Secondary structure of the 3′ UTR of bicoid mRNA
Introduction
The importance of the bcd mRNA 3′ UTR as a repository for signals determining bcd mRNA localization process, polyadenylation, translational regulation and time-triggered degradation has become apparent through abundant recent studies (reviewed in [1], [2]). The maternal bicoid gene plays a crucial role in the early development of Drosophila melanogaster. bcd mRNA is transcribed in nurse cells and transported to the oocyte during early stages of oocyte differentiation. Then, it follows a multi-step localization process until its anchoring at the anterior pole of the embryo. The translation of bcd mRNA is kept silenced until the anchoring step is activated, then producing a steep concentration gradient of Bicoid that establishes head and thoracic development (reviewed in [3]). bcd mRNA is transcribed in nurse cells and transported to the oocyte during early stages of oocyte differentiation. The 3′ UTR encompasses around 800 nucleotides and is organized into domains (I–V) that contain functional elements. Domains IV and V together can direct the earliest phase of transport, and are sufficient for normal localization until embryogenesis. Distinct determinants were identified to be important for the Exuperentia dependant step that governs localization of bcd mRNA at the anterior margin of the oocyte from stage 6 [4], [5]. In the latest stages, dimerization through intermolecular base pairing between two complementary loops of domain III, the hairpin loop IIIb and the interior loop IIIa, plays an essential role in the localization process in the embryo [6], [7]. The intermolecular interactions, referred as ‘hand-by-arm’ interactions (by opposition to ‘kissing’ loop interactions) were demonstrated by standard disruption/restoration mutation experiments [6], [7]. Dimerization was shown to be driven by a two-step mechanism, involving initiation and stabilization [7]. Recently, we showed that dimerization is initiated by a more limited number of intermolecular base pairs than expected, leading to a reversible single ‘hand-by-arm’ interaction (‘open’ dimer) [8]. This reversible open dimer is then converted into an irreversible dimer, involving a double ‘hand-by-arm’ interaction (‘closed dimer’). This stabilization step is probably driven by a kinetically controlled mechanism [8].
In the present paper, we investigated the secondary structure of the full length 3′ UTR of the bcd mRNA using a variety of chemical and enzymatic structural probes. Our data allowed us to give a detailed description of the 3′ UTR of the bcd mRNA and its organization into five well-defined and independent domains. This study was carried out on both monomeric and dimeric species, in order to get information about the structural impact of dimerization. Experimental information was provided on bulged nucleotides, interior loops, possible non-canonical base pairs and branching regions. Our results will be discussed on the light of present knowledge of the literature.
Section snippets
Buffers and plasmid construction
Buffer D1 (Hepes 50 mM (pH 7.5), 450 mM KCl, 5 mM MgCl2); Buffer M1 (Hepes 50 mM (pH 7.5), 50 mM KCl, 0.1 mM MgCl2); Buffer D2 (50 mM sodium borate (pH 8), 450 mM KCl, 5 mM MgCl2); Buffer M2 (50 mM sodium borate (pH 8), 50 mM KCl, 0.1 mM MgCl2); Buffer D3 (sodium cacodylate 50 mM (pH 7.5), 450 mM KCl, 5 mM MgCl2); Buffer M3 (sodium cacodylate 50 mM (pH 7.5), 50 mM KCl, 0.1 mM MgCl2). Plasmid 875′ used in this study has been described in [7].
RNA synthesis and dimerization
RNA 875′ was synthesized by T7 RNA polymerase
Experimental strategy: chemical and enzymatic probing and derivation of the secondary structure model
The conformation of RNA 875′ was probed using a variety of enzymatic and chemical probes (for details, see [11], [12]). RNase T1 was used to map unpaired guanines, RNase T2 unpaired adenines and to a lesser extend unpaired uridines, and RNase V1 double-stranded or stacked regions. Watson–Crick positions were tested with DMS (A(N1) and C(N3)), and CMCT (G(N1) and U(N3)). The effects induced by intermolecular RNA–RNA interaction were tested under two salt conditions, either preventing or favoring
Discussion
bcd 3′ UTR contains cis-acting elements involved in a variety of functions: mRNA localization, polyadenylation, translational regulation and time-triggered degradation (reviewed in [1], [2]). RNA probing allowed us to get the first experimental insight into the folding that frames bcd 3′ UTR. Our data confirm the partition of the bcd 3′ UTR into well-defined and structured domains. One prominent result is the rather unexpected high degree of flexibility regarding domains orientation, resulting
Acknowledgments
We thank P. Romby for helpful comments on the manuscript, C. Wagner, F. Brulé and C. Isel for valuable discussions. We thank Christian Massire and Eric Westhof for help with the COSEQ program. This work was supported by the CNRS.
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