Isolation and Sequence of Partial cDNA Clones of Human L1: Homology of Human and Rodent L1 in the Cytoplasmic Region

We have isolated cDNA clones coding for the human homologue of the neuronal cell adhesion molecule L1. The nucleotide sequence of the cDNA clones and the deduced primary amino acid sequence of the carboxy terminal portion of the human L1 are homologous to the corresponding sequences of mouse L1 and rat NILE glycoprotein, with an especially high sequences identity in the cytoplasmic regions of the proteins. There is also protein sequence homology with the cytoplasmic region of the Drosophila cell adhesion molecule, neuroglian. The conservation of the cytoplasmic domain argues for an important functional role for this portion of the molecule.     

A Nonneuronal Isoform of Cell Adhesion Molecule L1: Tissue-Specific Expression and Functional Analysis

Abstract: The cell adhesion molecule L1 is a multifunctional protein in the nervous system characterizing cell adhesion, migration, and neurite outgrowth. In addition to full-length L1, we found an alternatively spliced variant lacking both the KGHHV sequence in the extracellular part and the RSLE sequence in the cytoplasmic part of L1. This L1 variant was expressed exclusively in nonneuronal cells such as Schwann cells, astrocytes, and oligodendrocytes, in contrast to the expression of the full-length L1 in neurons and cells of neuronal origin. To investigate the functions of the L1 variant, we established cell lines transfected with a cytoplasmic short L1 (L1cs) cDNA that lacks only the 12-bp segment encoding for the RSLE sequence. The promoting activities of homophilic cell adhesion, neurite outgrowth, and neuronal cell migration of L1cs-transfected cells (L4-2) were similar to those of full-length L1-transfected cells (L3-1), but the cell migratory activity of L4-2 itself was clearly lower than that of L3-1. In conclusion, the short form of L1 is a nonneuronal type, in contrast to the neuronal type of the full-length L1. Deletion of the four amino acids RSLE in the cytoplasmic region of L1 markedly reduced cell migratory activity, suggesting an importance of the RSLE sequence for the signaling events of neuronal migration mediated by L1.     

Mechanistic control of carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) splice isoforms by the heterogeneous nuclear ribonuclear proteins hnRNP L, hnRNP A1, and hnRNP M

Carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) is expressed in a variety of cell types and is implicated in carcinogenesis. Alternative splicing of CEACAM1 pre-mRNA generates two cytoplasmic domain splice variants characterized by the inclusion (L-isoform) or exclusion (S-isoform) of exon 7. Here we show that the alternative splicing of CEACAM1 pre-mRNA is regulated by novel cis elements residing in exon 7. We report the presence of three exon regulatory elements that lead to the inclusion or exclusion of exon 7 CEACAM1 mRNA in ZR75 breast cancer cells. Heterologous splicing reporter assays demonstrated that the maintenance of authentic alternative splicing mechanisms were independent of the CEACAM1 intron sequence context. We show that forced expression of these exon regulatory elements could alter CEACAM1 splicing in HEK-293 cells. Using RNA affinity chromatography, three members of the heterogeneous nuclear ribonucleoprotein family (hnRNP L, hnRNP A1, and hnRNP M) were identified. RNA immunoprecipitation of hnRNP L and hnRNP A1 revealed a binding motif located central and 3' to exon 7, respectively. Depletion of hnRNP A1 or L by RNAi in HEK-293 cells promoted exon 7 inclusion, whereas overexpression led to exclusion of the variable exon. By contrast, overexpression of hnRNP M showed exon 7 inclusion and production of CEACAM1-L mRNA. Finally, stress-induced cytoplasmic accumulation of hnRNP A1 in MDA-MB-468 cells dynamically alters the CEACAM1-S:CEACAM1:L ratio in favor of the l-isoform. Thus, we have elucidated the molecular factors that control the mechanism of splice-site recognition in the alternative splicing regulation of CEACAM1.     

Regulation of the NEDD8 conjugation system by a splicing variant,NUB1L

NEDD8 is a ubiquitin-like protein that controls vital biological events through its conjugation to target proteins. We previously identified a negative regulator of the NEDD8 conjugation system, NUB1, which works by recruiting NEDD8 and its conjugates to the proteasome for degradation. Recently, we found its splicing variant, NUB1L. It possesses an insertion of 14 amino acids that codes for a UBA domain. Structural study revealed that NUB1 has a NEDD8-binding site at the C terminus, whereas NUB1L has an additional site at the newly generated UBA domain. Interestingly, the sequence A(X4)L(X10)L(X3)L was conserved in these NEDD8-binding sites among human and other mammals. Mutational studies revealed that at least three Leu residues in the conserved sequence are required for binding with NEDD8. Functional study suggested that the NEDD8-binding ability at the C terminus of NUB1 and NUB1L is mainly involved in the down-regulation of NEDD8, but the NEDD8-binding ability at the UBA2 domain of NUB1L is minimally or not involved at all. The NEDD8-binding ability at the UBA2 domain might be required for an unknown function of NUB1L.     

A common RNA recognition motif identified within a defined U1 RNA binding domain of the 70K U1 snRNP protein

We have defined the RNA binding domain of the 70K protein component of the U1 small nuclear ribonucleoprotein to a region of 111 amino acids. This domain encompasses an octamer sequence that has been observed in other proteins associated with RNA, but has not previously been shown to bind directly to a specific RNA sequence. Within the U1 RNA binding domain, an 80 amino acid consensus sequence that is conserved in many presumed RNA binding proteins was discerned. This sequence pattern appears to represent an RNA recognition motif (RRM) characteristic of a distinct family of proteins. By site-directed mutagenesis, we determined that the 70K protein consists of 437 amino acids (52 kd), and found that its aberrant electrophoretic migration is due to a carboxy-terminal charged domain structurally similar to two Drosophila proteins (su(wa) and tra) that may regulate alternative pre-messenger RNA splicing.     

Identification of a human homologue of the dendritic cell-associated C-type lectin-1, dectin-1

Previously we identified the novel type II lectin receptor, dectin-1, that is expressed preferentially by murine antigen presenting dendritic cells (DC) and is involved in co-stimulation of T cells by DC. To identify the human homologue (DECTIN-1), we employed degenerative PCR amplification of mRNA isolated from DC and subsequent cDNA cloning. DECTIN-1 is a type II lectin receptor with high homology to type II lectin receptors expressed by natural killer (NK) cells. It contains an immunoreceptor tyrosine-based activation motif within the cytoplasmic domain. Human DECTIN-1 mRNA is expressed predominantly by peripheral blood leukocytes and preferentially by DC. The mRNA likely encodes a 33 kDa glycoprotein. In human epidermis, the protein is expressed selectively by Langerhans cells, which are an epidermal subset of DC. A truncated form of DECTIN-1 RNA (termed T beta) encodes for a polypeptide lacking almost the entire neck domain, which is required for accessibility of the carbohydrate recognition domain to ligands. Genome analysis showed the deleted amino acid sequence in T beta to be encoded by an exon, indicating that T beta RNA is produced by alternative splicing. DECTIN-1 gene maps to chromosome 12, between p13.2 and p12.3, close to the NK gene complex (12p13.1 to p13.2) which contains genes for NK lectin receptors. Our results indicate that human DECTIN-1 shares many features with mouse dectin-1, including the generation of neck domain-lacking isoforms, which may down-regulate the co-stimulatory function of dectin-1.     

Allele-specific expression of the cytoplasmic exon of HLA-DQB1 gene

The chain of the HLA-DQ molecule is shorter by eight amino acid residues than other major histocompatibility complex class II chains due to elimination of the fifth exon coding for part of the cytoplasmic domain. This elimination is caused by one base substitution in the splice accepter site of the exon. We found that two HLA-DQB1 alleles, DQB1 ^* 0503 and DQB1 ^* 0601, did not have this substitution, and the exon was utilized in these two alleles. However, two forms of HLA-DQB mRNA, with or without exon 5, were generated in Epstein-Barr virus-transformed cell lines homozygous for DQB1 ^* 0503 or DQB1*0601, indicating alternative mRNA splicing. The alternative splicing of DQB1*0601 mRNA was also found in peripheral blood lymphocytes and L cell transfectants. To investigate the functional relevance of the allele-specific long cytoplasmic tail of HLA-DQ chain, we developed three types of L cell transfectants expressing exclusively the HLA-DQw6 molecules with short cytoplasmic tail, long cytoplasmic tail, or both forms of the chain, and used them as antigen presenting cells for streptococcal cell wall antigen-specific T cell lines. These three types of transfectants could function almost equally well as antigen presenting cells. It was thus demonstrated that both forms of HLA-DQ chain, with or without eight amino acid residues coded for by the exon 5, can be associated with the HLA-DQ chain, be expressed on the cell surface, and function as restriction molecules in antigen recognition by the CD4⁺ T cells.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M83920-83923.     

Specific regulation of mRNA splicing in vitro by a peptide from HIV-1 Rev

The Rev protein of HIV-1 regulates the synthesis of partially spliced forms of cytoplasmic viral mRNA by binding to a cis-acting RNA sequence, the Rev response element (RRE). We have investigated the regulation of splicing in vitro and have shown that Rev specifically inhibits splicing of pre-mRNAs containing an RRE by 3- to 4-fold. A synthetic peptide of 17 amino acids containing the RNA-binding domain of Rev is highly functional and specifically inhibits splicing by up to 30-fold. Other peptides that bind to the RRE with high affinity, but with low specificity, do not specifically inhibit splicing. Six repeated monomeric binding sites for the peptide can substitute for the RRE, indicating that regulation by Rev requires interactions with multiple sites. The peptide acts at a step in the assembly of splicing complexes, suggesting that one of the functions of the basic region of Rev is to prevent formation of a functional spliceosome.     

Fc receptor isoforms exhibit distinct abilities for coated pit localization as a result of cytoplasmic domain heterogeneity

Mouse macrophages and lymphocytes express two distinct isoforms of a single class of Fc receptor for IgG. The macrophage isoform (FcRII-B2) is identical to the lymphocyte isoform (FcRII-B1) except for an inframe insertion in the cytoplasmic tail of FcRII-B1 that increases its length from 47 to 94 amino acids. To determine the functional significance of this cytoplasmic domain variation, presumably the result of alternative mRNA splicing, we expressed both isoforms in receptor-negative fibroblasts. While FcRII-B2 mediated the efficient ligand internalization and delivery to lysosomes, endocytosis via FcRII-B1--and via a tailminus mutant--was relatively inefficient. This difference reflected the inability of FcRII-B1 (and the tailminus mutant) to accumulate in clathrin-coated pits. Thus, the FcRII-B2 cytoplasmic tail contains a domain needed for accumulation in coated pits, and this domain is disrupted by the 47 amino acid insertion in FcRII-B1.     

Plasma cell membrane glycoprotein PC-1. cDNA cloning of the human molecule, amino acid sequence, and chromosomal location

The murine cell membrane glycoprotein PC-1 is a homodimer with restricted tissue distribution, being first characterized in plasma cells. We now describe the isolation of cDNA clones encoding the human homolog of the murine PC-1 protein, its complete amino acid sequence, and its chromosomal location. Overall, the amino acid sequence of the human protein is about 80% identical to the murine protein, although the extent of homology varies in different domains. It had not been possible to assign a definitive amino terminus to the murine protein. Comparison of the murine and human sequence necessitates reassignment of the amino terminus, resulting in a cytoplasmic tail of 24 amino acids rather than 58 amino acids as previously published for the mouse. The sequence of several independently obtained cDNA clones indicates that the 3' end of the mRNA is subject to alternative splicing. Southern blots suggest a single copy gene. In situ chromosomal hybridization localizes the gene for human PC-1 to chromosome 6q22-q23, a common site for deletions in human lymphoid neoplasia.     

The neural cell adhesion molecule L1: genomic organisation and differential splicing is conserved between man and the pufferfish Fugu

The human gene for the neural cell adhesion molecule L1 is located on Xq28 between the ALD and MeCP2 loci. Mutations in the L1 gene are associated with four related neurological disorders, X-linked hydrocephalus, spastic paraplegia (SPG1), MASA syndrome, and X-linked corpus callosum agenesis. The clinical relevance of L1 has led us to sequence the L1 gene in human and to investigate its conservation in the vertebrate model genome of the pufferfish, Fugu rubripes (Fugu), a species with a compact genome of around 40 Mb. For this purpose we have sequenced a human and a Fugu cosmid clone containing the corresponding L1 genes. For comparison, we have also amplified and sequenced the complete Fugu L1 cDNA. We find that the genomic structure of L1 is conserved. The human and Fugu L1 gene both have 28 exons of nearly identical size. Differential splicing of exons 2 and 27 is conserved over 430 million years, the evolutionary time span between the teleost Fugu and the human L1 gene. In contrast to previously published Fugu genes, many introns are larger in the Fugu L1 gene, making it slightly larger in size despite the compact nature of the Fugu genome. Homology at the amino acid and the nucleotide level with 40% and 51%, respectively, is lower than that of any previously reported Fugu gene. At the level of protein structure, both human and Fugu L1 molecules are composed of six immunoglobulin (Ig)-like domains and five fibronectin (Fn) type III domains, followed by a transmembrane domain and a short cytoplasmic domain. Only the transmembrane and the cytoplasmic domains are significantly conserved in Fugu, supporting their proposed function in intracellular signalling and interaction with cytoskeletal elements in the process of neurite outgrowth and fascicle formation. Our results show that the cytoplasmic domain can be further subdivided into a conserved and a variable region, which may correspond to different functions. Most pathological missense mutations in human L1 affect conserved residues. Fifteen out of 22 reported missense mutations alter amino acids that are identical in both species.     

A U1 small nuclear ribonucleoprotein particle with altered specificity induces alternative splicing of an adenovirus E1A mRNA precursor.

We have altered the specificity of U1 small nuclear RNA by replacing its 5' splice site recognition sequence (nucleotides 3 to 11) with sequences complementary to other regions of either the adenovirus E1A or the rabbit beta-globin mRNA precursor. We then used a HeLa cell transient expression assay to test whether such altered U1 small nuclear ribonucleoprotein particles (snRNPs) could interfere with splicing of the targeted mRNA precursors. The altered U1 snRNPs were able to cause novel splicing of the E1A mRNA precursor, minor changes in the ratio of E1A 12 to 13S mRNAs, and modest nuclear accumulation of beta-globin mRNA precursors with either one of the two introns removed. Most of the altered U1 snRNPs did not affect the level of mature cytoplasmic mRNA significantly, but in one case an altered U1 snRNP (alpha 1) whose intended target was located downstream from the adenovirus E1A 12S 5' splice site was able to reduce the level of cytoplasmic 12S mRNA by approximately 60% and that of 13S mRNA by 90%. This alpha 1 snRNP induced an additional E1A splice, resulting in the appearance of 10 and 11S E1A mRNAs normally found only late in adenovirus infection. Thus, a trans-acting factor can induce alternative splicing. Surprisingly, the effects of alpha 1 on E1A splicing were not abolished by deleting the intended target sequence on the mRNA precursor.