Modulation of msl-2 5′ splice site recognition by Sex-lethal

Abstract

The protein Sex-lethal (SXL) controls dosage compensation in Drosophila by inhibiting splicing and subsequently translation of male-specific-lethal-2 (msl-2) transcripts. We have previously shown that SXL blocks the binding of U2 auxiliary factor (U2AF) to the polypyrimidine (Py)-tract associated with the 39 splice site of the regulated intron. We now report that a second pyrimidine-rich sequence containing 11 consecutive uridines immediately downstream from the 59 splice site is required for efficient splicing inhibition by SXL. Psoralen-mediated crosslinking experiments suggest that SXL binding to this uridine-rich sequence inhibits recognition of the 59 splice site by U1 snRNP in HeLa nuclear extracts. We also show that SXL interferes with the binding of the protein TIA-1 to the uridine-rich stretch. Because TIA-1 binding to this sequence is necessary for U1 snRNP recruitment to msl-2 59 splice site and for splicing of this pre-mRNA, we propose that SXL antagonizes TIA-1 activity and thus prevents 59 splice site recognition by U1 snRNP. Taken together with previous data, we conclude that efficient retention of msl-2 intron involves inhibition of early recognition of both splice sites by SXL.

An Intronic Polypyrimidine-rich Element Downstream of the Donor Site Modulates Cystic Fibrosis Transmembrane Conductance Regulator Exon 9 Alternative Splicing

“…In msl-2, the Drosophila regulator Sex-lethal (SLX) interferes with the binding of TIA-1 near the 5Ј-ss and, in this manner, prevents the U1 snRNP-mediated recognition of the 5Ј-ss (35). In in vitro experiments with CFTR exon 9, PTB competed with TIA-1 for binding to the PCE (Fig.…”

“…4). In the case of fas alternative splicing, binding of TIA-1 to the 5Ј-ss region of intron 5 induces exon 6 inclusion at the expense of transcripts in which exon 6 is skipped, suggesting that TIA-1 modulates the choice between 3Ј-splice sites (35). In the context of our CFTR exon 9 minigene, the 5Ј-ss region of the upstream exon does not contain polypyrimidine-rich sequences; therefore, TIA-1 cannot bind to this region and, as a result, selects the cryptic 3Ј-ss.…”

Sex-lethalsplicing autoregulation in vivo: interactions between SEX-LETHAL, the U1 snRNP and U2AF underlie male exon skipping

“…SXL also suppresses expression of the male-specific-lethal-2 (msl-2) gene by binding similar U-rich sequences (Bashaw and Baker, 1995;Kelley et al, 1995;Zhou et al, 1995). However, the mechanism by which SXL regulates msl-2 processing is more complex than for tra because it includes both translational repression and splicing inhibition (Bashaw and Baker, 1997;Forch et al, 2001;Gebauer et al, 1999;Gebauer et al, 1998;Kelley et al, 1997;Merendino et al, 1999). These two examples demonstrate that SXL is capable of controlling expression of its target pre-mRNAs by diverse mechanisms.…”

Sex lethal and U2 small nuclear ribonucleoprotein auxiliary factor (U2AF65) recognize polypyrimidine tracts using multiple modes of binding

“…SXL regulates tra through 3Ј-splice site switching by competing with the binding of U2AF 65 to the non-sex-specific (NSS) Py tract of tra, thereby diverting U2AF 65 to an otherwise weak, female-specific (FS), Py tract located further downstream, to which SXL does not bind (Sosnowski et al 1989;Inoue et al 1990;Valcarcel et al 1993;Granadino et al 1997). In the msl2 pre-mRNA, SXL competes for the binding of TIA-1 and U2AF 65 proteins to the uridine-rich sequences near the 5Ј-and 3Ј-splice sites, respectively, causing retention of an intron in the 5Ј-UTR (Merendino et al 1999;Forch et al 2001). Moreover, the binding of SXL to uridine-rich sequences in both 5Ј-and 3Ј-UTRs blocks the translation of msl2 (Bashaw and Baker 1997;Kelley et al 1997;Gebauer et al 1998).…”