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

Abstract

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.

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

“…Given that Hrp59 is an hnRNP protein that binds to nascent mRNPs cotranscriptionally (Kiesler et al, 2005) and given that Hrp59 interacts with Rrp4, Ski6/Rrp41, and Rrp6, we propose that Hrp59 mediates the association of the exosome to the nascent premRNPs. Several exosome subunits, including Rrp4, contain RNA-binding domains and can, in principle, bind RNA directly.…”

“…From these experiments we conclude that Rrp4 interacts, directly or indirectly, with Hrp59 in vivo. Hrp59 associates cotranscriptionally with transcripts from many different genes, as shown by the fact that an anti-Hrp59 antibody stains many bands in the polytene chromosomes ( Figure 6E and Kiesler et al, 2005). The nucleoli appear as dark spots in the Hrp59-stained cells (Nu in Figure 6E), which indicates that Hrp59 is not present in the nucleoli.…”

“…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).…”

“…The anti Pol-II CTD antibody 4H8 (ab5408) was from Abcam (Cambridge, MA), and the negative control anti-IgG antibody (Z0456) was from DakoCytomation (Carpenteria, CA). The following antibodies were used for Western blotting: polyclonal anti-Hrp59 (Kiesler et al, 2005), polyclonal against Rrp6 (kindly provided by E. Andrulis, Case Western Reserve University School of Medicine, Cleveland, OH), mAb 10:3G1 against Chironomus Hrp36/sqd (Kiseleva et al, 1997), mAb Bj6 against NonA (kindly provided by H. Saumweber, Humboldt University, Berlin, Germany), and mAb against PEP (kindly provided by S. Amero, Loyola University Chicago, Maywood, IL). The mAb Y12 was used for the detection of snRNP proteins (Lerner et al, 1981).…”

“…For experiments of RNA digestion, the chromosomes were incubated with 100 g/ml RNase A for 30 min and washed with TKM before being fixed and processed as above. Immunofluorescent staining of salivary glands was carried out as described by Kiesler et al (2005). …”