Functional Mapping of Destabilizing Elements in the Protein-coding Region of the Drosophila fushi tarazumRNA

Abstract

The instability of the fushi tarazu (ftz) mRNA is essential for the proper development of the Drosophila embryo. Previously, we identified a 201-nucleotide instability element (FIE3) in the 3 untranslated region (UTR) of the ftz mRNA. Here we report on the identification of two additional elements in the protein-coding region of the message: the 63-nucleotide-long FIE5-1 and the 69-nucleotide-long FIE5-2. The function of both elements was position-dependent; the same elements destabilized RNAs when present within the coding region but did not when embedded in the 3 UTR of the hybrid mRNAs. We conclude that ftz mRNA has three redundant instability elements, two in the protein-coding region and one in the 3 UTR. Although each instability element is sufficient to destabilize a heterologous mRNA, the destabilizing activity of the two 5-elements depended on their position within the message.Drosophila embryonic development depends on the precise temporal and spatial expression of maternal and zygotic pattern-forming genes (1). Maternal pattern-forming genes are transcribed during oogenesis, and their mRNA abundance decreases rapidly in the early embryo. Moreover, many mRNAs encoded by zygotic pattern-forming genes undergo dramatic changes in abundance and spatial distribution during early embryogenesis. To achieve these rapid changes, especially for rapid down-regulation, transcriptional control alone is insufficient, and regulation at the level of mRNA stability is essential. For instance, the maternal bicoid mRNA is completely stable during the first 2 h of embryogenesis but is rapidly destabilized at cellularization of the blastoderm (2). As discussed in the following text, the zygotic fushi tarazu (ftz) mRNA is one of the most unstable eukaryotic mRNAs known. Given that most mRNAs in the Drosophila embryo are constitutively stable (30), the question arises how selected mRNAs in the same embryo cytoplasm are targeted for degradation. Recognition of the targeted RNAs by the RNA degrading machinery must involve cis-acting sequences. These sequences are the focus of the present study.ftz is a member of the pair-rule class of segmentation genes and one of the best characterized early zygotic genes. In early embryos ftz mRNA is detected only from about 1.5 to 4.5 h after fertilization. When first expressed, ftz mRNA is uniformly distributed through the embryo (4). As development progresses, its distribution first becomes restricted to a region comprising from 15 to 65% of egg length, then to four broad bands, and finally to seven narrow stripes that encircle the embryo (4 -6). The seven stripes are short-lived, and no ftz mRNA is detected by 5 h after fertilization. This rapid change of expression pattern and formation of stripes in a short time span can be attributed to the termination of transcription in interstripe regions coupled with rapid mRNA turnover. The need for rapid mRNA turnover is emphasized by the fact that the FTZ protein activates its own transcription in a positive feedback loop (7). Thus, it is impor...

Drosophila Maternal Hsp83 mRNA Destabilization Is Directed by Multiple SMAUG Recognition Elements in the Open Reading Frame

“…For example, the R-H-R transcript could be unstable due to loss of such elements. rpA1 has been used previously as a backbone with which to map instability elements within the bicoid and fushi tarazu mRNAs (17,19,26,34). Since the bicoid and fushi tarazu instability elements function in the presence of the rpA1 5Ј UTR and/or ORF, we favor-and subsequent experiments presented below support-the interpretation that the Hsp83 mRNA contains instability elements.…”

Regulation and function of maternal mRNA destabilization during early Drosophila development

“…Other instability elements have been identified in the 5 0 -UTRs of yeast sdh1, sdh2, suc2, and ppr1 mRNAs, and in the ORFs of mammalian c-fos, c-myc, manganese superoxide dismutase (MnSOD), plasminogen activator inhibitor type 2 (pai-2), and interferon-b (ifn-b) mRNAs, yeast mat1a mRNA, and Drosophila fushi tarazu (ftz) mRNA (Shyu et al, 1989;Wisdom and Lee, 1991;Caponigro et al, 1993;Pierrat et al, 1993;Cereghino et al, 1995;Davis et al, 2001;Ito and Jacobs-Lorena, 2001;Tierney and Medcalf, 2001;de la Cruz et al, 2002;Paste et al, 2003). Several of the transcripts that contain elements in their ORFs also contain instability elements in their 3 0 -UTR (c-fos, c-myc, ifn-b, and pai-2 contain AREs while ftz contains a ftz instability element 3 [FIE3]).…”

“…In addition to the identification of SREs and NREs as cis ‐elements that direct maternal mRNA degradation, the mapping of instability elements in bcd and ftz mRNAs using transgenic reporter constructs has revealed additional elements that control mRNA instability in early embryos. A BIE maps to the first 43 nt of the bcd ‐3′‐UTR and is distinct from the NRE, while ftz mRNA contains three instability elements: FIE5–1, FIE5–2, and FIE3 (Riedl and Jacobs‐Lorena, 1996; Surdej and Jacobs‐Lorena, 1998; Fontes et al, 2001; Ito and Jacobs‐Lorena, 2001). Both BCD and FTZ proteins are critical for the proper patterning of the embryo and thus their precise temporal and spatial expression warrants tight regulation, presumably via multiple regulatory elements.…”

Regulatory sequence responsible for insulin destabilization of cytochrome P450 2B1 (CYP2B1) mRNA

“…Whereas most of the sequences involved in the regulation of mRNA stability were identified in the 3 -UTR, a few cases of regulatory sequences in the open reading frame have been reported in the mRNA of human c-fos, c-myc and interferon-β [27][28][29], mouse β-tubulin [30] and Saccharomyces cerevisiae (baker's yeast) PPR1 transcripts [31]. Moreover, two destabilizing elements in the protein-coding region of fushi tarazu mRNA are essential for Drosophila embryogenesis [32].…”

“…Complexes of cytoplasmic proteins from untreated Fao cells on full-length 32 P-CYP2B1 mRNAThe reaction was carried out in a final volume of 20 µl containing 10 5 c.p.m. of full-length32 P-CYP2B mRNA (lanes 1-6) and 40 µg of cytoplasmic proteins from Fao cells (lanes 3-6). For competition experiments, 3 µg of antisense oligonucleotides (ASns, lane 4; AS5-2B, lane 5;…”