Ribosome Fate during Decoding of UGA-Sec Codons

Decoding of genetic information into polypeptides occurs during translation, generally following the codon assignment rules of the organism’s genetic code. However, recoding signals in certain mRNAs can overwrite the normal rules of translation. An exquisite example of this occurs during translation of selenoprotein mRNAs, wherein UGA codons are reassigned to encode for the 21st proteogenic amino acid, selenocysteine. In this review, we will examine what is known about the mechanisms of UGA recoding and discuss the fate of ribosomes that fail to incorporate selenocysteine.

The expression of essential selenoproteins during zebrafish development requires SECIS binding protein 2-like

The dietary requirement for selenium is based on its incorporation into selenoproteins, which contain the amino acid selenocysteine (Sec). The Sec insertion sequence (SECIS) is an RNA structure found in the 3' UTR of all selenoprotein mRNAs, and it is required to convert in-frame UGA codons from termination to Sec-incorporating codons. There are two proteins that bind to SECIS elements, but only one, SECIS binding protein 2 (Sbp2), has been shown to be required for Sec incorporation. The Sbp2 paralogue, SECIS binding protein 2-like (Secisbp2l) is conserved in all vertebrates and shares many features with Sbp2, but its function is unknown. Here we set out to determine the relative roles of Sbp2 and Secisbp2l by introducing CRISPR mutations in both genes in zebrafish. By monitoring selenoprotein synthesis with 75Se labeling during embryogenesis, we found that sbp2-/- embryos still make a select subset of selenoproteins but secisbp2l-/- embryos retain the full complement. Abrogation of both genes completely prevents selenoprotein synthesis and juveniles die at 14 days post fertilization. Embryos lacking Sbp2 are sensitive to oxidative stress and express the stress marker Vtg1. We propose a model where Secisbp2l is required to promote essential selenoprotein synthesis during stress.

The polypyrimidine tract binding protein, PTBP1, regulates selenium homeostasis via the Selenoprotein P 3′ untranslated region

Selenoproteins contain the 21st amino acid, selenocysteine (Sec), which is incorporated at select UGA codons when the encoding mRNA contains a specialized hairpin sequence in its 3′ UTR. This hairpin, the so-called Sec insertion sequence (SECIS) element, is found in all selenoprotein mRNAs, but the sequence surrounding these elements is widely variable and in many cases of considerable length. In order to determine the function of one such SECIS context, we chose to focus on the plasma selenoprotein, SELENOP, that is required to maintain selenium homeostasis. It is unique in that its mRNA contains two SECIS elements that lie in the context of a highly conserved 843-nucleotide 3′ UTR. Prior work has attempted to examine the functions of the SECIS context but none were identified. Here we have used CRISPR/Cas9 genome editing to delete the region between the two SECIS elements. We found that this sequence is required to mediate an increase in SELENOP synthesis under conditions of peroxide stress. Using RNA affinity chromatography, we have identified PTBP1 as the major RNA binding protein that specifically interacts with this region.

The Interaction between Dietary Selenium Intake and Genetics in Determining Cancer Risk and Outcome

There is considerable interest in the trace element selenium as a possible cancer chemopreventive dietary component, but supplementation trials have not indicated a clear benefit. Selenium is a critical component of selenium-containing proteins, or selenoproteins. Members of this protein family contain selenium in the form of selenocysteine. Selenocysteine is encoded by an in-frame UGA codon recognized as a selenocysteine codon by a regulatory element, the selenocysteine insertion sequence (SECIS), in the 3′-untranslated region of selenoprotein mRNAs. Epidemiological studies have implicated several selenoprotein genes in cancer risk or outcome based on associations between allelic variations and disease risk or mortality. These polymorphisms can be found in or near the SECIS or in the selenoprotein coding sequence. These variations both function to control protein synthesis and impact the efficiency of protein synthesis in response to the levels of available selenium. Thus, an individual’s genetic makeup and nutritional intake of selenium may interact to predispose them to acquiring cancer or affect cancer progression to lethality.

A Versatile Strategy to Reduce UGA-Selenocysteine Recoding Efficiency of the Ribosome Using CRISPR-Cas9-Viral-Like-Particles Targeting Selenocysteine-tRNA[Ser]Sec Gene

The translation of selenoprotein mRNAs involves a non-canonical ribosomal event in which an in-frame UGA is recoded as a selenocysteine (Sec) codon instead of being read as a stop codon. The recoding machinery is centered around two dedicated RNA components: The selenocysteine insertion sequence (SECIS) located in the 3′ UTR of the mRNA and the selenocysteine-tRNA (Sec-tRNA[Ser]Sec). This translational UGA-selenocysteine recoding event by the ribosome is a limiting stage of selenoprotein expression. Its efficiency is controlled by the SECIS, the Sec-tRNA[Ser]Sec and their interacting protein partners. In the present work, we used a recently developed CRISPR strategy based on murine leukemia virus-like particles (VLPs) loaded with Cas9-sgRNA ribonucleoproteins to inactivate the Sec-tRNA[Ser]Sec gene in human cell lines. We showed that these CRISPR-Cas9-VLPs were able to induce efficient genome-editing in Hek293, HepG2, HaCaT, HAP1, HeLa, and LNCaP cell lines and this caused a robust reduction of selenoprotein expression. The alteration of selenoprotein expression was the direct consequence of lower levels of Sec-tRNA[Ser]Sec and thus a decrease in translational recoding efficiency of the ribosome. This novel strategy opens many possibilities to study the impact of selenoprotein deficiency in hard-to-transfect cells, since these CRISPR-Cas9-VLPs have a wide tropism.

A Zebrafish Model for Selenoprotein Synthesis and Function (OR11-01-19)

Objectives We have established a zebrafish model system that will allow unprecedented access to the role of selenoprotein function during development. The work described here focuses on a poorly characterized RNA binding protein that is similar to SECIS binding protein 2 (SBP2), which is required for the co-translational insertion of selenocysteine at select UGA codons in selenoprotein mRNAs. This protein, SECISBP2L, shares many features with SBP2 but is has no known function. We hypothesize that the zebrafish model system will reveal a selenoprotein-synthesis related function for SECISBP2L. Methods Using CRISPR/Cas9, we generated zebrafish with a disruption in one of the conserved domains in SECISBP2L. When bred out to a homozygous mutant animal, we verified that SECISBP2L protein expression was eliminated. To analyze selenoprotein synthesis, we metabolically labeled wild-type and mutant embryos with radioactive selenium (Se-75). Results In terms of overt phenotypes in SECISBP2L null fish, we observed no defects in growth, mobility or fertility. However, we noticed a significant sensitivity to oxidative stress as measured by lethality associated with peroxide exposure. In order to detect changes in selenoprotein expression that may have resulted from SECISBP2L loss, we performed Se-75 labeling in embryos. We also began an investigation of the effect of oxidative stress on selenoprotein expression during development. As such, one set of embryos was treated for 24 hours with 100 nM Se-75 and the other with Se-75 plus 200 μM H2O2. In general, we did not observe an overall alteration of selenoprotein expression as a result of SECISBP2L loss. We did, however, observe a significant spike of expression for a 50 kDa selenoprotein that did not occur in the SECISBP2L null animals. Based on this molecular weight, we predict that this band corresponds to selenophosphate synthase (SEPHS2). In addition, subsequent labeling at later time points revealed a general reduction of selenoprotein expression that may result from reduced SEPHS2 expression because it is essential for selenocysteine-tRNA synthesis. Conclusions We conclude that SECISBP2L is required for optimal selenoprotein expression and its function may be induced by oxidative stress. We have also demonstrated the value of a zebrafish model system for studying the mechanism of selenoprotein synthesis. Funding Sources Funded by the National Institutes of Health.

Selenium-Related Transcriptional Regulation of Gene Expression

Selenium is a trace metal essential to human health, and its deficiency has been related to, for instance, cardiovascular and myodegenerative diseases, infertility and osteochondropathy Kashin-Beck disease. It is incorporated as selenocysteine to selenoproteins, which protect against reactive oxygen and nitrogen species. They also participate in the activation of thyroid hormone, and play a role in immune system functioning. The synthesis and incorporation of selenocysteine occurs via a special mechanism, which differs from the one used for standard amino acids. The codon for selenocysteine is the regular in-frame stop codon, which can be passed by specific complex machinery participating in translation elongation and termination. This includes the presence of selenocysteine insertion sequence (SECIS) in the 3’-untranslated part of the selenoprotein mRNAs. Selenium deficiency is known to control both selenoprotein and non-selenoprotein transcriptomes. Nonsense-mediated decay is involved in the regulation of selenoprotein mRNA levels, both other mechanisms are also possible.