA substrates
Multiple myeloma-associated hDIS3 mutations cause perturbations in cellular RNA metabolism and suggest hDIS3 PIN domain as a potential drug target
hDIS3 is a mainly nuclear, catalytic subunit of the human exosome complex, containing exonucleolytic (RNB) and endonucleolytic (PIN) active domains. Mutations in hDIS3 have been found in ∼10% of patients with multiple myeloma (MM). Here, we show that these mutations interfere with hDIS3 exonucleolytic activity. Yeast harboring corresponding mutations in DIS3 show growth inhibition and changes in nuclear RNA metabolism typical for exosome dysfunction. Construction of a conditional DIS3 knockout in the chicken DT40 cell line revealed that DIS3 is essential for cell survival, indicating that its function cannot be replaced by other exosome-associated nucleases: hDIS3L and hRRP6. Moreover, HEK293-derived cells, in which depletion of endogenous wild-type hDIS3 was complemented with exogenously expressed MM hDIS3 mutants, proliferate at a slower rate and exhibit aberrant RNA metabolism. Importantly, MM mutations are synthetically lethal with the hDIS3 PIN domain catalytic mutation both in yeast and human cells. Since mutations in PIN domain alone have little effect on cell physiology, our results predict the hDIS3 PIN domain as a potential drug target for MM patients with hDIS3 mutations. It is an interesting example of intramolecular synthetic lethality with putative therapeutic potential in humans