The Drosophila RNA-binding protein RBP1 is localized to transcriptionally active sites of chromosomes and shows a functional similarity to human splicing factor ASF/SF2.

Abstract

An RNA-binding protein gene (rbpl) from Drosophila melanogaster, encoding an RNA recognition motif and an Arg-Ser rich (RS) domain, has been characterized. The predicted amino acid sequence of rbpl is similar to those of the human splicing factor ASF/SF2, the Drosophila nuclear phosphoprotein SRp55, and the Drosophila puff-associated protein B52. Northern and immunohistochemical analyses showed that rbpl is expressed at all stages in all tissues and that the RBP1 protein is localized to the nucleus. Consistent with a role in mRNA metabolism, indirect immunofluorescence reveals that the RBP1 protein colocalizes with RNA polymerase II on larval salivary gland polytene chromosomes. RBP1 protein made in Escherichia coli was tested for splicing activity using human cell extracts in which ASF has been shown previously both to activate splicing and to affect the choice of splice sites in alternatively spliced pre-mRNAs. In these assays, RBP1 protein, like ASF, is capable of both activating splicing and switching splice site selection. However, in each case, clear differences in the behavior of the two proteins were detected, suggesting that they have related but not identical functions. The general nuclear expression pattern, colocalization on chromosomes with RNA polymerase II, the similarity to ASF/SF2, SRp55, and B52, along with the effect on alternative splicing shown in vitro, suggest that rbpl is involved in the processing of precursor mRNAs.

Distribution of pre-mRNA splicing factors at sites of RNA polymerase II transcription.

“…Where particular cytological methods have been available, splicing factors have been detected at active sites of transcription. On Drosophila polytene chromosomes, SRp55 (B52) and RBP1 (possibly the homolog of SRp20; see Manley and Tacke 1996) were detected at transcriptionally active puffs as well as bands reactive with antibodies against RNA Pol II (Champlin et al 1991;Kim et al 1992). Importantly, a range of immunostaining intensities relative to anti-Pol II was observed in both studies, raising the possibility that these two SR proteins accumulate differently on nascent transcripts in this experimental system.…”

“…This has led to the suggestion that speckles contain the highest concentrations of splicing factors--perhaps serving as storage or assembly sites--and that lower concentrations may be present where splicing occurs (Fakan et al 1984;Fakan 1994;Xing et al 1995;Huang and Spector 1996). Interestingly, SR proteins and splicing snRNPs are detected at active sites of transcription on polytene and lampbrush chromosomes, which are particularly well-suited for cytological observation (Roth et al 1989(Roth et al , 1991Champlin et al 1991;Amero et al 1992;Kim et al 1992;Wu et al 1993;Bauren et al 1996). Although splicing factors accumulate at very large and highly active viral transcription units in infected mammalian cells (cf.…”

A complementary transposon tool kit for Drosophila melanogaster using P and piggyBac

“…Because exons in P elements can disrupt gene function by acting as ectopic splice acceptors 13,14 , we designed the RB vector with an exon from the D. melanogaster gene Rbp1 (ref. 19) as a second splice trap XP and piggyBac elements had comparable numbers of hot spots, although more than twice as many piggyBac inserts were generated.…”

The splicing factors 9G8 and SRp20 transactivate splicing through different and specific enhancers

“…Pre-mRNA splicing requires five small nuclear ribonucleoprotein particles (U1, U2, U4, U5, and U6 snRNPs), as well as a large number of protein factors (see Moore et al+, 1993;Krämer, 1996 for review)+ Selection of the splice sites and branch site, and the precise pairing of the corresponding 59 and 39 splice sites occur via multiple RNA-RNA, RNA-protein and protein-protein interactions+ A number of factors involved in the earliest steps of the spliceosome formation have been extensively studied (Krämer, 1996), in particular a family of related factors, called SR proteins (Fu, 1995;Manley & Tacke, 1996)+ To date, ten SR proteins have been identified in human, with or without known homologs in Drosophila: ASF/SF2, also known as SRp30a (Ge et al+, 1991;Krainer et al+, 1991), SC35, also called PR264 or SRp30b Vellard et al+, 1992), another SRp30 factor, 9G8 (Cavaloc et al+, 1994), SRp20, also RBP1 in Drosophila (Kim et al+, 1992;Zahler et al+, 1992), SRp75 (Zahler et al+, 1993b), SRp40, SRp55, and SRp30c (Screaton et al+, 1995), p54 (Zhang & Wu, 1996), and finally SRp46, a recently identified SR species (Soret et al+, 1998)+ All these factors contain at their amino terminus one or two copies of an RNA binding domain (RBD) including the conserved RNP-1 and RNP-2 submotifs (Birney et al+, 1993)+ At their carboxy terminus, they contain a region rich in arginine (R) and serine (S) residues, with extensive repetition of R-S dipeptides (the RS domain)+ Several lines of evidence indicate that the SR proteins play an important role at several stages of the splicing reaction+ First, it has been shown that all SR proteins can complement a splicing-deficient S100 cytoplasmic extract, raising the possibility that these factors may be interchangeable in the splicing reaction (Fu, 1995)+ Secondly, SR proteins are required to stabilize the binding of U1 snRNP to the 59 splice site (Kohtz et al+, 1994), and to form the early E complex (Stacknis & Reed, 1994), in agreement with the fact that the interaction of the SR proteins with the pre-mRNA is a prerequisite for the other steps of the spliceosome assembly (Fu, 1993;)+ Finally, SR proteins are involved in the recruitment of the U4/ U6-U5 tri-snRNP to the spliceosome (Roscigno & Garcia-Blanco, 1995)+ Involvement of SR proteins in alternative splicing, specifically their ability to influence in vitro the selection of alternative 59 splice sites in a ...…”