The Drosophila Heterogeneous Nuclear Ribonucleoprotein M Protein, HRP59, Regulates Alternative Splicing and Controls the Production of Its Own mRNA

Abstract

The Drosophila heterogeneous nuclear ribonucleoprotein M, HRP59, is a nuclear protein that associates co-transcriptionally with pre-mRNA and is necessary for the correct expression of a subset of mRNAs. We show here that the hrp59 pre-mRNA is alternatively spliced to generate two different mRNAs that differ in the presence of exon 3. Exon 3-containing transcripts make up the majority of hrp59 transcripts and encode for the functional protein, HRP59-1. Transcripts that lack exon 3 contain a premature translation termination codon and are targeted to the nonsense mediated decay pathway. We show that exon 3 inclusion is itself inhibited by HRP59 and that changes in the HRP59 protein levels affect the splicing activity of the cell. We propose that the ability of HRP59 to regulate the alternative splicing of its own pre-mRNA serves in a negative feedback loop that controls the levels of the HRP59 protein and maintains the homeostasis of the splicing environment.Pre-mRNAs associate co-transcriptionally with a variety of RNA-binding proteins known as hnRNP 2 proteins or mRNP proteins (reviewed in Refs. 1 and 2). hnRNP proteins exist in all eukaryotes and are evolutionarily conserved. More than 20 hnRNP proteins have been identified in human cells, and these have been designated from A1 to U (reviewed in Ref. 2). Although the degree of homology at the amino acid level is not very high, hnRNP proteins from different organisms share the same types of domain and the same domain organization. In Drosophila melanogaster, more than 10 hnRNP proteins have been identified (3-6).hnRNP proteins are abundant nuclear proteins and they play a general role in the packaging of nascent transcripts into ribonucleoprotein complexes (pre-mRNP). Many hnRNP proteins also have specific functions in gene expression. For instance, in the cell nucleus the mammalian hnRNP K interacts with the transcription machinery and stimulates transcription (7), while in the cytoplasm hnRNP K controls translation initiation of specific mRNAs (8). hnRNP I, also known as polypyrimidine tract-binding protein, plays a role in mRNA stability and mRNA localization (9, 10).hnRNP proteins are modular and characterized by the presence of one or more RNA-binding motifs and auxiliary domains of different types (reviewed in Ref. 11). Several hnRNP genes generate multiple isoforms through alternative splicing. The significance of these alternatively spliced forms has been elucidated for members of the hnRNP families A/B, D, and I. For example, the stabilities of human hnRNP D isoforms are differentially controlled by the insertion of an alternate exon that regulates their ubiquitin targeting activities (12). Different isoforms of hrp40, a Drosophila hnRNP A/B homolog, play distinct roles in Gurken localization during oogenesis (13). Alternative skipping of exon 11 in the human hnRNP I transcript leads to the production of a mRNA that is degraded by the nonsense-mediated decay (NMD) pathway (14). Exon 11 skipping is promoted by hnRNP I itself, and thus it has been propose...

The Exosome Associates Cotranscriptionally with the Nascent Pre-mRNP through Interactions with Heterogeneous Nuclear Ribonucleoproteins

“…Hrp59 associates cotranscriptionally with transcripts from many different genes, as shown by immunostaining of polytene chromosomes, and it is probable that Hrp59 has a general role in the packaging of mRNPs (Kiesler et al, 2005). Hrp59 has also a specific role in the regulating the alternative splicing of some transcripts (Hase et al, 2006). We have now shown that pre-mRNAs synthesized in the absence of Hrp59 are defective in the recruitment of the surveillance machinery.…”

“…In all the experiments, Mascot searches identified Hrp59, the product of the CG9373 gene, as a protein significantly enriched in the immunoprecipitates ( Figure 5B, Supplemental Tables S3 and S4), which indicates that Hrp59 and Rrp4 interact physically in vivo. Hrp59, also known as Rumpelstiltskin, is the hnRNP M protein of D. melanogaster (Kiesler et al, 2005;Hase et al, 2006;Jain and Gavis, 2008).…”

“…RNA interference (RNAi) was carried out essentially as described by Hase et al (2006). Double-strand RNAs (dsRNAs) against Hrp59 and green fluorescent protein (GFP) were prepared by in vitro transcription from PCR products with T7 promoters on both ends of the amplimers, using the Megascript RNAi kit (Ambion, Austin, TX).…”

Autoregulation of Fox protein expression to produce dominant negative splicing factors

“…The prototypical examples are the SR and SR-like proteins, where often a stop codon containing ''poison exon'' is activated by the SR protein through an ESE (Jumaa and Nielsen 1997;Sureau et al 2001;Kumar and Lopez 2005;Lareau et al 2007;Ni et al 2007). Other splicing regulators can repress a critical exon within their pre-mRNA to induce a frameshift and NMD (Stoilov et al 2004;Wollerton et al 2004;Dredge et al 2005;Hase et al 2006;Saltzman et al 2008). Indeed, nearly all hnRNP and related proteins show evidence in the EST databases for a NMD targeted isoform (P Stoilov and DL Black, unpubl.).…”

Regulatory Roles of Heterogeneous Nuclear Ribonucleoprotein M and Nova-1 Protein in Alternative Splicing of Dopamine D2 Receptor Pre-mRNA

“…hnRNPs are known to be negative splicing regulators by directly antagonizing the recognition of splice sites or interfering with the binding of splicing regulators to splicing enhancer sequences (28). More recently, Drosophila hnRNP M, HRP59, has been shown to bind to its own mRNA, inhibiting HRP59-mediated exon 3 inclusion (29,30). In addition, hnRNP M binds to the intronic splicing enhancer/intronic splicing silencer-3 of FGFR2 (FGF receptor 2) pre-mRNA and promotes FGFR2 exon IIIc skipping and also enhances the excision of preprotachykinin exon 4 (31).…”