A 69-kD protein that associates reversibly with the Sm core domain of several spliceosomal snRNP species

Hackl, Wolfgang; Fischer, Utz; Lührmann, Reinhard

doi:10.1083/jcb.124.3.261

Cited by 26 publications

(38 citation statements)

References 42 publications

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“…TLS is the high nuclear ribonucleoprotein (hnRNP) p2 that has been identified in a protein complex assembled on adenovirus pre-mRNA (21). TLS is engaged in a complex with the hnRNPs A1 and C1/C2 (9) and associates with several spliceosomal small nuclear ribonucleoproteins (22). TLS is complexed to RNA polymerase II transcripts in UV-irradiated HeLa cell extracts (23), which are consistent with the location of the Drosophila homologue of TLS (SARFH/CAZ) (24, 25) on actively transcribed regions in the polytene chromosomes of salivary glands.…”

Section: Tls (Translocated Insupporting

confidence: 59%

Identification of an RNA Binding Specificity for the Potential Splicing Factor TLS

Lerga

Hallier

Delva

et al. 2001

Journal of Biological Chemistry

166

170

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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...

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Section: Tls (Translocated Insupporting

confidence: 59%

Identification of an RNA Binding Specificity for the Potential Splicing Factor TLS

Lerga

Hallier

Delva

et al. 2001

Journal of Biological Chemistry

166

170

View full text Add to dashboard Cite

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“…The identity of these proteins is not known. The 70 kDa protein is not phosphorylated and was not recognized by antibodies against the U1-specific 70 kDa phosphoprotein or against the recently described 69 kDa snRNP core protein [27,35]. Both antibodies immunoblot and immunoprecipitate nuclear snRNP proteins but do not recognize a 70 kDa protein in the cytoplasmic 20S complex in either the L929 cells or HeLa cells.…”

Section: Two Anti-sm Monoclonal Antibodies Immunoprecipitate All the mentioning

confidence: 76%

“…Antibodies directed against the 69 kDa snRNP associated protein [27] were provided by Reinhard Lü hrmann (Philipps University, Marburg, Germany).…”

Section: Antisera Immunoprecipitations and Western Blotsmentioning

confidence: 99%

Thirteen Anti-Sm Monoclonal Antibodies Immunoprecipitate the Three Cytoplasmic snRNP Core Protein Precursors in Six Distinct Subsets

Fury

Andersen

Ponda

et al. 1999

Journal of Autoimmunity

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“…32 P-labelled U5 snRNA was prepared by in vitro transcription as described above and incubated under standard reconstitution conditions. Immunoprecipitations were performed with rabbit sera directed against the U5 116-kDa protein (12), essentially as previously described (15). Briefly, protein A-Sepharose (PAS)-bound antibody was incubated for 2 h at 4°C with 12.5 l of a splicing reaction mixture containing 10 5 cpm (10 ng) of 32 P-labelled U5 snRNA in 200 l of IPP 150 buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.05% [vol/vol] Nonidet P-40) and subsequently washed four times with IPP buffer containing 300 mM NaCl.…”

Section: Methodsmentioning

confidence: 99%

Conserved Loop I of U5 Small Nuclear RNA Is Dispensable for Both Catalytic Steps of Pre-mRNA Splicing in HeLa Nuclear Extracts

Ségault

Will

Polycarpou‐Schwarz

et al. 1999

Molecular and Cellular Biology

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The function of conserved regions of the metazoan U5 snRNA was investigated by reconstituting U5 small nuclear ribonucleoprotein particles (snRNPs) from purified snRNP proteins and HeLa or Xenopus U5 snRNA mutants and testing their ability to restore splicing to U5-depleted nuclear extracts. Substitution of conserved nucleotides comprising internal loop 2 or deletion of internal loop 1 had no significant effect on the ability of reconstituted U5 snRNPs to complement splicing. However, deletion of internal loop 2 abolished U5 activity in splicing and spliceosome formation. Surprisingly, substitution of the invariant loop 1 nucleotides with a GAGA tetraloop had no effect on U5 activity. Furthermore, U5 snRNPs reconstituted from an RNA formed by annealing the 5 and 3 halves of the U5 snRNA, which lacked all loop 1 nucleotides, complemented both steps of splicing. Thus, in contrast to yeast, loop 1 of the human U5 snRNA is dispensable for both steps of splicing in HeLa nuclear extracts. This suggests that its function can be compensated for in vitro by other spliceosomal components: for example, by proteins associated with the U5 snRNP. Consistent with this idea, immunoprecipitation studies indicated that several functionally important U5 proteins associate stably with U5 snRNPs containing a GAGA loop 1 substitution.Nuclear pre-mRNA splicing proceeds via a two-step mechanism. In the first step, the pre-mRNA is hydrolyzed at the 5Ј splice site and the 5Ј end of the intron interacts concomitantly with an adenosine at the so-called branch point. The splicing intermediates thus generated include exon 1 and a lariat structure comprised of the intron and exon 2. In the second step, hydrolysis at the 3Ј splice site and the concomitant ligation of exons 1 and 2 give rise to the mRNA and the excised intron in the form of a lariat. Both reactions are catalyzed by the spliceosome, a large ribonucleoprotein complex formed by the ordered interaction of numerous splicing factors and the four small nuclear ribonucleoprotein particles (snRNPs), U1, U2, U5, and U4/U6, with conserved regions of the pre-mRNA (reviewed in references 19, 27, and 34). Spliceosome assembly is initiated by the interaction of the U1 and U2 snRNPs with the 5Ј splice site and branch site, respectively, thereby generating the so-called prespliceosome, or complex A. Mature spliceosomes (i.e., complexes B and C) are ultimately formed by the subsequent interaction of the U4/U6 and U5 snRNPs, in the form of a preassembled U4/U6.U5 tri-snRNP complex (reviewed in references 19 and 34).The assembly of a catalytically active spliceosome requires the formation of a network of RNA-RNA interactions which favorably position the chemically reactive groups of the premRNA for catalysis (for reviews, see references 26 and 38). The U5 snRNP has been proposed to play a central role in recognizing and aligning the 5Ј and 3Ј splice sites for catalysis, and its function appears to be mediated, at least in part, by base pairing interactions between the U5 small nuclear RNA (snRNA) ...

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A 69-kD protein that associates reversibly with the Sm core domain of several spliceosomal snRNP species

Cited by 26 publications

References 42 publications

Identification of an RNA Binding Specificity for the Potential Splicing Factor TLS

Identification of an RNA Binding Specificity for the Potential Splicing Factor TLS

Thirteen Anti-Sm Monoclonal Antibodies Immunoprecipitate the Three Cytoplasmic snRNP Core Protein Precursors in Six Distinct Subsets

Conserved Loop I of U5 Small Nuclear RNA Is Dispensable for Both Catalytic Steps of Pre-mRNA Splicing in HeLa Nuclear Extracts

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