Diagnostic challenges during pretreatment long-term follow-up in a patient with FIP1L1-PDGFRA-positive eosinophilia.

Obtaining a precise characterization of eosinophilia is crucial, as successful treatment relies on the underlying etiology of the disease. Platelet-derived growth factor receptor alpha-related disorders were first specified in 2008 as a distinct group of clonal eosinophilic disorders with exceptional responsiveness to imatinib. We herein present the case of a man with myeloid neoplasm and eosinophilia in whom a definitive diagnosis could not be adequately made based on histopathological features who was ultimately diagnosed only after extensive molecular analyses and successfully treated with imatinib. In addition, we discuss the diagnostic and therapeutic approaches to treating patients presenting with eosinophilia.

Production, purification, and assay of recombinant proteins for in vitro biochemical analyses of the plant polyadenylation complex.

In the post-genomic era where gene sequences are available for many organisms, attention has shifted from DNA to the workhorses of the cell-RNA and protein. A number of proteins, as recent studies indicate, seem to possess RNA-binding and RNA cleavage activities. In order to understand the events that comprise RNA processing such as splicing, 3' end processing, and even RNA turnover, well established methods are necessary. Bacterial recombinant proteins afford an invaluable opportunity to produce proteins in an economical and reproducible fashion in order to study these activities. This chapter describes various experimental protocols to begin the elucidation of the many events that surround RNA processing at the 3' end of a transcript.

A serendipitous discovery that in situ proteolysis is essential for the crystallization of yeast CPSF-100 (Ydh1p).

The cleavage and polyadenylation specificity factor (CPSF) complex is required for the cleavage and polyadenylation of the 3'-end of messenger RNA precursors in eukaryotes. During structural studies of the 100 kDa subunit (CPSF-100, Ydh1p) of the yeast CPSF complex, it was serendipitously discovered that a solution that is infected by a fungus (subsequently identified as Penicillium) is crucial for the crystallization of this protein. Further analyses suggest that the protein has undergone partial proteolysis during crystallization, resulting in the deletion of an internal segment of about 200 highly charged and hydrophilic residues, very likely catalyzed by a protease secreted by the fungus. With the removal of this segment, yeast CPSF-100 (Ydh1p) has greatly reduced solubility and can be crystallized in the presence of a minute amount of precipitant.

Genome-wide analysis of mRNAs bound to the histone stem-loop binding protein.

The replication-dependent histone mRNAs are cell-cycle-regulated and expressed only during S phase. In contrast to all other eukaryotic mRNAs, the histone mRNAs end in a highly conserved 16-nucleotide stem-loop rather than a poly(A) tail. The stem-loop is necessary and sufficient for the post-transcriptional regulation of histone mRNA during the cell cycle. The histone mRNA 3' stem-loop is bound by the stem-loop binding protein (SLBP) that is involved in pre-mRNA processing, translation, and stability of histone mRNA. Immunoprecipitation (IP) of RNA-binding proteins (RBPs) followed by microarray analysis has been used to identify the targets of RNA-binding proteins. This method is sometimes referred to as RIP-Chip (RNA IP followed by microarray analysis). Here we introduce a variation on the RIP-Chip method that uses a recombinant RBP to identify mRNA targets in a pool of total RNA; we call this method recombinant, or rRIP-Chip. Using this method, we show that recombinant SLBP binds exclusively to all five classes of histone mRNA. We also analyze the messages bound to the endogenous SLBP on polyribosomes by immunoprecipitation. We use two different microarray platforms to identify enriched mRNAs. Both platforms demonstrate remarkable specificity and consistency of results. Our data suggest that the replication-dependent histone mRNAs are likely to be the sole target of SLBP.

Association of polyadenylation cleavage factor I with U1 snRNP.

Splicing and polyadenylation factors interact for the control of polyadenylation and the coupling of splicing and polyadenylation. We document an interaction between the U1 snRNP and mammalian polyadenylation cleavage factor I (CF Im), one of several polyadenylation factors needed for the cleavage of the pre-mRNA at the polyadenylation site. Sucrose density gradient centrifugation demonstrated that CF Im separated into two fractions, a light fraction which contained the known CF Im subunits (72, 68, 59, and 25 kD), and a heavy fraction, rich in snRNPs, which contained predominately the 68- and 25-kD CF Im subunits. Using specific antibodies we found that the heavy fraction contains U1 snRNP/CF Im coprecipitable complexes. These complexes were insensitive to RNase treatment, suggesting that the coprecipitation is not due to RNA tethering. In vitro binding experiments show that both the 68- and 25-kD subunits bind to and comigrate with U1 snRNP. In addition, the 25-kD CF Im subunit binds specifically to the 70K protein of U1 snRNP (U1 70K). This binding may account for the CF Im/U1 snRNP interaction. During these studies we found that mAb 2.73 (mAb 2.73), an established U1 70K antibody, efficiently precipitates the bulk of the CF Im from cellular extracts. Because mAb 2.73 has been used in a number of previous studies related to the U1 snRNP and the U1 70K protein, the precipitation of CF Im must be considered in evaluating past and future data based on the use of mAb 2.73.