Biomedical subjects

Bastiaan B J Tops

Publications and source records attributed to Bastiaan B J Tops.

4 recordsLinked to original sources

PubMed2005

RDE-2 interacts with MUT-7 to mediate RNA interference in Caenorhabditis elegans.

In Caenorhabditis elegans, the activity of transposable elements is repressed in the germline. One of the mechanisms involved in this repression is RNA interference (RNAi), a process in which dsRNA targets cleavage of mRNAs in a sequence-specific manner. The first gene found to be involved in RNAi and transposon silencing in C.elegans is mut-7, a gene encoding a putative exoribonuclease. Here, we show that the MUT-7 protein resides in complexes of approximately 250 kDa in the nucleus and in the cytosol. In addition, we find that upon triggering of RNAi the cytosolic MUT-7 complex increases in size. This increase is independent of the presence of target RNA, but does depend on the presence of RDE-1 and RDE-4, two proteins involved in small interfering RNA (siRNA) production. Finally, using a yeast two-hybrid screen, we identified RDE-2/MUT-8 as one of the other components of this complex. This protein is encoded by the rde-2/mut-8 locus, previously implicated in RNAi and transposon silencing. Using genetic complementation analysis, we show that the interaction between these two proteins is required for efficient RNAi in vivo. Together these data support a role for the MUT-7/RDE-2 complex downstream of siRNA formation, but upstream of siRNA mediated target RNA recognition, possibly indicating a role in the siRNA amplification step.

ATP-Binding Cassette Transporters↗

PubMed2004

Processing of primary microRNAs by the Microprocessor complex.

Mature microRNAs (miRNAs) are generated via a two-step processing pathway to yield approximately 22-nucleotide small RNAs that regulate gene expression at the post-transcriptional level. Initial cleavage is catalysed by Drosha, a nuclease of the RNase III family, which acts on primary miRNA transcripts (pri-miRNAs) in the nucleus. Here we show that Drosha exists in a multiprotein complex, the Microprocessor, and begin the process of deconstructing that complex into its constituent components. Along with Drosha, the Microprocessor also contains Pasha (partner of Drosha), a double-stranded RNA binding protein. Suppression of Pasha expression in Drosophila cells or Caenorhabditis elegans interferes with pri-miRNA processing, leading to an accumulation of pri-miRNAs and a reduction in mature miRNAs. Finally, depletion or mutation of pash-1 in C. elegans causes de-repression of a let-7 reporter and the appearance of phenotypic defects overlapping those observed upon examination of worms with lesions in Dicer (dcr-1) or Drosha (drsh-1). Considered together, these results indicate a role for Pasha in miRNA maturation and miRNA-mediated gene regulation.

Animals↗

PubMed2003

A micrococcal nuclease homologue in RNAi effector complexes.

RNA interference (RNAi) regulates gene expression by the cleavage of messenger RNA, by mRNA degradation and by preventing protein synthesis. These effects are mediated by a ribonucleoprotein complex known as RISC (RNA-induced silencing complex). We have previously identified four Drosophila components (short interfering RNAs, Argonaute 2 (ref. 2), VIG and FXR) of a RISC enzyme that degrades specific mRNAs in response to a double-stranded-RNA trigger. Here we show that Tudor-SN (tudor staphylococcal nuclease)--a protein containing five staphylococcal/micrococcal nuclease domains and a tudor domain--is a component of the RISC enzyme in Caenorhabditis elegans, Drosophila and mammals. Although Tudor-SN contains non-canonical active-site sequences, we show that purified Tudor-SN exhibits nuclease activity similar to that of other staphylococcal nucleases. Notably, both purified Tudor-SN and RISC are inhibited by a specific competitive inhibitor of micrococcal nuclease. Tudor-SN is the first RISC subunit to be identified that contains a recognizable nuclease domain, and could therefore contribute to the RNA degradation observed in RNAi.

Animals↗

PubMed2002

Biochemical characterization and subcellular localization of human copper transporter 1 (hCTR1).

The human copper transporter 1 gene (hCTR1) was previously identified by functional complementation in ctr1-deficient yeast. Overexpression of hCTR1 in wild-type yeast leads to increased sensitivity to copper toxicity, and mice with a homozygous disruption at the Ctr1 locus die early during embryogenesis. It is proposed that hCTR1 is responsible for high-affinity copper uptake into human cells, but the underlying molecular mechanisms are unknown. To begin to investigate the biochemical characteristics of hCTR1, a polyclonal antiserum was raised against recombinant hCTR1-fusion peptides. Biosynthetic studies using this antiserum revealed that hCTR1 was synthesized as a precursor protein of 28 kDa containing N-linked oligosaccharides, and is then converted to a mature protein of approx. 35 kDa, which is ubiquitously expressed. Immunofluorescence studies showed that subcellular hCTR1 localization differed markedly between cell types. In some cell lines, hCTR1 was located predominantly in an intracellular vesicular perinuclear compartment, and in others hCTR1 was located predominantly at the plasma membrane. In contrast with the copper export P-type ATPases mutated in Wilson disease and Menkes disease, the localization of hCTR1 was not influenced by copper concentrations. Inhibition of endocytosis by methyl-beta-cyclodextrin caused a partial redistribution of hCTR1 to the cell surface of HeLa cells. Taken together, the results in this study suggest a cell-specific control of copper uptake, which involves subcellular localization of the hCTR1 protein.

Base Sequence↗