Biomedical subjects

Joseph Milone

Publications and source records attributed to Joseph Milone.

3 recordsLinked to original sources

PubMed2004

Characterization of deadenylation in trypanosome extracts and its inhibition by poly(A)-binding protein Pab1p.

The stability of mRNAs is an important point in the regulation of gene expression in eukaryotes. The mRNA turnover pathways have been identified in yeast and mammals. However, mRNA turnover pathways in trypanosomes have not been widely studied. Deadenylation is the first step in the major mRNA turnover pathways of yeast and mammals. To better understand mRNA degradation processes in these organisms, we have developed an in vitro mRNA turnover system that is functional for deadenylation. In this system, addition of poly(A) homopolymer activates the deadenylation of poly(A) tails. The trypanosomal deadenylase activity is a 3'-->5' exonuclease specific for adenylate residues, generates 5'-AMP as a product, is magnesium dependent, and is inhibited by neomycin B sulfate. These characteristics suggest similarity with other eukaryotic deadenylases. Furthermore, this activity is cap independent, indicating a potential difference between the trypanosomal activity and PARN, but suggesting similarity to Ccr4p/Pop2p activities. Extracts immunodepleted of Pab1p required the addition of poly(A) competition to activate deadenylation. Trypanosomal Pab1p functions as an inhibitor of the activity under in vitro conditions. Pab1p appears to be one of several mRNA stability proteins in trypanosomal extracts.

Adenosine↗

PubMed2004

Assessing messenger RNA decapping in cellular extracts.

Removal of the 5' cap from a messenger RNA (mRNA) is an integral part of all mRNA decay pathways and can be a highly regulated event. Assays designed to assess decapping in vitro need to effectively resolve four products of mRNA decay: 7meGpppG produced by 3'-5' shortening of the transcript by the exosome, 7meGMP produced by the scavenger decapping enzyme DcpS acting on the product of exosomal decay, 7meGDP produced by the Dcp1/2 decapping enzyme, and free phosphate, which can be generated by phosphatases in the extract acting upon either of the two products of decapping noted above. We have outlined both thin-layer chromatography and acrylamide-gel based approaches that can be used to assess decapping activities.

Chromatography, Thin Layer↗

PubMed2002

Identification of mRNA decapping activities and an ARE-regulated 3' to 5' exonuclease activity in trypanosome extracts.

mRNA turnover is a regulated process that contributes to the steady state level of cytoplasmic mRNA. The amount of each mRNA determines, to a large extent, the amount of protein produced by that particular transcript. In trypanosomes, there is little transcriptional regulation; therefore, differential mRNA stability significantly contributes to mRNA levels in each stage of the parasite life cycle. To investigate the enzymatic activities that contribute to mRNA turnover, we developed a cell-free system for mRNA turnover using the trypanosome Leptomonas seymouri. We identified a decapping activity that removed m(7)GDP from mRNAs that contain an m(7)GpppN cap at their 5' end. In yeast, the release of m(7)GDP by the pyrophosphatase Dcp1p/Dcp2p is a rate-limiting step in mRNA turnover. A secondary enzymatic activity, similar to the human cap scavenger activity, was identified in the trypanosome extracts. Both the human and trypanosome scavenger activities generate m(7)GMP from short capped RNA and are inhibited by addition in trans of m(7)GpppG. A third enzymatic activity uncovered in the parasite extracts functioned as a 3' to 5' exonuclease. Importantly, this exonuclease activity was stimulated by an AU-rich element present in the RNA. In summary, the cell-free system has defined several RNA turnover steps that likely contribute to regulated mRNA decay in trypanosomes.

Animals↗