The TLS/FUS gene is involved in a recurrent chromosomal translocation in human myxoid liposarcomas. We previously reported that TLS is a potential splicing regulator able to modulate the 5-splice site selection in an E1A pre-mRNA. Using an in vitro selection procedure, we investigated whether TLS exhibits a specificity with regard to RNA recognition. The RNAs selected by TLS share a common GGUG motif. Mutation of a G or U residue within this motif abolishes the interaction of TLS with the selected RNAs. We showed that TLS can bind GGUG-containing RNAs with a 250 nM affinity. By UV cross-linking/competition and immunoprecipitation experiments, we demonstrated that TLS recognizes a GGUG-containing RNA in nuclear extracts. Each one of the RNA binding domains (the three RGG boxes and the RNA recognition motif) contributes to the specificity of the TLS⅐RNA interaction, whereas only RRM and RGG2-3 participate to the E1A alternative splicing in vivo. The specificity of the TLS⅐RNA interaction was also observed using as natural pre-mRNA, the G-rich IVSB7 intron of the ␤-tropomyosin pre-mRNA. Moreover, we determined that RNA binding specificities of TLS and high nuclear ribonucleoprotein A1 were different. Hence, our results help define the role of the specific interaction of TLS with RNA during the splicing process of a pre-mRNA. TLS (Translocated in LipoSarcoma)1 or FUS has been first characterized as a rearranged gene in chromosomal translocations specific of human myxoid liposarcoma (1, 2). The resulting fusion protein contains the N-terminal part of TLS fused to a transcription factor of the CAAT/enhancer-binding protein family of proteins: CHOP. In an acute myeloid leukemia, TLS is also involved in a chromosomal breakpoint that juxtaposes the same N-terminal region of TLS to a transcription factor of the ETS proteins family: ERG-1 (3, 4). TLS is highly similar to EWS, a gene implicated in chromosomal translocations that are specific of the tumors of the Ewing family (5, 6). In most of these sarcoma, the N-terminal region of EWS is fused to the DNA binding domain of either ERG-1 or FLI-1, which are two closely related ETS proteins.Both TLS and EWS have in common a similar structural organization. Their C terminus part contains multiple domains that are involved in RNA⅐protein interactions: an RNA recognition motif (RRM) flanked by two regions rich in Arg-Gly-Gly repeats (RGG domains) and a C 2 C 2 zinc finger. In their N terminus domain, they contain a glutamine-, serine-, and tyrosine-rich region that functions as a transcriptional activation domain when fused to a heterologous DNA binding domain (7-9). In oncogenic chimera, the adjunction of this N-terminal region of TLS or EWS to the transcriptional regulators CHOP, FLI-1, or ERG-1 generates proteins with transcriptional activities that differ from those of the wild-type counterpart (8,10). From these data it has been proposed that the oncogenic fusion proteins disturb the expression of genes that are regulated by CHOP, FLI-1, or ERG-1. However, no cellular targe...

Spi-1/PU.1 is an Ets protein deregulated by insertional mutagenesis during the murine Friend erythroleukemia. The overexpression of the normal protein in a proerythroblastic cell prevents its terminal differentiation. In normal hematopoiesis Spi-1/PU.1 is a transcription factor that plays a key role in normal myeloid and B lymphoid differentiation. Moreover, Spi-1/PU.1 binds RNA and interferes in vitro with the splicing process. Here we report that Spi-1 interacts in vivo with TLS (translocated in liposarcoma), a RNA-binding protein involved in human tumor-specific chromosomal translocations. This interaction appears functionally relevant, since TLS is capable of reducing the abilities of Spi-1/ PU.1 to bind DNA and to transactivate the expression of a reporter gene. In addition, we observe that TLS is potentially a splicing factor. It promotes the use of the distal 5 splice site during the E1A pre-mRNA splicing. This effect is counterpoised in vivo by Spi-1. These data suggest that alteration of pre-mRNA alternative splicing by Spi-1 could be involved in the transformation of an erythroblastic cell.The transcription factor Spi-1/PU.1 (1) is an Ets protein that plays a central role in the differentiation of macrophages and B cells during normal hematopoiesis. Indeed, the disruption of the Spi-1/PU.1 gene in mouse (2, 3) induces an early lethality of animals that lack mature macrophages, neutrophils, B and T cells, and also osteoclasts (4). These animals present no alteration in erythrocytic and megacaryocytic lineages. Consistent with this phenotype, Spi-1-responsive elements have been identified in transcriptional promoters and enhancers of many myeloid and B lymphoid genes (for review, see Ref. 5). In murine Friend acute erythroleukemia, the mutation of the spi-1 gene by retroviral insertional mutagenesis induces its transcriptional activation. The erythroleukemic process developed in spi-1 transgenic mice reveals that Spi-1 is involved in blocking the differentiation of the proerythroblast (6). The molecular mechanisms that lead Spi-1 to arrest the erythroid differentiation are not yet understood. The up-regulation of Spi-1 related to the transformation of the proerythroblast suggests that Spi-1 may induce a transcriptional dysregulation of some erythroid genes or/and may be abnormally associated with erythroid partners.Some nuclear proteins interacting with Spi-1 have already been identified such as the transactivator NF-IL6␤/C/EBP␦ (7), the transcription factor Pip/NF-EM5 (8), the retinoblastoma protein (9), and the basal transcription factor TFIID (9). We reported previously that Spi-1 interacts with the RNA-binding protein p54 nrb (nuclear RNA-binding protein, 54 kDa), binds to RNA by its DNA-binding domain and interferes in vitro with the splicing of a ␤-globin minigene (10). Here, we describe that Spi-1/PU.1 interacts with another RNA-binding protein TLS and that this association impedes the transcriptional functions of Spi-1. In addition, TLS influences, in vivo, the selection of the 5Ј splice site dur...

To rescue stalled ribosomes, eubacteria employ a molecule, transfer messenger RNA (tmRNA), which functions both as a tRNA and as an mRNA. With the help of small protein B (SmpB), tmRNA restarts protein synthesis and adds by the trans-translation mechanism a peptide tag to the stalled protein to target it for destruction by cellular proteases. Here, the cellular location and expression of endogenous SmpB were monitored in vivo. We report that SmpB is associated with 70S ribosomes and not in the soluble fraction, independently of the presence of tmRNA. In vitro, SmpB that is pre-bound to a stalled ribosome can trigger initiation of trans-translation. Our results demonstrate the existence of a novel pathway for the entry of tmRNA to the ribosome and for the trans-transfer of a nascent peptide chain from peptidyl-tRNA to charged tmRNA.