Functional Characterization of Alternatively Spliced Human SECISBP2 Transcript Variants

Signal Transduction Laboratory, Queensland Institute of Medical Research, Herston, Queensland, Australia.
Nucleic Acids Research (Impact Factor: 9.11). 12/2008; 36(22):7192-206. DOI: 10.1093/nar/gkn829
Source: PubMed


Synthesis of selenoproteins depends on decoding of the UGA stop codon as the amino acid selenocysteine (Sec). This process requires the presence of a Sec insertion sequence element (SECIS) in the 3'-untranslated region of selenoprotein mRNAs and its interaction with the SECIS binding protein 2 (SBP2). In humans, mutations in the SBP2-encoding gene Sec insertion sequence binding protein 2 (SECISBP2) that alter the amino acid sequence or cause splicing defects lead to abnormal thyroid hormone metabolism. Herein, we present the first in silico and in vivo functional characterization of alternative splicing of SECISBP2. We report a complex splicing pattern in the 5'-region of human SECISBP2, wherein at least eight splice variants encode five isoforms with varying N-terminal sequence. One of the isoforms, mtSBP2, contains a mitochondrial targeting sequence and localizes to mitochondria. Using a minigene-based in vivo splicing assay we characterized the splicing efficiency of several alternative transcripts, and show that the splicing event that creates mtSBP2 can be modulated by antisense oligonucleotides. Moreover, we show that full-length SBP2 and some alternatively spliced variants are subject to a coordinated transcriptional and translational regulation in response to ultraviolet type A irradiation-induced stress. Overall, our data broadens the functional scope of a housekeeping protein essential to selenium metabolism.

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    • "Since we have identified both of these motifs in lizard (Anolis carolinensis) SBP2, we speculate that the LSAD15-26 and PFVQ44-56 motifs were lost in the lineages giving rise to fish and amphibians. In addition, Papp et al [16] recently identified several splice variants of human SBP2, the most abundant of which encodes a mitochondrial targeted protein (mtSBP2). Interestingly, this splice isoform lacks both the LSAD15-26 and PFVQ44-56 motifs but retains the SBP2-specific motifs presented below. "
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    ABSTRACT: The co-translational incorporation of selenocysteine into nascent polypeptides by recoding the UGA stop codon occurs in all domains of life. In eukaryotes, this event requires at least three specific factors: SECIS binding protein 2 (SBP2), a specific translation elongation factor (eEFSec), selenocysteinyl tRNA, and a cis-acting selenocysteine insertion sequence (SECIS) element in selenoprotein mRNAs. While the phylogenetic relationships of selenoprotein families and the evolution of selenocysteine usage are well documented, the evolutionary history of SECIS binding proteins has not been explored. In this report we present a phylogeny of the eukaryotic SECIS binding protein family which includes SBP2 and a related protein we herein term SBP2L. Here we show that SBP2L is an SBP2 paralogue in vertebrates and is the only form of SECIS binding protein in invertebrate deuterostomes, suggesting a key role in Sec incorporation in these organisms, but an SBP2/SBP2L fusion protein is unable to support Sec incorporation in vitro. An in-depth phylogenetic analysis of the conserved L7Ae RNA binding domain suggests an ancestral relationship with ribosomal protein L30. In addition, we describe the emergence of a motif upstream of the SBP2 RNA binding domain that shares significant similarity with a motif within the pseudouridine synthase Cbf5. Our analysis suggests that SECIS binding proteins arose once in evolution but diverged significantly in multiple lineages. In addition, likely due to a gene duplication event in the early vertebrate lineage, SBP2 and SBP2L are paralogous in vertebrates.
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    • "Several possibilities exist that would pertain to a more complex selenoprotein synthesis mechanism in higher eukaryotes: (i) the N-terminal extension could participate in fine-tuning selenoprotein expression with a direct or indirect role in the SBP2 nuclear/cytoplasmic shuttling, as it contains a nuclear localization signal (46); (ii) we established by structure prediction and experimental data that SBP2 is an Intrinsically Disordered Protein and that the N-terminal extension is widely unstructured (Olieric et al., manuscript in preparation). It is very possible that this region acquires its proper folding in the presence of yet to be discovered protein partners, consistent with the role of the Hsp90 chaperone and co-chaperones in the folding and assembly of proteins bearing an L7Ae RNA-binding module (47) and (iii) finally, the N-terminal domain could be involved in an SBP2 function different from selenocysteine incorporation, as inferred very recently from the finding that several SBP2 isoforms arise from splice variants in the N-terminal region (48). "
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    ABSTRACT: Selenoproteins contain the amino acid selenocysteine which is encoded by a UGA Sec codon. Recoding UGA Sec requires a complex mechanism, comprising the cis-acting SECIS RNA hairpin in the 3′UTR of selenoprotein mRNAs, and trans-acting factors. Among these, the SECIS Binding Protein 2 (SBP2) is central to the mechanism. SBP2 has been so far functionally characterized only in rats and humans. In this work, we report the characterization of the Drosophila melanogaster SBP2 (dSBP2). Despite its shorter length, it retained the same selenoprotein synthesis-promoting capabilities as the mammalian counterpart. However, a major difference resides in the SECIS recognition pattern: while human SBP2 (hSBP2) binds the distinct form 1 and 2 SECIS RNAs with similar affinities, dSBP2 exhibits high affinity toward form 2 only. In addition, we report the identification of a K (lysine)-rich domain in all SBP2s, essential for SECIS and 60S ribosomal subunit binding, differing from the well-characterized L7Ae RNA-binding domain. Swapping only five amino acids between dSBP2 and hSBP2 in the K-rich domain conferred reversed SECIS-binding properties to the proteins, thus unveiling an important sequence for form 1 binding.
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