Identification of Human Common Nuclear‐Matrix Proteins as Heterogeneous Nuclear Ribonucleoproteins H and H′ by Sequencing and Mass Spectrometry

Abstract

Nuclear-matrix proteins were prepared from different rat and human cells and separated by twodimensional gel electrophoresis. By computer-assisted analysis of the images, two of the proteins were identified as ubiquitously occurring (common) nuclear-matrix proteins, which appeared in tissue-dependent concentrations. The two proteins that originated from human blood mononuclear cells were analyzed further. Tryptic digests of the blotted proteins were analyzed by partial peptide sequencing and matrixassisted laser-desorption ionization-time-of-flight mass spectrometry. The two human common nuclearmatrix proteins were identified as heterogeneous nuclear ribonucleoproteins (hnRNP) H and H' or their variants. Furthermore, mass analysis revealed details on the N terminus of hnRNP H.Keywords: nuclear matrix ; common nuclear-matrix protein ; heterogeneous nuclear ribonucleoprotein ; tissue specificity ; peptide sequencing.The nuclear matrix is operationally defined as the residual nuclear structure that is yielded by sequential treatment of isolated nuclei with detergents, nucleases and buffers of high ionic strength 11 1. It is considered to represent the three-dimensional fibrillar protein structure constituting the framework of the interphase nucleus. In addition to its role in maintaining the nuclear architecture and the higher order structure of chromatin, the nuclear matrix has been reported as being involved in various nuclear activities, such as DNA replication, DNA transcription, RNA processing and steroid-hormone action. Studies on selected cell types and tissues have indicated that some nuclearmatrix proteins are cell and differentiation specific, while others are shared among various cell types [2]. Stuurman et al. [3] have presented evidence for the presence of a common set of polypeptides termed minimal matrix proteins in the nuclear matrices of mouse cells. Nakayasu and Berezney [4] have reported that eight proteins named nuclear matrins are major constituents of the internal nuclear matrix of rat liver cells. Belgrader et al. were the first to determine the sequence of a defined nuclear-matrix protein, matrin 3, by cloning and sequencing [5]. The detection of other common nuclear-matrix proteins, such as nuclear lamin B [3, 41, nucleolar protein B23 [3, 41, or the nuclear-mitotic apparatus protein [6, 71, was mainly based on immunological methods. However, the number and identity of common nuclearmatrix proteins have not been established unequivocally (reviewed in [8, 91).In systematic studies, we are investigating the nuclear-matrix proteins of various human and rat cells and tissues. ComputerCorrespondence to G. Sauermann,

Differential nuclear localization and nuclear matrix association of the splicing factors PSF and PTB

“…Leukocytes of healthy human donors were purified as described previously [Holzmann et al, 1997]. Human tissue and tumor samples were kindly provided by Dr. P. Obrist and Dr. C. Ensinger, University of Innsbruck, Austria.…”

Reassembling proteins and chaperones in human nuclear matrix protein fractions

“…Amino acid sequencing and matrix-assisted laser-desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry of trypsin hydroly-sates was performed as reported previously for hnRNPs H and HЈ [Holzmann et al, 1997]. For Western blotting, proteins were electrophoretically transferred onto nitrocellulose membranes (0.2 µm).…”

Androgen Receptor Is Targeted to Distinct Subcellular Compartments in Response to Different Therapeutic Antiandrogens

“…The nuclear fraction (N) contained polypyrimidine tract binding proteinassociated splicing factor and nucleolin, proteins involved in transcriptional regulation and RNA maturation (27,28), as well as structural proteins. The NM and U fractions contained proteins found in the cell cytoskeleton and intermediate filaments, such as actin (29), myosin (30), keratins (31,32), and vimentin (32), as well as classical NM proteins such as lamins (21) and heterogeneous nuclear ribonucleoprotein H (33).…”