Serine-arginine (SR) protein-like factors that antagonize authentic SR proteins and regulate alternative splicing.

We have characterized two RNA-binding proteins, of apparent molecular masses of approximately 40 and 35 kDa, which possess a single N-terminal RNA-recognition motif (RRM) followed by a C-terminal domain rich in serine-arginine dipeptides. Their primary structures resemble the single-RRM serine-arginine (SR) protein, SC35; however their functional effects are quite distinctive. The 40-kDa protein cannot complement SR protein-deficient HeLa cell S100 extract and showed a dominant negative effect in vitro against the authentic SR proteins, SF2/ASF and SC35. Interestingly, the 40- and 35-kDa proteins antagonize SR proteins and activate the most distal alternative 5' splice site of adenovirus E1A pre-mRNA in vivo, an activity that is similar to that characterized previously for the heterogeneous nuclear ribonucleoprotein particles A/B group of proteins. A series of recombinant chimeric proteins consisting of domains from these proteins and SC35 in various combinations showed that the RRM, but not the C-terminal domain rich in serine-arginine dipeptides, has a dominant role in this activity. Because of the similarity to SR proteins we have named these proteins SRrp40 and SRrp35, respectively, for SR-repressor proteins of approximately 40 and approximately 35 kDa. Both factors show tissue- and cell type-specific patterns of expression. We propose that these two proteins are SR protein-like alternative splicing regulators that antagonize authentic SR proteins in the modulation of alternative 5' splice site choice.

Influence of intron length on alternative splicing of CD44.

Although the splicing of transcripts from most eukaryotic genes occurs in a constitutive fashion, some genes can undergo a process of alternative splicing. This is a genetically economical process which allows a single gene to give rise to several protein isoforms by the inclusion or exclusion of sequences into or from the mature mRNA. CD44 provides a unique example; more than 1,000 possible isoforms can be produced by the inclusion or exclusion of a central tandem array of 10 alternatively spliced exons. Certain alternatively spliced exons have been ascribed specific functions; however, independent regulation of the inclusion or skipping of each of these exons would clearly demand an extremely complex regulatory network. Such a network would involve the interaction of many exon-specific trans-acting factors with the pre-mRNA. Therefore, to assess whether the exons are indeed independently regulated, we have examined the alternative exon content of a large number of individual CD44 cDNA isoforms. This analysis shows that the downstream alternatively spliced exons are favored over those lying upstream and that alternative exons are often included in blocks rather than singly. Using a novel in vivo alternative splicing assay, we show that intron length has a major influence upon the alternative splicing of CD44. We propose a kinetic model in which short introns may overcome the poor recognition of alternatively spliced exons. These observations suggest that for CD44, intron length has been exploited in the evolution of the genomic structure to enable tissue-specific patterns of splicing to be maintained.

Lymphocyte inhibitor of TRAIL (TNF-related apoptosis-inducing ligand): a new receptor protecting lymphocytes from the death ligand TRAIL.

Apoptosis can be triggered by the engagement of cell surface receptors by their ligands. A growing number of receptors belonging to the TNF receptor family have been identified that contain a conserved cytoplasmic death domain. These include Fas, TNF-R1, lymphocyte-associated receptor of death (LARD), DR4, and TNF-related apoptosis-inducing ligand receptor inducer of cell killing-2 (TRICK2). The latter two are receptors for the cytotoxic ligand TNF-related apoptosis-inducing ligand (TRAIL), and one of the paradoxes raised by the cloning of these molecules was why do most cells not die upon contact with the widely expressed TRAIL molecule? This is a particular problem for lymphocytes that express DR4 and TRICK2 and are in constant circulation through TRAIL-expressing tissues. We have cloned LIT (lymphocyte inhibitor of TRAIL), which lacks a death domain. LIT is expressed predominantly on PBL, where it can competitively inhibit TRAIL-induced apoptosis through DR4/TRICK2, and may function to modulate lymphocyte sensitivity to TRAIL.

TRICK2, a new alternatively spliced receptor that transduces the cytotoxic signal from TRAIL.

A subset of the tumour necrosis factor (TNF) receptor family contain a conserved intracellular motif, the death domain. Engagement of these receptors by their respective ligands initiates a signalling cascade that rapidly leads to cell death by apoptosis. We have cloned a new member of this family, TRICK2, the TRAIL (TNF-related apoptosis-inducing ligand) receptor inducer of cell killing 2. TRICK2 is expressed in a number of cell types, and to particularly high levels in lymphocytes and spleen. Two isoforms of the TRICK2 mRNA are generated by alternative pre-mRNA splicing and differ by a 29 amino-acid extension to the extracellular domain. Overexpression of TRICK2 rapidly induced apoptosis in 293T cells; this induction was dependent upon the presence of the death domain of TRICK2. Using a soluble molecule containing the TRICK2 extracellular domain, we demonstrated that TRICK2, like DR4 [1], is a receptor for TRAIL/APO-2L [2,3] and could inhibit TRAIL-induced killing of lymphocyte lines, such as the Jurkat T-cell line. TRAIL is upregulated upon lymphocyte activation, as is the intensively studied ligand for Fas, FasL [4]. TRAIL and its receptors might therefore provide another system for the regulation of lymphocyte selection and proliferation, as well as providing an additional weapon in the armoury of cytotoxic lymphocytes.

LARD: a new lymphoid-specific death domain containing receptor regulated by alternative pre-mRNA splicing.

Fas and TNF-R1 are cysteine-rich cell surface receptors related to the low-affinity nerve growth factor receptor family. Engagement of these receptors by their respective ligands, FasL and tumor necrosis factor, leads to apoptosis that is signaled through a conserved intracellular portion of the receptor termed the "death domain." We have cloned a new member of this family, lymphocyte-associated receptor of death (LARD), which leads to spontaneous apoptosis when expressed in 293T cells. The expression of LARD is more tightly regulated than that of either Fas or TNF-R1 as it is found predominantly on lymphocytes (T and B cells) but not on macrophages or a number of transformed lymphocyte cell lines. Alternative pre-mRNA splicing generates at least 11 distinct isoforms of LARD. The full-length isoform, LARD-1, extends to include the transmembrane and death domains, whereas the other isoforms encode potentially secreted molecules. Naive B and T cells express very little LARD-1 but express combinations of the other isoforms. Upon T cell activation, a programmed change in alternative splicing occurs so that the full-length, membrane-bound LARD-1 predominates. This may have implications for the control of lymphocyte proliferation following activation.