Biomedical subjects

Andrzej Dziembowski

Publications and source records attributed to Andrzej Dziembowski.

8 recordsLinked to original sources

PubMed2006

A single subunit, Dis3, is essentially responsible for yeast exosome core activity.

The conserved core of the exosome, the major eukaryotic 3' --> 5' exonuclease, contains nine subunits that form a ring similar to the phosphorolytic bacterial PNPase and archaeal exosome, as well as Dis3. Dis3 is homologous to bacterial RNase II, a hydrolytic enzyme. Previous studies have suggested that all subunits are active 3' --> 5' exoRNases. We show here that Dis3 is responsible for exosome core activity. The purified exosome core has a hydrolytic, processive and Mg(2+)-dependent activity with characteristics similar to those of recombinant Dis3. Moreover, a catalytically inactive Dis3 mutant has no exosome core activity in vitro and shows in vivo RNA degradation phenotypes similar to those resulting from exosome depletion. In contrast, mutations in Rrp41, the only subunit carrying a conserved phosphorolytic site, appear phenotypically not different from wild-type yeast. We observed that the yeast exosome ring mediates interactions with protein partners, providing an explanation for its essential function.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Subunit architecture of multimeric complexes isolated directly from cells.

Recent developments in purification strategies, together with mass spectrometry (MS)-based proteomics, have identified numerous in vivo protein complexes and suggest the existence of many others. Standard proteomics techniques are, however, unable to describe the overall stoichiometry, subunit interactions and organization of these assemblies, because many are heterogeneous, are present at relatively low cellular abundance and are frequently difficult to isolate. We combine two existing methodologies to tackle these challenges: tandem affinity purification to isolate sufficient quantities of highly pure native complexes, and MS of the intact assemblies and subcomplexes to determine their structural organization. We optimized our protocol with two protein assemblies from Saccharomyces cerevisiae (scavenger decapping and nuclear cap-binding complexes), establishing subunit stoichiometry and identifying substoichiometric binding. We then targeted the yeast exosome, a nuclease with ten different subunits, and found that by generating subcomplexes, a three-dimensional interaction map could be derived, demonstrating the utility of our approach for large, heterogeneous cellular complexes.

Chromatography, Affinity↗

PubMed2004

Proteomic analysis identifies a new complex required for nuclear pre-mRNA retention and splicing.

Using the proteomic tandem affinity purification (TAP) method, we have purified the Saccharomyces cerevisie U2 snRNP-associated splicing factors SF3a and SF3b. While SF3a purification revealed only the expected subunits Prp9p, Prp11p and Prp21p, yeast SF3b was found to contain only six subunits, including previously known components (Rse1p, Hsh155p, Cus1p, Hsh49p), the recently identified Rds3p factor and a new small essential protein (Ysf3p) encoded by an unpredicted split ORF in the yeast genome. Surprisingly, Snu17p, the proposed yeast orthologue of the seventh human SF3b subunit, p14, was not found in the yeast complex. TAP purification revealed that Snu17p, together with Bud13p and a newly identified factor, Pml1p/Ylr016c, form a novel trimeric complex. Subunits of this complex were not essential for viability. However, they are required for efficient splicing in vitro and in vivo. Furthermore, inactivation of this complex causes pre-mRNA leakage from the nucleus. The corresponding complex was named pre-mRNA REtention and Splicing (RES). The presence of RES subunit homologues in numerous eukaryotes suggests that its function is evolutionarily conserved.

Amino Acid Sequence↗

PubMed2004

Identification of a novel human nuclear-encoded mitochondrial poly(A) polymerase.

We report here on the identification of a novel human nuclear-encoded mitochondrial poly(A) polymerase. Immunocytochemical experiments confirm that the enzyme indeed localizes to mitochondrial compartment. Inhibition of expression of the enzyme by RNA interference results in significant shortening of the poly(A) tails of the mitochondrial ND3, COX III and ATP 6/8 transcripts, suggesting that the investigated protein represents a bona fide mitochondrial poly(A) polymerase. This is in agreement with our sequencing data which show that poly(A) tails of several mitochondrial messengers are composed almost exclusively of adenosine residues. Moreover, the data presented here indicate that all analyzed mitochondrial transcripts with profoundly shortened poly(A) tails are relatively stable, which in turn argues against the direct role of long poly(A) extensions in the stabilization of human mitochondrial messengers.

Amino Acid Sequence↗

PubMed2004

Recent developments in the analysis of protein complexes.

The goal of this review is to analyse how recent technical developments contributed to the biochemical characterisation of protein complexes. Improvement of tags used for protein purification, including in our own laboratory, and the development of new strategies have allowed the use of generic procedures for the purification of a wide variety of protein complexes. Together with increased mass spectrometry sensitivity and automation, this made high throughput studies of protein complexes possible and allowed proteome-wide analyses of protein complexes. However, knowledge of protein complex composition, even at the cellular level, will not be sufficient to understand their function. We suggest that the next level of analysis in this area will be the definition of internal subunit arrangement in complexes as a first step toward more detailed structural analyses.

Affinity Labels↗

PubMed2003

Human polynucleotide phosphorylase, hPNPase, is localized in mitochondria.

The human gene encoding a polynucleotide phosphorylase (hPNPase) has been recently identified as strongly up-regulated in two processes leading to irreversible arrest of cell division: progeroid senescence and terminal differentiation. Here, we demonstrate that the hPNPase is localized in mitochondria. Our finding suggests the involvement of mitochondrial RNA metabolism in cellular senescence.

Cloning, Molecular↗

PubMed2002

Localisation of the human hSuv3p helicase in the mitochondrial matrix and its preferential unwinding of dsDNA.

We characterised the human hSuv3p protein belonging to the family of NTPases/helicases. In yeast mitochondria the hSUV3 orthologue is a component of the degradosome complex and participates in mtRNA turnover and processing, while in Caenorhabditis elegans the hSUV3 orthologue is necessary for viability of early embryos. Using immunofluorescence analysis, an in vitro mitochondrial uptake assay and sub-fractionation of human mitochondria we show hSuv3p to be a soluble protein localised in the mitochondrial matrix. We expressed and purified recombinant hSuv3p protein from a bacterial expression system. The purified enzyme was capable of hydrolysing ATP with a K(m) of 41.9 micro M and the activity was only modestly stimulated by polynucleotides. hSuv3p unwound partly hybridised dsRNA and dsDNA structures with a very strong preference for the latter. The presented analysis of the hSuv3p NTPase/helicase suggests that new functions of the protein have been acquired in the course of evolution.

Adenosine Triphosphatases↗

PubMed2002

The yeast mitochondrial degradosome. Its composition, interplay between RNA helicase and RNase activities and the role in mitochondrial RNA metabolism.

The yeast mitochondrial degradosome (mtEXO) is an NTP-dependent exoribonuclease involved in mitochondrial RNA metabolism. Previous purifications suggested that it was composed of three subunits. Our results suggest that the degradosome is composed of only two large subunits: an RNase and a RNA helicase encoded by nuclear genes DSS1 and SUV3, respectively, and that it co-purifies with mitochondrial ribosomes. We have found that the purified degradosome has RNA helicase activity that precedes and is essential for exoribonuclease activity of this complex. The degradosome RNase activity is necessary for mitochondrial biogenesis but in vitro the degradosome without RNase activity is still able to unwind RNA. In yeast strains lacking degradosome components there is a strong accumulation of mitochondrial mRNA and rRNA precursors not processed at 3'- and 5'-ends. The observed accumulation of precursors is probably the result of lack of degradation rather than direct inhibition of processing. We suggest that the degradosome is a central part of a mitochondrial RNA surveillance system responsible for degradation of aberrant and unprocessed RNAs.

Amino Acid Sequence↗