TheDrosophilaSuppressor of sable Protein Binds to RNA and Associates with a Subset of Polytene Chromosome Bands

Abstract

Mutations of the Drosophila melanogaster suppressor of sable [su(s)] gene, which encodes a 150-kDa nuclear protein [Su(s)], increase the accumulation of specific transcripts in a manner that is not well understood but that appears to involve pre-mRNA processing. Here, we report biochemical analysis of purified, recombinant Su(s) [rSu(s)] expressed in baculovirus and in Escherichia coli as maltose binding protein (MBP) fusions and immunocytochemical analysis of endogenous Su(s). This work has shown that purified, baculovirus-expressed rSu(s) binds to RNA in vitro with a high affinity and limited specificity. Systematic evolution of ligands by exponential enrichment was used to identify preferred RNA targets of rSu(s), and a large proportion of RNAs isolated contain a full or partial match to the consensus sequence UCAGUAGUCU, which was confirmed to be a high-affinity rSu(s) binding site. An MBP-Su(s) fusion protein containing the N-terminal third of Su(s) binds RNAs containing this sequence with a higher specificity than full-length, baculovirus-expressed rSu(s). The consensus sequence resembles both a cryptic 5 splice site and a sequence that is found near the 5 end of some Drosophila transcripts. Immunolocalization studies showed that endogenous Su(s) is distributed in a reticulated pattern in Drosophila embryo and salivary gland nuclei. In salivary gland cells, Su(s) is found both in the nucleoplasm and in association with a subset of polytene chromosome bands. Considering these and previous results, we propose two models to explain how su(s) mutations affect nuclear pre-mRNA processing.The Drosophila melanogaster suppressor of sable [su(s)] gene is one of a group of recessive suppressors that, when mutated, modify phenotypes associated with transposon insertion mutations at particular genes (reviewed in reference 38). Upon further examination at the molecular level, it has become apparent that these suppressor genes encode proteins that function more generally in regulating specific aspects of transcription and pre-mRNA processing. For example, suppressor of white-apricot [su(w a )] encodes an alternative splicing factor (47), and the protein encoded by suppressor of forked [su(f)] is homologous to a human polyadenylation factor subunit (40). The su(s) gene encodes a 150-kDa nuclear protein [Su(s)] with relatively little homology to other known proteins. Voelker et al. (43) identified a region within su(s) encoding 77 amino acids that is 28.6% identical to a region in the Drosophila U1 70K protein (23). This region of U1 70K is thought to be an Arg-Ser domain, which is found in numerous splicing factors (2, 11). Although the corresponding region of Su(s) contains two Argrich clusters, the characteristic Arg-Ser dipeptides are virtually absent from Su(s). Thus, the observed similarity between Su(s) and U1 70K may or may not be functionally significant. Voelker et al. (43) also identified a region of Su(s) with weak homology to an RNA recognition motif (RRM). More recently, this region of su(s) was reexamined, ...

Similar but distinct effects of the tristetraprolin/TIS11 immediate-early proteins on cell survival

“…Precedents set by various other Cys 3 His zinc ®nger proteins predict that TTP/TIS11 proteins are likely to have RNA-associated functions (Barabino et al, 1997;Guedes and Priess, 1997;Murray et al, 1997;Rudner et al, 1998;Tabara et al, 1999;Tronchere et al, 1997), and evidence indicates that TTP can bind and in¯uence the stability of TNF-a and other ARE-containing cytokine mRNAs (Carballo et al, 1998;Lai et al, 1999). We have determined that TTP/TIS11 proteins in¯uence cell growth and survival mechanisms, suggesting that they may also act on mRNAs that are involved in those pathways.…”

“…Each of these three proteins, which we refer to as the TTP/TIS11 proteins, is induced rapidly by multiple dierent agents, although they vary with respect to their baseline mRNA levels and induction by particular stimuli (Corps and Brown, 1995;Gomperts et al, 1992;Varnum et al, 1991). Other Cys 3 His zinc ®nger proteins are involved in mRNA binding, cleavage, or processing, or have been implicated in post-transcriptional gene regulation (Barabino et al, 1997;Batchelder et al, 1999;Guedes and Priess, 1997;Murray et al, 1997;Rudner et al, 1998;Tabara et al, 1999;Tronchere et al, 1997) predicting that TTP/TIS11 proteins also perform mRNA-associated functions.…”

Suppressor of sable [Su(s)] and Wdr82 down-regulate RNA from heat-shock-inducible repetitive elements by a mechanism that involves transcription termination

“…Thus, endonucleolytic cleavage could be part of the mechanism by which Su(s)-Wdr82 induces termination as well. As an RNA-binding protein (Murray et al 1997;Turnage et al 2000), Su(s) might recognize specific RNA sequences within the regulatory regions that we have delineated and promote termination and RNA degradation by cleaving the RNA or interacting transiently with another protein that performs this function.…”

“…Su(s) of Drosophila melanogaster is a nuclear RNA-binding protein (Voelker et al 1991;Murray et al 1997) that inhibits the accumulation of certain aberrant mRNAs. The best characterized targets of this regulation are mutant alleles of protein-coding genes that have transposable elements (TEs) inserted a short distance downstream from the transcription start site (Fridell et al 1990;Geyer et al 1991;Kim et al 1996).…”

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

“…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).…”