Post-transcriptional Regulation

Regulation of RNA metabolism plays a major role in controlling gene expression during developmental processes. The Drosophila RNA-binding protein Held out wing (HOW), regulates an array of developmental processes in embryonic and adult growth. The primary sequence and secondary structural requirements for the HOW response element (HRE) has been characterized; this site (ACUAA) is necessary and sufficient for HOW binding. Based on this analysis, the Drosophila TGFß homolog, dpp, was identified as a novel direct target for HOW negative regulation in the wing imaginal disc. The binding of the repressor isoform HOW(L) to the dpp 3' untranslated region (UTR) leads to a reduction of GFP-dpp3'UTR reporter levels in S-2 cells, in an HRE site-dependent manner. Moreover, co-expression of HOW(L) in the wing imaginal disc with a dpp-GFP fusion construct led to a reduction in DPP-GFP levels in a dpp-3'UTR-dependent manner. Conversely, a reduction of the endogenous levels of HOW by targeted expression of HOW-specific double-stranded RNA led to a corresponding elevation in dpp mRNA level in the wing imaginal disc. Thus, by characterizing the RNA sequences that bind HOW, a novel aspect has been demonstrated of regulation, at the mRNA level, of Drosophila DPP (Israeli, 2007).

It has been shown that HOW binds directly to the 3'UTR of stripe. To characterize the HOW-binding sites further, the stripe 3'UTR 1.2 kb sequence was truncated into smaller fragments, which were individually transcribed in vitro and labeled with biotin. These fragments were tested for HOW binding by adding in vitro-translated HOW tagged with hemagglutinin (HA) to the biotin-labeled RNA followed by precipitation of the RNA complexes using avidin-conjugated magnetic beads. The presence of HOW on the beads was then tested by western blot analysis using anti-HA antibodies. As a control for non-specific binding, a mutant HOW variant (HOWm) was used, that carries a mis-sense mutation in the KH domain exchanging arginine at position 185 to cysteine, mimicking the severe loss-of-function howe44 allele. HOWm does not exhibit RNA-binding activity. This analysis allowed selection of two HOW-binding fragments (a and b) in which the sequence ACUAA, which was similar, but not identical, to the GLD-1 hexanucleotide-binding site in tra-2, was identified. In fragment a, there are three repeats of this sequence, and fragment b contains one such sequence (Israeli, 2007).

It is concluded that the sequence ACUAA represents the primary HRE. Importantly, one of the HRE sequences (at position 766) is conserved in the 3'UTR of stripe in Drosophila pseudoobscura. Moreover, three repeats of the pentamer AAUAA (which also binds HOW, but to a lesser extent) were identied that are conserved between the two Drosophila species. Thus the HOW-binding site NA(C>A)UAA closely resembles that of STAR proteins from other species, although it is not identical. The binding of HOW was studied in the context of the entire stripe 3'UTR, and it was demonstrated that deletion of these four sites indeed abrogates the responsiveness of the stripe 3'UTR to HOW (Israeli, 2007).

Because a pentanucleotide sequence would be relatively abundant within the 3'UTRs of many RNAs, it was suspected that additional restrictions might exist in addition to the primary sequence ACUAA. Analysis of the distinct HOW-binding sites in the stripe 3'UTR using the Mfold program showed that high-affinity binding for HOW occurs when the binding site (ACUAA) is included within a single-stranded loop. However, secondary-structure predictions of large RNA fragments (larger than 30-40 nucleotides) using the Mfold program resulted in numerous alternatives. To test whether a loop secondary structure is essential for the binding of HOW, HRE-containing loops of distinct sizes were constructed, fused to the 3' end of the stripe 3'UTR fragment (1-225), which does not bind HOW. It was found that single-stranded loops that are larger than 12 nucleotides and contain a single HRE site exhibit significant binding, whereas loops smaller than 12 nucleotides did not exhibit specific binding to HOW. Presumably, these loops are too small to allow this binding (Israeli, 2007).

Structural studies helped identify a novel HOW target, namely dpp mRNA, in the wing imaginal disc. Normally, the repressor isoform of HOW, HOW(L), reduces dpp mRNA levels in the wing imaginal disc and in the pupal wing, leading to reduced DPP protein levels during the establishment of the anteroposterior axis, and later during wing vein formation. Presumably, in the absence of HOW(L), higher DPP levels at the source would alter the overall shape of the DPP gradient, thus expanding the Spalt expression domain. The phenotype of ectopic veins obtained by continuous expression of HOW(L) dsRNA in the pupal wings supports an additional role for HOW(L) in repressing dpp mRNA at later stages of wing development (Israeli, 2007).

The sensitivity of the embryo to DPP levels has been demonstrated by the DPP haplo-insufficient phenotype. This sensitivity is also exhibited in the wing imaginal disc by the observation that endogenous dpp can be replaced by UAS-GFP-dpp driven by dpp-gal4 only at low temperatures (16°C or 19°C), at which the Gal4 protein is significantly less active. Because the responsiveness of the cells to DPP levels is highly sensitive, it is necessary to tightly regulate the levels of DPP protein; for example, by constitutive reduction of its mRNA levels in DPP-secreting cells by the HOW(L) protein (Israeli, 2007).

decapentaplegic: Biological Overview | Evolutionary Homologs | Transcriptional regulation | Protein Interactions | Targets of activity | Developmental Biology | Effect of mutation | References

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