Mutations in the hrp48 Gene, Which Encodes a Drosophila Heterogeneous Nuclear Ribonucleoprotein Particle Protein, Cause Lethality and Developmental Defects and Affect P-Element Third-Intron Splicing In Vivo

Abstract

The Drosophila melanogaster hnRNP protein, hrp48, is an abundant heterogeneous nuclear RNA-associated protein. Previous biochemical studies have implicated hrp48 as a component of a ribonucleoprotein complex involved in the regulation of the tissue-specific alternative splicing of the P-element third intron (IVS3). We have taken a genetic approach to analyzing the role of hrp48. Mutations in the hrp48 gene were identified and characterized. hrp48 is an essential gene. Hypomorphic mutations which reduce the level of hrp48 protein display developmental defects, including reduced numbers of ommatidia in the eye and morphological bristle abnormalities. Using a P-element third-intron reporter transgene, we found that reduced levels of hrp48 partially relieve IVS3 splicing inhibition in somatic cells. This is the first direct evidence that hrp48 plays a functional role in IVS3 splicing inhibition.The hnRNP (heterogeneous nuclear ribonucleoprotein particle) proteins are abundant eukaryotic nuclear RNA-binding proteins that bind nascent transcripts (heterogeneous nuclear RNAs or pre-mRNAs) as they emerge from the transcriptional machinery. hnRNP complexes from vertebrates, where there are over 20 abundant, highly conserved, nuclear hnRNP proteins, designated A to U (10), have been extensively studied. These proteins are important for the packaging, processing, and intracellular transport of cellular mRNAs (5). hnRNP proteins are also found in invertebrates, such as Drosophila melanogaster. In Drosophila, several hnRNP proteins have been isolated by immunopurification and characterized by molecular cloning (20,21). There are at least 10 major hnRNP proteins in Drosophila, the most abundant being hrp36, hrp40, and hrp48. These proteins have been shown to associate with the majority of nascent transcripts (19,21).All the abundant Drosophila heterogeneous nuclear RNAbinding proteins characterized so far are similar in structure to the vertebrate A and B hnRNP proteins, containing two Nterminal RBD-type RNA-binding domains and a glycine-rich carboxyl-terminal domain (21). The RBD (or RNP-CS) type is a distinct class of RNA recognition motif (5). The RBD domains are necessary for RNA binding, and the glycine-rich region is thought to mediate RNA-binding, RNA-RNA-annealing, or protein-protein interactions (10). Proteins with these motifs are referred to as 2ϫ RBD-Gly proteins (5). Alignment of RNA-binding domains from evolutionarily diverse 2ϫ RBD-Gly proteins reveals a high degree of conservation of these domains among members of this protein family and establishes a specific consensus sequence for 2ϫ RBD-Gly proteins (5).Early evidence for a role for hnRNP proteins in pre-mRNA splicing came from antibody inhibition experiments implicating the mammalian hnRNP C protein as a splicing factor (9). More recent evidence for the role of an hnRNP protein in pre-mRNA splicing was obtained through the purification of an activity that influences alternative 5Ј splice site selection in vitro and its identification as hnRNP A1 (22). By u...

Hrb27C, Sqd and Otu cooperatively regulategurkenRNA localization and mediate nurse cell chromosome dispersion inDrosophilaoogenesis

“…Hrb27C contains two RNA recognition motifs (RRMs) (Matunis et al, 1992a) (Fig. 1A) and has been previously studied for its role in the regulation of the tissuespecific inhibition of splicing of the P-element third intron (IVS3) (Siebel et al, 1994;Hammond et al, 1997). As Sqd affects grk RNA localization and translational control, our findings raise the possibility that the splicing of grk is directly linked to localization, or, the model we favor, that Hrb27C has functions in oogenesis other than splicing.…”

“…The following alleles are lethal P-element insertions at various distances from the coding region: hrb27C 10280 (1.5 kb), hrb27C rF680 (1.8 kb), hrb27C k16303 (2.2 kb), hrb27C k10413 (2.2 kb) and hrb27C 02647 (3.3 kb) (Hammond et al, 1997). There are no molecular data on hrb27C k02814 ; hrb27C 377 is an EMS allele produced by Mary Lilly.…”

Long-Term and Short-Term Evolutionary Impacts of Transposable Elements onDrosophila

“…We found that the amino acid sequence divergence between D. melanogaster and D. willistoni orthologs (Clark et al 2007) for most candidate genes is greater than the genome-wide median ( Figure 5). The only two exceptions [Hrb27C (1.7%) and Psi (16.4%)] both have other essential host functions (Hammond et al 1997;Labourier et al 2002;Goodrich et al 2004;Huynh et al 2004;Yano et al 2004;Blanchette et al 2005) and their evolution is expected to be strongly constrained. These observations therefore do not support the scenario of D. melanogaster preadaptation to P elements.…”

Distinct Sets of Adjacent Heterogeneous Nuclear Ribonucleoprotein (hnRNP) A1/A2 Binding Sites Control 5′ Splice Site Selection in the hnRNP A1 mRNA Precursor

“…Using model pre-mRNAs carrying competing 5Ј splice sites, important shifts toward distal 5Ј splice sites can be obtained by the addition of purified or recombinant hnRNP A1 to a HeLa nuclear extract (23,24). The Drosophila hrp48 protein, which is similar to hnRNP A1, is required in collaboration with P-element somatic inhibitor and the U1 snRNP to repress splicing of the P-element pre-mRNA in somatic tissues (25). hnRNP A1 can elicit exon skipping, but not all pre-mRNAs are responsive to variations in the concentration of hnRNP A1 in vitro (26).…”