SBP2 is a critical factor for selenoprotein synthesis, with roles in SECIS element binding, ribosome binding and Sec incorporation. Much of recent work has elaborated on understanding its role in the translation of 25 mammalian selenoproteins, by detailed molecular and biochemical characterization. Mutations in the SECISBP2
gene that either alter the reading frame through splicing defects or alter the interaction of SBP2 with a subset of SECIS elements due to amino acid substitutions, result in abnormal thyroid hormone function in humans (15
). In this study, we presented the first detailed in vivo
and in silico
characterization of human SECISBP2
alternative splicing and highlighted a new mode of transcriptional and translational regulation. Interestingly and intriguingly, the most abundant SBP2 variant after the full-length SBP2 contains a MTS, which we showed is functional and required for targeting of the mtSBP2 protein to mitochondria. A small proportion of the endogenous SBP2 pool is also localized to the mitochondria, however, only a small fraction of cells appeared to have strong mitochondrial SBP2 localization. This suggests that the mitochondrial localization of SBP2 may be tightly regulated and may occur predominantly during certain, yet unidentified conditions.
The identification of a potential mitochondrial human-specific SBP2 isoform was an intriguing and somewhat puzzling finding when viewed against the known SBP2 function in decoding UGA as Sec during selenoprotein translation. This process is unlikely to occur within the inner mitochondria for the following reasons: (i) analysis of the mitochondrial genome using the SECISearch program (34
) suggests that the human mitochondrial genome does not contain any genes with SECIS elements and hence does not encode selenoproteins; (ii) UGA codes for tryptophan in the human mitochondrial genome; and (iii) mitochondrial matrix translation uses a mechanism similar to that of prokaryotes. These facts would argue against the requirement of a mitochondrial SBP2 isoform with canonical function in Sec incorporation. However, emerging evidence suggests that translationally active cytoplasmic ribosomes, or polysomes, are present on the outer membrane of the mitochondria and that about 50% of mRNAs coding for mitochondria-localized products are primarily associated with mitochondria-bound polysomes (35–37
). Targeting mRNAs to the mitochondria is mediated by conserved secondary structures in their 3′ UTR (36
). Although this process is poorly understood, it is possible that the conservation of localized translation might assist in the co-translational import of hydrophobic proteins into mitochondria to prevent their aggregation in the cytoplasm, or to simply provide a more efficient way of translation, as one mRNA molecule can serve in several rounds of translation. The mitochondrial SBP2 isoform could thus function in the translation of selenoproteins targeted to mitochondria such as thioredoxin reductase 2 (TxnRd2/TR3) (1
), mitochondrial form of phospholipid glutathione peroxidase (PHGPx/GPx4) (39
) and glutathione peroxidase 1 (GPx1) (40
) on mitochondria-bound cytoplasmic ribosomes. Alternatively, mtSBP2 could be involved in binding to mRNA structures, possibly also in non-selenoprotein encoding mRNAs that target the message to the outer mitochondrial ribosomes. The high-resolution images we obtained of both over-expressed mtSBP2-GFP and endogenous mtSBP2 pointed to a localization of the mtSBP2 isoform on the outer mitochondrial surface, in specific punctate clusters. This localization would indeed be in agreement with the functional localization of SBP2 at polysomes. The fact that we could not detect mtSBP2 in mitochondrial subfractions of cells by western blotting argues for a possible loose interaction between mtSBP2 and the organelle, which may be easily disrupted during the fractionation procedure. But if mtSBP2 is localized on the outer surface of the mitochondria, why would it require a classical type of MTS that generally targets proteins to the inner mitochondrial matrix? Although this question remains to be answered, our findings are relevant to a recent report showing that DAKAP1, a multifunctional protein with roles in cAMP dependent protein kinase (PKA) regulation and mRNA binding, can be targeted to the cytosolic side of the outer membrane of the mitochondria by use of a classical MTS. DAKAP1 contains a bi-functional targeting motif that switches to encode either an MTS, such as the one present in mtSBP2, or an ER targeting signal (41
). Although the precise mechanism of how this protein attaches to the mitochondria outer membrane remains unknown at present, it is possible to reconcile the idea that proteins that localize on the cytosolic side of the mitochondrial membrane can be targeted there by classical MTSs. The MTS in mtSBP2 may thus facilitate the localization of the protein to the cytosolic side of the mitochondria via active mitochondrial import. More detailed studies will be required to investigate this aspect of mtSBP2 localization as well as the precise role of this isoform in the cell. The low expression of mtSBP2 isoform in the cell lines tested indicates that its synthesis could be dependent on tissue-specific translation factors and may be maximized in tissues or organs with high demand for mitochondrial selenoproteins. A prime candidate would be testis, where PHGPx is the most abundant selenoprotein. Indeed, the expression of both full-length SECISBP2
transcripts was highest in testis when compared to twelve other tissues, providing additional proof to strengthen this hypothesis.
This study established that human SECISBP2
has an extensive alternative splicing pattern in the 5′-region. Some of the splicing events provoke ORF alterations, which lead to premature termination codons, thus promoting translation from downstream ATG start codons. Our in vivo
splicing assay and treatment with ASOs showed that at least four additional ATG start codons in exons 2, 3a, 3b and 5 are used to produce proteins with different N-terminal amino acid sequence such as mtSBP2, or N-terminally truncated SBP2 isoforms. Because all alternative splicing events within SBP2 are confined within the region dispensable for Sec incorporation in vitro
, it is reasonable to postulate that these events are unlikely to affect RNA binding per se
, however, this level of regulation may play a fine-tuning or regulatory role in SBP2-dependent Sec incorporation function in vivo
. The role of the N-terminal region of SBP2 is a puzzling, yet unanswered, question in the field. So far, the only characterized motif within this region is a nuclear localization signal (NLS) that enables SBP2 to shuttle between the nucleus and cytoplasm (14
), however, its requirement for selenoprotein synthesis in vivo
has not been determined. Interestingly, the NLS is located within exon 8, which is common to all alternatively spliced SBP2 isoforms suggesting that this motif may indeed play an important role in the function of SBP2 in the nucleus in vivo
. Consistent with this, recent studies have suggested a new role of SBP2 in protection of selenoprotein encoding mRNAs from nonsense mediated decay by binding to these mRNAs in the nucleus (42
The human selenoproteome consists of 25 selenoproteins (44
) but the SECIS core element and the binding site for SBP2 are well conserved. As a result, Sec incorporation with regard to SECIS element binding would not require variability within the SBP2 RNA binding region which could explain the lack of alternate splicing in the C-terminal region of SBP2. In contrast, the hierarchy of selenoprotein synthesis is expected to involve specific factors that may dictate the differential binding of SBP2 to different selenoprotein mRNAs (25
). The high sequence variability in the N-terminal region of SBP2 could thus serve as a protein–protein interaction domain and function in the recruitment of such factors prior to interaction with the SECIS element.
In a previous study we showed that acute oxidative stress caused by H2
and sodium selenite had an inhibitory effect on selenoprotein synthesis, and that this was mediated by oxidation of SBP2 redox-sensitive cysteine residues and its depletion from the ribosomes (14
). In the current study, using UVA-irradiation as a cause of oxidative stress we found that SBP2 responded by coordinated transcriptional and translational changes following stress. Importantly, SECISBP2
transcripts were simultaneously and immediately induced during treatment, and stabilized during the recovery phase. At the protein level, SBP2 was degraded and levels remained approximately half of the initial levels for ~16 h post-irradiation. The SBP2 isoforms that we could monitor using the minigene showed similar down-regulation as full-length SBP2, suggesting that their intracellular function is most likely related to the function of full-length SBP2 in SECIS binding and Sec incorporation. However, our data does not exclude the possibility that mtSBP2 or any other SBP2 isoforms may perform additional functions unrelated to selenium metabolism.
This is the first report validating the occurrence and significance of alternative splicing associated with SECISBP2
, an essential gene linked to selenium metabolism. Although, we have focussed mainly on one spliced variant, mtSECISBP2
, there remain other variants of significant interest. Having shown that an ASO-based approach could be used to generate specific spliced variants, future studies could be tailored to examine their functions. Recent years have seen a dramatic evolution in our understating of translational regulation by microRNAs, a class of small RNA that could be synthesized by a stem-loop containing transcript generated from RNA Polymerase II (45
). To a broader significance, it remains to be seen if some of the poorly translatable SECISBP2
spliced variants including mtSBP2 are meant to generate microRNAs and/or serve as potential targets of microRNAs.