A Human Common Nuclear Matrix Protein Homologous to Eukaryotic Translation Initiation Factor 4A Klaus Holzmann,* ,1 Christopher Gerner,* Angelika Po ¨ltl,* Romana Scha ¨ fer,* Peter Obrist,† Christian Ensinger,† Rudolf Grimm,‡ ,2 and Georg Sauermann* *Institute of Tumor Biology-Cancer Research, University of Vienna, Borschkegasse 8a, A-1090 Vienna, Austria; †Institute of Pathology, University of Innsbruck, Mu ¨ llerstrasse 44, A-6020 Innsbruck, Austria; and ‡Hewlett-Packard, Chemical Analysis Group Europe, D-76337 Waldbronn, Germany Received December 2, 1999 Amino acid sequencing and mass spectrometry re- vealed identity of a human nuclear matrix protein, termed hNMP 265, with a predicted protein of gene KIAA0111. Two-dimensional electrophoresis and Northern hybridization showed the protein to ubiqui- tously occur in various human cell types. Exhibiting DEAD-box motifs characteristic for RNA helicases, hNMP 265 is highly similar to the human initiation factors eIF4A-I and -II. On the other hand, hNMP 265 greatly differs from the initiation factors by a N-terminal sequence rich in charged amino acids. Se- quence searches and alignments indicate proteins re- lated to hNMP 265 in other eukaryotes. Chimeras be- tween hNMP 265 and green fluorescence protein or hapten appeared as speckles in extranucleolar regions in the nucleus, but not in the cytoplasm. Experiments with tagged deletion mutants indicated that the N-terminal amino acid sequence is necessary for nu- clear localization. A putative role of hNMP 265 in pre- mRNA processing is discussed. © 2000 Academic Press In the cytoplasmic compartment of eukaryotic cells initiation of protein synthesis is regulated by several translation initiation factors (eIFs) that are involved in the binding of the 40S and 60S ribosomal subunits to a messenger RNA molecule. Factor eIF4A with RNA heli- case activity is required for melting of intramolecular secondary structures within the mRNA [for review, see (1)]. eIF4A—more abundant than ribosomes [for review, see (2)]—is a member of the DEAD-box family of proteins (3). These are characterized by eight motifs, with motif I responsible for ATP binding, motif II for ATPase activity and coupling to helicase, motif III for helicase and motif VI for RNA binding activity [for reviews, see (4, 5)]. Most of the DEAD-box proteins are presumed ATP-dependent helicases that are involved in RNA synthesis, RNA pro- cessing and RNA transport. Being highly dependent on the presence of specific factors and substrates, helicase activity has only been documented for some of the known DEAD-box proteins (5). The filamentous protein framework maintaining the structure of the interphase nucleus has been desig- nated as nuclear matrix [for reviews, see (6, 7)]. In technical terms the nuclear matrix has been described as the insoluble material that resists exposure of iso- lated nuclei to nucleases, detergents and highly con- centrated salt solutions (8). Basic nuclear processes have been reported to proceed in tight association with the nuclear matrix. Thus, the nuclear matrix has been shown to play a role in DNA replication, RNA synthe- sis, RNA processing, RNA transport, steroid hormone action and signal transduction (6, 7). In this communication a human nuclear matrix pro- tein (hNMP) is described that is highly similar to the translation initiation factor eIF4A. hNMP 265 was de- tected in the course of our systematic studies on nu- clear matrix proteins (9 –14). Contrasting from eIF-4A by its terminal amino acid sequence, the eIF4A-like protein was found localized in the nonnucleolar nu- clear region of the cell, and thus divergent from a related yeast protein, Fal1p, known for a role in nucle- olar processing of pre-ribosomal RNA (15). Using tagged deletion mutants the influence of the N-terminus of hNMP 265 on its intracellular localiza- tion was investigated. MATERIALS AND METHODS Cells. Leukocytes of healthy human donors were purified, and human HeLa S3 (ATCC CCL-2.2, cervix adenocarcinoma) cells cul- tured as described previously (10). Human tissue samples were from Abbreviations used: GFP, green fluorescence protein; HA, hapten; hNMP, human nuclear matrix protein; MALDI-TOF, matrix- assisted laser-desorption ionization-time-of-flight. 1 To whom correspondence should be addressed. Fax: +43-1-4277- 9651. E-mail: klaus.holzmann@univie.ac.at. 2 Current address: Hexal Biotech ForschungsGmbH, Indus- triestrasse 25, D-83607 Holzkirchen, Germany. Biochemical and Biophysical Research Communications 267, 339 –344 (2000) doi:10.1006/bbrc.1999.1973, available online at http://www.idealibrary.com on 339 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.