The Drosophila heterogeneous nuclear ribonucleoprotein M protein, HRP59, regulates alternative splicing and controls the production of its own mRNA.

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.

Hrp59, an hnRNP M protein in Chironomus and Drosophila, binds to exonic splicing enhancers and is required for expression of a subset of mRNAs.

Here, we study an insect hnRNP M protein, referred to as Hrp59. Hrp59 is relatively abundant, has a modular domain organization containing three RNA-binding domains, is dynamically recruited to transcribed genes, and binds to premRNA cotranscriptionally. Using the Balbiani ring system of Chironomus, we show that Hrp59 accompanies the mRNA from the gene to the nuclear envelope, and is released from the mRNA at the nuclear pore. The association of Hrp59 with transcribed genes is not proportional to the amount of synthesized RNA, and in vivo Hrp59 binds preferentially to a subset of mRNAs, including its own mRNA. By coimmunoprecipitation of Hrp59-RNA complexes and microarray hybridization against Drosophila whole-genome arrays, we identify the preferred mRNA targets of Hrp59 in vivo and show that Hrp59 is required for the expression of these target mRNAs. We also show that Hrp59 binds preferentially to exonic splicing enhancers and our results provide new insights into the role of hnRNP M in splicing regulation.

Direct interaction with nup153 mediates binding of Tpr to the periphery of the nuclear pore complex.

Tpr is a 267-kDa protein forming coiled coil-dominated homodimers that locate at the nucleoplasmic side of the nuclear pore complex (NPC). The proteins that tether Tpr to this location are unknown. Moreover, the question whether Tpr itself might act as a scaffold onto which other NPC components need to be assembled has not been answered to date. To assess Tpr's role as an architectural element of the NPC, we have studied the sequential disassembly and reassembly of NPCs in mitotic cells, paralleled by studies of cells depleted of Tpr as a result of posttranscriptional tpr gene silencing by RNA interference (RNAi). NPC assembly and recruitment of several nucleoporins, including Nup50, Nup93, Nup96, Nup98, Nup107, and Nup153, in anaphase/early telophase is shown to precede NPC association of Tpr in late telophase. In accordance, cellular depletion of Tpr by RNAi does not forestall binding of these nucleoporins to the NPC. In a search for proteins that moor Tpr to the NPC, we have combined the RNAi approach with affinity-chromatography and yeast two-hybrid interaction studies, leading to the identification of nucleoporin Nup153 as the binding partner for Tpr. The specificity of this interaction is demonstrated by its sensitivity to Tpr amino acid substitution mutations that abolish Tpr's ability to adhere to the NPC and affect the direct binding of Tpr to Nup153. Accordingly, cellular depletion of Nup153 by RNAi is shown to result in mislocalization of Tpr to the nuclear interior. Nup153 deficiency also causes mislocalization of Nup50 but has no direct effect on NPC localization of the other nucleoporins studied in this investigation. In summary, these results render Tpr a protein only peripherally attached to the NPC that does not act as an essential scaffold for other nucleoporins.

The evolutionarily conserved single-copy gene for murine Tpr encodes one prevalent isoform in somatic cells and lacks paralogs in higher eukaryotes.

Vertebrate Tpr and its probable homologs in insects and yeast are heptad repeat-dominated nuclear proteins of M(r) 195,000 to M(r) 267,000 the functions of which are still largely unknown. Whereas two homologs exist in Saccharomyces cerevisiae, it has remained uncertain whether metazoans possess different paralogs or isoforms of Tpr that might explain controversial reports on the subcellular localization of this protein. To address these possibilities, we first determined the sequence and structure of the murine tpr gene, revealing a TATA box-less gene of approximately 57 kb and 52 exons. Southern hybridization of genomic DNA and radiation hybrid mapping showed that murine tpr exists as a single-copy gene on chromosome 1; RNA blotting analyses and EST (expressed sequence tag) database mining revealed that its expression results in only one major mRNA in embryonic and most adult tissues. Accordingly, novel antibodies against the N- and C-terminus of Tpr identified the full-length protein as the major translation product in different somatic cell types; reinvestigation of Tpr localization by confocal microscopy corroborated a predominant localization at the nuclear pore complexes in these cells. Antibody specificity and reliability of Tpr localization was demonstrated by post-transcriptional tpr gene silencing using siRNAs that eliminated the Tpr signal at the nuclear periphery but did not affect intranuclear staining of Tpr-unrelated proteins. Finally, we defined several sequence and structural features that characterize Tpr polypeptides in different species and used these as a guideline to search whole-genome sequence databases for putative paralogs of Tpr in higher eukaryotes. This approach resulted in identification of the Tpr orthologs in Arabidopsis thaliana and Caenorhabditis elegans, but also in the realization that no further paralogs of Tpr exist in several metazoan model organisms or in humans. In summary, these results reveal Tpr to be a unique protein localized at the nuclear periphery of the somatic cell in mammals.