p68 RNA helicase: identification of a nucleolar form and cloning of related genes containing a conserved intron in yeasts.

Abstract

The human p68 protein is an RNA-dependent ATPase and RNA helicase which was first identified because of its immunological cross-reaction with a viral RNA helicase, simian virus 40 large T antigen. It belongs to a recently discovered family of proteins (DEAD box proteins) that share extensive regions of amino acid sequence homology, are ubiquitous in living organisms, and are involved in many aspects of RNA metabolism, including splicing, translation, and ribosome assembly. We have shown by immunofluorescent microscopy that mammalian p68, which is excluded from the nucleoli during interphase, translocates to prenucleolar bodies during telophase. We have cloned 55% identical genes from both Schizosaccharomyces pombe and Saccharomyces cerevisiae and shown that they are essential in both yeasts. The human and yeast genes contain a large intron whose position has been precisely conserved. In S. cerevisiae, the intron is unusual both because of its size and because of its location near the 3' end of the gene. We discuss possible functional roles for such an unusual intron in an RNA helicase gene.Manipulation of RNA secondary structure is essential for the proper execution of a large number of processes in the cell. The importance that the cell attaches to this problem has only recently come to light with the discovery of a large family of proteins with putative ATP-dependent RNA helicase activity. These proteins are readily identified because they all possess a core region of highly conserved motifs in their primary amino acid sequences. One of the most conserved motifs is the DEAD box (LDEADxxL), which gives the family its name (34). DEAD box proteins form a subset of a more loosely defined family, identified by Hodgman (27) and Gorbalenya et al. (21), of proteins with known or putative helicase activity.RNA helicase activity has been demonstrated in vitro for only two DEAD box proteins: p68 (26), in which the DEAD motifs were first identified (18), and eIF4A (44), a translation initiation factor. Although the biological processes in which the other members are involved may prove very diverse, it seems reasonable to expect that all of them will show RNA helicase activity, at least in strand displacement assays in vitro. The family appears to be very large, since DEAD box proteins have been found in all prokaryotes and eukaryotes examined, and even the relatively small genome of Saccharomyces cerevisiae contains genes encoding at least 12 different members We are interested in p68 because of its specific immunological cross-reaction with the simian virus 40 large T antigen (31). We have taken two approaches to determine the function of p68. First, we have used immunofluorescent microscopy and antibody microinjection to study the cell biology of p68 in mammalian cells. Using antibodies to * Corresponding author. t Present address:

A Novel Helicase-Type Protein in the Nucleolus: Protein NOH61

“…In addition, this approach will allow us to identify and further characterize the functional domains present in this molecule. Surprisingly, in higher eukaryotics only a few nucleolar RNA helicases have been described and their precise functions are largely unknown: Xenopus An3 protein (Gururajan et al, 1994;Gururajan and Weeks, 1997), protein Dbp45A from Drosophila (Lavoie et al, 1993), and the human proteins p68 (Iggo et al, 1991), Ski2w (Qu et al, 1998), and the Gu/RH-II helicase (Flores-Rozas and Hurwitz, 1993;Valdez et al, 1996Valdez et al, , 1997. Protein NOH61 represents a new member of widespread occurrence in this fast growing family of proteins.…”

Domain Structure of Human Nuclear DNA Helicase II (RNA Helicase A)

“…In addition to the central core motif, many DEX(D/H) proteins contain further nucleic acid binding domains, such as the arginine-serine (RS)-rich domain of two other human RNA helicases (34,35), a motif for binding to ribosomal RNA of yeast PRP22 (36), and finally a glycine-rich motif, called RGG-box (17)(18)(19)(20)(21)(22)(23). NDH II contains, in addition to the DEIH core, two copies of a dsRNA binding domain at its NH 2 terminus (6) and an RGG-box at its COOH terminus.…”

“…The carboxylterminal 100 amino acids of NDH II consist of a consecutive stretch of glycines that is regularly interrupted by either aromatic amino acids or arginine. Similar RGG-rich sequences (RGG-boxes) have been found as part of many nucleic acidbinding proteins, such as the heterogeneous nuclear ribonucleoproteins hnRNP A1 (13) and hnRNP U (14), nucleolin (15), yeast single-strand DNA-binding protein 1 (16), as well as of other proteins from the superfamily of DEX(D/H) helicases (17)(18)(19)(20)(21)(22)(23). Except hnRNP U, where the RGG-box is the only nucleic acid binding domain (14), RGG-boxes cooperate with other domains to achieve an increased affinity for nucleic acids.…”

The rRNA-Processing Function of the Yeast U14 Small Nucleolar RNA Can Be Rescued by a Conserved RNA Helicase-Like Protein

“…This gene encodes a DEAD box protein of 875 amino acids, and the two protein sequences are 78% similar (62% identical), with highest relatedness in the DEAD box segments. Because the yeast gene conforms so well to the consensus sequence for DEAD-DEXH box proteins, we prefer to designate it DBP4 (DEAD box protein 4); three other yeast DBP genes have already been described (17,18,33).…”