Identification and characterization of multiple isoforms of a mouse ribosome receptor

Abstract

We isolated multiple cDNA clones encoding various isoforms of a mouse ribosome receptor protein (mRRp). The cDNAs were isolated from a 13.5-day-old mouse conceptus cDNA library by polymerase chain reaction-based screening. The predicted proteins encoded by these cDNAs showed significant homology with ribosome receptors present in dogs (83%), humans (80%), and chickens (45%). The cDNA isoforms had highly identical N- and C-terminal sequences but differed in their central sequences, suggesting that these cDNA isoforms may be derived by alternative splicing from a single gene. Genomic Southern blot analysis confirmed the existence of only a single mouse ribosome receptor gene. Alignments of the deduced amino acid sequences of the mRRp cDNA isoforms revealed that they differ in the number of decapeptide repeats present in the central domain of the protein. These repeats have been previously suggested to mediate ribosome binding and thus differences in repeat number may translate to different ribosome binding abilities. The longest mRRp isoform had 61 tandem repeats. This is of interest because in the human and canine ribosome receptor proteins there are only 54 tandem repeats, suggesting that humans and dogs may also have larger ribosome receptor protein isoforms. Surprisingly, mRRp has a very short basic C-terminal sequence of only 35 amino acids, while in contrast, the known human and canine forms of this protein have acidic C-terminal regions comprised of 803 and 798 amino acid residues, respectively. Although the function of the C-terminal region is currently unknown, it may be that those C-terminal sequences that are present in human and canine RRp proteins but missing in mRRp do not play critical roles in RRp function. The cDNA of the ES/130 isoform, which lacks tandem repeats and presumably are unable to bind ribosomes, could be isolated by reverse transcriptase-PCR from E 13.5 mouse embryos. mRRp mRNAs were expressed in all tissues examined but expression levels of each isoform differed between tissues. The identification of multiple mRRp isoforms in the mouse will allow us to study the regulation and function of ribosome receptors on a genetic level.

Introduction

The ribosome receptor anchors ribosomes to the membrane of the endoplasmic reticulum (ER) during translocation process (Savitz and Meyer, 1990, Savitz and Meyer, 1993, Savitz and Meyer, 1997). This is one of the crucial first steps in the transport and secretion of intracellular proteins in mammalian cells (reviewed in Brodsky, 1998). cDNAs encoding the 180-kDa ribosome receptor (p180) have been cloned from dogs and humans (Wanker et al., 1995, Langley et al., 1998). The predicted amino acid sequences of the dog and human p180 proteins share 95% identity, indicating that p180 may be highly conserved. The primary structure of p180 is comprised of three distinct domains: a hydrophobic N-terminus that includes the membrane-anchoring domain followed by a highly conserved basic tandem repeat domain and a large uninterrupted acidic C-terminal region (Wanker et al., 1995). The tandem repeat domain is likely to be a ribosome-binding domain whereby the ribosome receptor mediates the interaction between ribosomes and the ER membrane (Wanker et al., 1995). In humans and dogs, there are a total of 54 consecutive tandem repeats (Wanker et al., 1995, Langley et al., 1998). In chicks and humans, however, there is also another form of the ribosome receptor, denoted ES/130 (Rezaee et al., 1993, Basson et al., 1996, Langley et al., 1998). While this form bears the membrane anchor and the C-terminal regions present in p180, it entirely lacks the tandem repeat region. Thus, ES/130 may not be able to bind ribosomes and most likely has completely different functions to the p180 form. The large acidic C-terminal region constitutes half of the entire sequence of ES130/p180 and is composed of heptad repeats that are predicted to form an α -helical double-stranded coiled-coil rod (Leung et al., 1996, Langley et al., 1998). The function of this secondary structure is, however, currently unknown.

Interestingly, multiple spliced isoforms of p180, including the ES130 form, have been identified in humans (Langley et al., 1998). The splicing occurs at the tandem repeat sequence. The effects of the splicing can be quite dramatic, resulting in, for example, the conversion of p180-like forms into ES/130-like forms. That the different isoforms vary in their tandem repeat domain length suggests they may also differ in their ribosome-binding affinities. Northern blot analysis has revealed, however, that transcripts of the p180 splice variants, including ES/130, are rare and probably constitute only the minor transcripts detected in most tissues (Langley et al., 1998).

During our efforts to clone novel homeobox genes expressed in murine osteoblast cells, we instead isolated a number of cDNA clones encoding ribosome receptor proteins. The deduced amino acid sequences indicate that the mouse ribosome receptor bears 45–83% homology with ribosome receptor proteins from other species and that it consists of a central tandem repeat domain that is variable in length, an N-terminus that anchors the receptor in the membrane, and a basic C-terminal region that is very short compared to that seen in humans and dogs. These observations indicate that a mouse model that can be manipulated genetically is now available for investigation into ribosome receptor function.