Suppressor of sable, a Putative RNA-Processing Protein, Functions at the Level of Transcription

Abstract

The Drosophila melanogaster su(s) gene product negatively regulates the expression of mutant alleles with transposon insertions in the 5-transcribed region by an unknown mechanism. We have investigated here su(s) function through in vivo structure-function analysis, heterologous reporter gene assays, and in vivo transcriptional induction experiments. We have shown that mutations of two arginine-rich motifs (ARMs), an acidic region, or two CCCH zinc fingers affect the ability of Su(s) to downregulate the expression of an insertion mutant allele and to autoregulate genomic su(s) transgenes. Using yeast and HeLa cell assays, we found that, when tethered to the promoter region, the N-and C-terminal regions of Su(s) can repress reporter gene expression, and all three motifs, but most significantly the ARMs, contribute to the repression activity. Finally, we showed that, in vivo, Su(s) inhibits the transcriptional induction of a transgene with an insertion in the first exon but does not affect induction of a similar transgene with a consensus 5 splice site near the upstream boundary of the insertion. Together, these results reveal a link between Su(s), transcription, and pre-mRNA processing.In eukaryotes, control mechanisms operate at various stages of gene expression to generate specific and dynamic patterns of protein accumulation. Efficient mRNA production depends on complex interactions between a large number of components that regulate pre-mRNA synthesis and processing in time and space. The current understanding of eukaryotic gene expression regulation has been derived primarily from experiments performed in relatively simple systems, e.g., in vitro, cultured cell lines or single-cell eukaryotes. Although much progress has been made and important insights have come from these studies, the view of how various mRNA metabolic pathways are coordinated and integrated under normal physiological conditions and during development is incomplete. The analysis of genetic regulatory processes in model eukaryotic organisms such as Drosophila melanogaster can contribute to the understanding of more complex aspects of regulation that cannot be studied in simpler model systems.Several pre-mRNA transcription and processing regulators in D. melanogaster have been identified by virtue of the fact that mutations in genes encoding these proteins can suppress or enhance the effects of transposon-induced mutations. One such gene is su(s). It encodes a 150-kDa nuclear protein that binds to RNA in vitro (28,39 ϩ than su(s) mutant flies. However, a v k derivative with a consensus (instead of a cryptic) 5Ј splice site at the upstream boundary of the insertion produces the same, high level of v RNA in the presence or absence of su(s) product (15). These results suggest that the efficiency of splicing complex assembly in the 5Ј region can influence Su(s)-mediated regulation of v k RNA levels. Since modulation of RNA levels by Su(s) depends on transcribed sequences, our lab and others concluded that Su(s) most likely influences RNA stability...

Drosophila Suppressor of Sable Protein [Su(s)] Promotes Degradation of Aberrant and Transposon-Derived RNAs

“…However, the mechanism by which Su(s) regulates RNA accumulation has not been previously established. The prior studies were consistent with possible roles for this protein in pre-mRNA splicing, RNA degradation, or transcription (15,17,27), and direct evidence for a role in any one of these processes has been lacking.…”

“…We previously made Su(s) derivatives with alterations in the RNA-binding domains and generated transformant lines that stably express the mutant proteins in the su(s) null mutant background (27). To test whether these domains are required for the association of Su(s) with the 87C region, we performed immunofluorescence analysis on chromosomes from transgenic larvae expressing Su(s) derivatives with either a deletion of both arginine-rich motifs [Su(s) ⌬ARM ] or with both zinc fingers mutated [Su(s) ZF ].…”

“…The wild-type stock used for RNA analysis was yw, the progenitor of su(s) R39 . Transformant lines carrying the su(s) wild-type, zinc finger, and arginine-rich motif mutant transgenes in the su(s) R39 background were generated previously (27). In the arginine-rich motif mutant, sequences encoding amino acids (aa) 151 to 168 and aa 269 to 294 have been deleted.…”

“…Salivary glands were dissected from wandering third-instar larvae. The procedures used for preparing chromosome squashes and antibody staining have been described previously (27). DAPI staining was performed as described by Weeks et al (51).…”

“…The CCCH zinc fingers of Tis11 bind AU-rich instability elements in the 3Ј untranslated region (UTR) of its target mRNAs (6). Both the arginine-rich motifs and the zinc fingers are important for the inhibitory activity of Su(s) (27). The RNA-binding activity of Su(s) together with the observation that this protein localizes to distinct sites on Drosophila larval salivary gland polytene chromosomes (34) suggest that Su(s) associates with nascent transcripts and acts cotranscriptionally.…”