Daniel R. Schoenberg

Ph.D. - University of Wisconsin

Post Doctoral - Baylor College of Medicine

The overall theme of research in the Schoenberg lab is how extracellular stimuli alter gene expression through changes in the processing and metabolism of mRNAs. These concepts are addressed in project areas which study the steps involved in the activation of mRNA decay by the female sex hormone estrogen, biochemical and molecular analysis of a ribonuclease which selectively targets a specific group of mRNAs for destabilization following estrogen stimulation, analysis of an element which regulates the length of poly(A) on mRNAs targeted for degradation by the estrogen-regulated ribonuclease.

Estrogen activation of mRNA decay:>
The life of any given mRNA begins with its transcription and its co-transcriptional processing involving the removal of intervening sequences (introns) and addition of a homopolymeric poly(A) tail prior to nuclear export to the cytoplasm.  Once in the cytoplasm mRNAs can be targeted to specific subcellular locations, translated, and ultimately, degraded. All of the steps in mRNA metabolism serve as potential regulatory points. Over the past 17 years we have worked to define the molecular mechanisms by which estrogen effects one of the most dramatic changes in the translational profile of any tissue thus far studied. When Xenopus laevis receive estrogen the liver ceases production of its normal complement of serum proteins, switching instead to the elaboration of large quantities of the yolk protein precursor vitellogenin. This loss in serum protein production is brought about by the destruction of all of the serum protein mRNAs, not by inhibition of their transcription. This process is dependent on the action of the nuclear estrogen receptor, but is independent of the synthesis of a new protein product. Several years ago we identified a ribonuclease activity with sequence specificity for serum protein mRNAs whose appearance on polysomes correlated with the degradation of these mRNAs. Recent work showed that this ribonuclease, termed PMR-1, exists in a latent form in an complex with its substrate mRNA bound to polysomes, and estrogen selectively activates the polysome-bound enzyme to initiate the process of mRNA degradation. We are currently working to identify the proteins that both bind to PMR-1 and constitute the mRNP complex to better understand the processes involved in endonuclease- mediated mRNA decay. Another line of research focuses on the signal transduction pathway(s) responsible for activating PMR-1 and mRNA decay using a cell line that lacks estrogen receptor but contains PMR-1. By transfecting estrogen receptor expression vectors into these cells we can activate mRNA decay upon addition of estradiol to the medium. This will provide a powerful tool for understanding the steps involved between estrogen binding to its receptor and mRNA decay.

Biochemical and genetic analysis of the messenger RNase PMR-1:
The sequence-selective Mr 60,000 RNase was purified from liver polysomes of estrogen-stimulated frogs and its cDNA cloned. Surprisingly it bears no sequence similarity to any known RNases. Rather, it is a member of the peroxidase gene family, showing the greatest sequence similarity to human myeloperoxidase. We named this enzyme PMR-1, for polysomal ribonuclease 1. Unlike enzymes of the peroxidase gene family, PMR-1 lacks both heme and N-linked oligosaccharide. This enzyme is the first vertebrate mRNA endonuclease to be cloned, thus making it a valuable tool in deciphering the processes of mRNA decay. Using purified PMR-1 and the multi-KH-domain protein vigilin we recently demonstrated for the first time the ability of an RNA-binding protein binding over an endonuclease cleavage site to specifically block cleavage by an mRNA endonuclease. Currently we are working to define the portion(s) of PMR-1 involved in its catalytic activity and to clone the human homologue. A long term goal of this work will be to apply gene array technology to identify the substrates of PMR-1 in human cells and to determine whether cell type-specific proteins guide the selection of target mRNAs by this mRNA endonuclease.

Regulated polyadenylation of nuclear pre-mRNA:
A feature all of the estrogen-destabilized serum protein mRNAs has in common is a very short, discrete poly(A) tail of 17-20 nt in length. This contrasts markedly with most somatic mRNAs, whose poly(A) tails are usually 100-200 nt long. Poly(A) addition onto nuclear pre-mRNA occurs in a two-step process in which 10+ residues are added in a slow, distributive reaction, followed by the rapid and processive addition of ~200 adenosine residues. We showed that the short poly(A) tail on albumin mRNA is also present on unprocessed albumin pre-mRNA, thus implicating either regulation of poly(A) addition, or the rapid removal of poly(A) in the nucleus as possible mechanisms for this phenomenon. We have replicated poly(A) length regulation in transfected cells, and using this approach mapped the sequence elements responsible for this (the PLE or poly(A)- limiting element) to the terminal exon of albumin pre-mRNA. The PLE is a conserved element that regulates the length of poly(A) on numerous mRNAs. We are currently working to identify the protein(s) which bind to the PLE, to determine the mechanism responsible for the regulation of poly(A) tail length, and to determine the functional consequences of regulated nuclear polyadenylation.

Recent Publications: