Ets-related protein PU.1 regulates expression of the immunoglobulin J-chain gene through a novel Ets-binding element.

Abstract

A c/s-acting negative regulator of splicing (NRS) within the gag gene of RSV is involved in control of the relative levels of spliced and unspliced viral mRNAs. Insertion of the NRS into the intron of an adenovirus pre-mRNA resulted in inhibition of splicing in vitro before the first cleavage step. Analyses of spliceosome assembly with this substrate showed that it formed large RNP complexes that did not migrate like mature spliceosomes on native gels. Affinity selection of the RNP complexes formed on NRS-containing pre-mRNAs showed an association with U l l and U12 snRNPs, as well as with the spliceosomal snRNPs. Immunoprecipitation with antisera specific for U1 and U2 snRNPS showed binding of both snRNPs to NRS RNA. A 7-nucleotide missense mutation in the NRS that prevented binding of U l l and U12 snRNPs impaired NRS activity in vivo, suggesting a functional role for U l l and U12 snRNPS in the inhibition of splicing mediated by the RSV NRS RNA.[Key Words: Retrovirus; RNA splicing; snRNPs] Received June 23, 1993; revised version accepted July 28, 1993.Retroviral replication involves reverse transcription of the RNA genome into DNA, which integrates into the host genome and is transcribed by cellular RNA polymerase II (for review, see Coffin 1990). While the viral RNA transcripts are processed, transported, and translated by cellular machinery, they yield both spliced and unspliced cytoplasmic mRNAs, unlike cellular premRNAs. The majority of the viral transcripts are not spliced but are transported to the cytoplasm, where they become full-length mRNA for Gag and Gag-Pol polyproteins, as well as packaged virion RNA. In the case of Rous sarcoma virus (RSV), the remainder of the primary transcripts are alternatively spliced in the nucleus to yield mRNAs for the env and src gene products. Production of infectious virus requires a balance of spliced and unspliced mRNAs.Splicing of mRNA precursors occurs in a dynamic macromolecular complex called a spliceosome; the reaction requires ATP, functional splice site and branchpoint sequences, small nuclear ribonucleoproteins (snRNPs), heterogeneous ribonucleoproteins (hnRNPs), and essential non-snRNP splicing factors (for review, see Krainer and Maniatis 1988; Green 1991). The spliceosomal snRNP particles U1, U2, U4/U6, and U5 are abundant in animal cells (10s-10 6 copies per cell) and participate in both steps of pre-mRNA splicing (for review, see Steitz et al. 1988). Genetic experiments have confirmed the sig- nificance of RNA-RNA base-pairing between U1 snRNA and the 5' splice site (Zhuang and Weiner 1986) and U2 snRNA and the branchpoint (Wu and Manley 1989; Zhuang and Weiner 1989). The U4/U6 and U5 snRNPs, which appear to associate as a multi-snRNP complex (Konarska and Sharp 1987), have also been identified as components of the mature spliceosome (Frendewey and Keller 1985; Bindereif and Green 1987). Other less abundant snRNPs have been identified whose functions have not been defined (Kramer 1987; Reddy and Busch 1988). For example, Wassarman and Steitz (1992...

Isolation and characterization of a chicken homologue of the Spi-1/PU.1 transcription factor

“…Two proteins of 40 kDa and 35 kDa were detected by SDS ± PAGE and were both recognized by antisera directed against the 110 N-terminal amino- Figure 7 Electrophoretic mobility shift assays (EMSA) were performed with Spi-1/PU.1 proteins from programmed reticulocyte lysates (2 ml) or HD11 nuclear extract puri®ed as described (Sambrook et al, 1989) (5 mg). The oligonucleotide use as probe is the Spi-1/PU.1 site within the Ig-J chain promoter (Shin and Koshland, 1993). Complexes were visualized on 6% non-denaturing polyacrylamide gel.…”

“…This binding site was reported to be speci®c for Spi-1/PU.1 proteins since other members of the ETS family failed to recognize it (Shin and Koshland, 1993).…”

“…Another band speci®c of the antiserum treatment can be also observed, coming probably from the supershifting of part of the faster migrating bands. Using this probe, Shin and Koshland (1993) had shown a major single complex with nuclear extracts from S194 myeloma cells, but speci®ed that a longer exposure of the gel revealed additional slower migrating species which did not react with the anti-PU.1 antibody. And strikingly, two antibody-reacting complexes from HD11 cells were also shown to bind to a Spi-1/PU.1 binding site in the enhancer of the chicken lysozyme gene (Ahne and StraÈ tling, 1994).…”

“…However, in accordance to its expression pattern, numerous target genes have been reported in di erent hematopoietic lineages (reviewed in Moreau-Gachelin, 1994). This is for example the case for genes with expression restricted to B lymphocytes, like genes coding for immunoglobulin chains: kappa (Pongubala et al, 1992(Pongubala et al, , 1993 and lambda (Eisenbeis et al, 1993) lightchains, mu heavy (Nelsen et al, 1993;Rivera et al, 1993) and J chains (Shin and Koshland, 1993); and genes regulated by Spi-1/PU.1 in myeloid lineages like interleukin lb (Kominato et al, 1995), CD18 (Rosmarin et al, 1995), c-Fes/ c-Fps tyrosine kinase (Ray-Gallet et al, 1995). In many cases, cell-type restricted expression of target genes was shown to result from combinatorial and cooperative action of various transcription factors, including Spi-1/PU.1.…”

Mutations of the transcription factor PU.1 are not associated with acute lymphoblastic leukaemia

“…The crucial and exclusive role of PU.1 in haematopoietic differentiation is established by the following observations. A number of genes are regulated by PU.1 in both myeloid and B-cell lineages, including those encoding colony-stimulating factor receptors and immunoglobulin subunits (Shin and Koshland, 1993;Zhang et al, 1994;Hohaus et al, 1995;Smith et al, 1996). Overexpression of PU.1 early in erythroid development blocks erythroblast differentiation and induces murine erythroleukaemia (Moreau-Gachelin et al, 1988Paul et al, 1991).…”