Book of Abstracts: Albany 2007
June 19-23 2007
Investigating the role of the SECIS RNA apical loop in selenoprotein synthesis
In eukaryotes, incorporation of the 21st amino acid selenocysteine requires the SECIS element, a stem-loop structure residing in 3' untranslated regions of selenoprotein mRNAs, and several RNA and protein trans-acting factors. Among those that have been characterized so far in great detail are (i) SBP2, the specific SECIS-binding protein; (ii) the specialized translation elongation factor eFSec; (iii) the Sec-tRNA; (iv) ribosomal protein L30. The SECIS hairpin contains only two conserved functional motifs: (1) a quartet of non-Watson-Crick base pairs with a central tandem of sheared G.A/A.G base pairs, and (2) an AAA sequence in the apical loop. The sheared base pairs form a recurrent RNA motif found in many RNAs, the kink-turn, that is sufficient for binding to a C-terminal version of SBP2. The function of the AAA sequence is still unknown, although it has been shown to be necessary for Sec incorporation in vivo. Having expressed and purified the recombinant full-length (FL) version of SBP2, we asked whether it would be a candidate for contacting the stretch of As.
Thorough mutagenesis of the apical loop (various combinations changing the wt sequence or dramatically shortening it by introduction of a UNCG stable tetraloop) and truncated versions of SBP2 were performed, followed by gel shift assays and enzymatic and chemical footprinting. To map the putative contacts, we also designed and constructed a SECIS RNA containing a single photoactivatable 4-thio-uridine residue (instead of a residing U) adjacent to the conserved AAA sequence. None of the experiments enabled us to provide evidence that FL-SBP2 interacts specifically with the SECIS apical loop. In agreement with this finding is our and earlier observations from others that both the full length SBP2 and its C-terminal fragment can equally stimulate selenocysteine incorporation in reticulocyte lysate. Pursuing our investigations, we asked whether eFSec could bind the AAA sequence, directly or through an SBP2-mediated interaction. Upon irradiation of the triple complex SBP2-eFSec-SECIS, the apical loop was able to crosslink to both proteins. However, competition experiments did not confirm the specificity of this interaction.
The AAA sequence is crucial for selenoprotein synthesis in reticulocyte lysates and in vivo. This conserved element might therefore interact with another, as yet unidentified component of the selenoprotein-specialized or general translation machinery. Alternatively, the AAA-dependent reactions could reflect fast kinetic interactions not detected under our in vitro conditions.
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