Selenoproteins are proteins containing selenium in the form of the 21st amino acid, selenocysteine. Members of this protein family have many diverse functions, but their synthesis is dependent on a common set of cofactors and on dietary selenium. Although the functions of many selenoproteins are unknown, several disorders involving changes in selenoprotein structure, activity or expression have been reported. Selenium deficiency and mutations or polymorphisms in selenoprotein genes and synthesis cofactors are implicated in a variety of diseases, including muscle and cardiovascular disorders, immune dysfunction, cancer, neurological disorders and endocrine function. Members of this unusual family of proteins have roles in a variety of cell processes and diseases.
Abbreviations: AD, Alzheimer's disease; COX, cyclo-oxygenase; CVB3, coxsackie virus B3; DIO, iodothyronine deiodinase; 15d-PGJ2, 15-deoxy-Δ12,14-prostaglandin J2; EFsec, selenocysteine tRNA-specific elongation factor; ER, endoplasmic reticulum; GPX, glutathione peroxidase; IκB, inhibitor of NF-κB; IKKβ, IκB kinase β; IL-1β, interleukin 1β; IL-2R, interleukin 2 receptor; LDL, low-density lipoprotein; NF-κB, nuclear factor κB; NPC, Nutritional Prevention of Cancer; PACAP, pituitary adenylate cyclase-activating polypeptide; PD, Parkinson's disease; PTZ, pentylentetrazol; ROS, reactive oxygen species; SBP2, selenocysteine insertion sequence-binding protein 2; Sec, selenocysteine; SECIS, selenocysteine insertion sequence; SELECT, Selenium and Vitamin E Cancer Prevention Trial; SelH (etc.), selenoprotein H (etc.); Sep15, 15 kDa selenoprotein; SIRS, systemic inflammatory response syndrome; SPS, selenophosphate synthetase; SR, sarcoplasmic reticulum; SRE, selenium-response element; Tat, transactivator of transcription; TCR, T-cell receptor; TNFα, tumour necrosis factor α; TPM, Topiramate; tRNASec, selenocysteine tRNA; TRX, thioredoxin; TRXR, thioredoxin reductase; UTR, untranslated region; VCP, valosin-containing protein
- © The Authors Journal compilation © 2009 Biochemical Society
Selenium (Se) is an essential trace element that is attained through consumption of a wide variety of dietary components . While cases of toxicity or extreme deficiency are rare in humans, less overt changes in dietary Se levels have been shown to significantly affect human health [2,3]. The effects ascribed to marginal changes in dietary Se are multifaceted and include supporting cardiovascular health, thyroid hormone metabolism, inflammation and immune function, as well as protection against neurodegeneration, cancer, and viral infection.
Se is incorporated into selenoproteins in the form of the amino acid selenocysteine (Sec), and the biological effects of Se are exerted primarily through the function of different selenoproteins. Defined by the incorporation of Sec and broadly classified as antioxidants, selenoproteins exhibit tissue and cell-specific expression patterns, act on a variety of substrates, and have multiple functions . Among these are control of the cellular redox state and protection from oxidative damage and stress. Selenoproteins also have roles in thyroid hormone metabolism , intracellular Ca++ mobilization [5,6,7], protein folding , Se transport , and they can even catalyze intermediates in the synthesis of Sec [10,11].
The incorporation of Se into selenoproteins occurs through a complex Sec biosynthesis pathway in which this amino acid is synthesized on a dedicated tRNA (tRNA[Ser]Sec) . The Sec moiety is then transferred to the nascent selenoprotein from Sec-tRNA[Ser]Sec by decoding a UGA codon within the selenoprotein mRNA [12,13]. Se status impacts the efficiency of Sec biosynthesis , Um34 methylation of Sec-tRNA[Ser]Sec [15,16], redefinition of the UGA codon for Sec incorporation , and the abundance of selenoprotein mRNA [17,18,19,20]. Under conditions of moderate Se-deficiency, it has been shown that nonessential selenoproteins involved in stress-related processes (e.g., glutathione peroxidase (Gpx) 1 and selenoprotein W (Sepw1)) are preferentially lost, whereas expression of the essential housekeeping selenoproteins (e.g., Gpx4 and thioredoxin reductase (Txnrd) 1) is preserved [21,22,23,24]. Likewise, available Se is not equally supplied to all tissues [25,26]. Under conditions of Se-deficiency, brain, testes and thyroid maintain near normal Se levels, whereas other tissues, such as liver, lung and those of the immune system, exhibit a significant decline in Se and selenoprotein synthesis.
The consequences on human health may vary greatly depending on the specific biochemical, cellular, and tissue functions that are most affected by the loss of selenoproteins in combination with interacting environmental factors such as exposure to chemical insult or viral infection. For example, several studies have found an association between Se-deficiency and an increased susceptibility to viral infection and progression of pathogenesis . Studies into the mechanism by which coxsackie-viral infection, the purported etiological agent of Keshan’s disease, which is affected by Se-deficiency, suggest that the resulting changes in oxidative stress levels, and perhaps the host immune response, allow for the selection of viruses with more virulent phenotypes [28,29]. Se-deficiency has also been shown to increase the pathology of influenza viral infections in animal models [30,31], and a rare study in humans revealed that Se supplements increased the cellular immune response to poliovirus vaccination through increased production of cytokines, T-cell proliferation, and more rapid viral clearance .
Multiple experimental studies in animal models have revealed that Se-deficiency impairs immune response to infection, cancer, and other stimuli. Examples include a reduction in CD4+ T-cell response in Se-deficient mice challenged with a peptide/adjuvant , increased tumor growth and spread in a mouse model of breast cancer , enhanced type I allergic response in a mouse model of active cutaneous anaphylaxis , and increased immunotoxicity resulting from arsenic exposure . In each case, the levels of interferon-γ and other cytokines were reduced in Se-deficient mice. Additional studies in mouse knockouts have demonstrated that the selenoproteome [37,38,39], as well as individual selenoproteins such as Selk  and Sep15 , appear to play direct roles in supporting immune function. While additional research is needed, the important roles that selenoproteins play in regulating cellular oxidation states, catalyzing redox reactions with protein and chemical substrates, Ca++ signaling, protein folding, and the downstream effects of these functions will likely be identified as the molecular mechanisms to explain the importance of dietary Se in supporting the immune systems , as well as other health-related physiological functions.
To fully understand the health effects of Se, it is important to identify both the changes in selenoprotein expression levels as well as changes to downstream targets that contribute to the health consequences of Se-deficiency. In this study, we have applied microarrays, RNA-Seq, qPCR, and ribosome profiling to examine the genome-wide consequences of Se-deficiency in mice. As expected from previous studies, RNA-Seq and microarray analysis identified a subset of selenoprotein genes (e.g., Gpx1, selenoprotein H (Selh), Sepw1 and selenoprotein P (Sepp1)) in which mRNA abundance was significantly reduced, while the expression of other essential selenoproteins (e.g., Gpx4, selenophosphate synthetase 2 (Sephs2), and Txnrd1) was preserved. Ribosome profiling further indicated that translation of non-essential selenoprotein mRNAs, and particularly Sec incorporation efficiency, was also significantly affected by the availability of Se. Pathway and gene ontology analysis of genes showing increased expression in the liver and lung of Se-replete mice identified a number of inflammation-related genes that are regulated by interferon-γ. This result was confirmed by serum cytokine analyses, which showed that Se-replete mice have increased levels of circulating interleukin (IL)-6 and interferon-γ.
Although excessive releases of IL-6 and interferon-γ have been associated with inflammatory and autoimmune diseases, moderate increases of these cytokines, which are known for both their pro-inflammatory and anti-inflammatory properties, play a pivotal role in host defense through the ability to activate macrophage cell functions with important implications in health and disease states . Thus, our results support previous studies suggesting that adequate Se levels are required to support a healthy and robust immune response; much of it may be mediated by interferon-γ-regulation.
2. Experimental Section
2.1. Accession Codes
Microarray and ribosome profiling data are accessible through the Gene Expression Omnibus database, accession #GSE70160 .
TRIzol reagent was purchased from Invitrogen (Carlsbad, CA, USA), iScript cDNA Synthesis kit and SYBR Green Supermix from Bio-Rad Laboratories (Hercules, CA, USA). Real-time qPCR primers were from Sigma-Genosys (St. Louis, MO, USA). Se-deficient (0 ppm sodium selenite; TD.04484) and Se-supplemented diets (0.1 ppm sodium selenite; TD.10645) were obtained from Harlan-Teklad Laboratories, Inc. (Madison, WI, USA). RNase I and RNase Inhibitor were obtained from Invitrogen/Thermo Fisher Scientific (Waltham, MA, USA). Antarctic Phosphatase was obtained from New England Biolabs (Ipswich, MA, USA). The TruSeq Small RNA Sample Prep Kit, TruSeq Stranded Total RNA Sample Preparation Kit, and Ribo-Zero Human/Mouse/Rat were obtained from Illumina (San Diego, CA, USA). Other reagents were of the highest commercially-available quality.
2.3. Mice, Diets, Tissue Preparations and Selenium Concentrations
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the NCI-Bethesda Animal Care and Use Committee at the National Institutes of Health (permit number for this study is BRL-002). Upon weaning, three week-old male wild-type mice in a FVB/N background were given Se-deficient diets supplemented with 0 ppm or 0.1 ppm Se (sodium selenite) and maintained on the diets for 6 weeks prior to euthanasia via CO2 asphyxiation. Livers and lungs were rapidly excised, washed in PBS and immediately frozen in liquid nitrogen. Selenium concentrations in the torula yeast-based diets yeast-based diets given to animals were determined fluorometrically by the South Dakota Agricultural Laboratories (Brookings, SD, USA). Selenium concentrations were also determined in liver, lung and plasma tissues (N = 3 for each tissue) by this laboratory.
2.4. mRNA Analysis from Mouse Liver and Lung Tissue
Total RNA was isolated from tissues using TRIzol reagent following the manufacturer’s recommendation. 500 ng of total RNA were reverse transcribed using iScript cDNA synthesis kit. qPCR was performed in triplicate using iTaq Universal SYBR Green Supermix according to the manufacturer’s instructions. Primer sequences used are shown in Table S1.
2.5. Microarray Analysis
Total mRNA was isolated from mouse liver and lung tissues with the phenol-chloroform extraction method using TRIzol. Microarray analysis was performed on Affymetrix (Cleveland, OH, USA) Mouse 430_2.0A gene chips. Three arrays were analyzed from three different mRNA samples per Se diet. The MAS5 statistical algorithm, which normalizes each array independently and sequentially, was used for background and noise correction in the conversion of probe level to gene expression data, and absent calls were excluded from analysis. Mice on deficient dietary Se and controls (adequate Se diet) were compared by t-test.
2.6. Ribosomal Profiling
Livers were rapidly excised, and frozen in liquid nitrogen. Approximately 100 mg of tissue were pulverized by rapid agitation in a Mini-Beadbeater-8 (Biospec Products Inc., Bartlesville, OK, USA) in 1.5 mL pre-chilled lysis buffer (10 mM Tris-Cl (pH 7.5), 300 mM KCl, 10 mM MgCl2, 200 μg/mL cycloheximide (Sigma-Aldrich, MO, USA), 1 mM DTT, and 1% Triton X-100). 1 mL of crude lysate was incubated with 500 units of RNase I for 30 min at 25 °C. Monosomes were isolated by centrifugation through a sucrose cushion at 48,000 RPM (100,000 max. rcf) for 3 h in a TL100 ultracentrifuge. Ribonuclease-resistant RNA fragments were isolated from the pellet by TRIzol extraction and electrophoresis on a 15% polyacrylamide, 8 M urea gel. The region of the gel containing ~26–36 nt size RNA fragments was excised, and RNA isolated by passive elution prior to library construction.
Gel purified ribosome footprints were treated with 5 units of Antarctic Phosphatase in the presence of 40 units of RNase inhibitor for 30 min at 37 °C followed by 5 min at 65 °C to deactivate the enzyme. Small RNA sequencing libraries were constructed using the Illumina TruSeq Small RNA Sample Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. Following limited PCR amplification (11 cycles), the PCR-amplified library was resolved on a 6% Novex TBE PAGE gel (Invitrogen/Thermo Fisher Scientific, Grand Island, NY, USA) and a gel fragment representing the size range expected for ligation of ribosome footprints was excised from the gel. Small RNA library molecules were eluted by soaking the crushed gel fragment overnight in ultra-pure water at room temperature. Libraries were subjected to 50 cycle single-end sequencing on an Illumina HiSeq 2000 Instrument.
2.7. RNA-Sequencing Analysis (RNA-Seq)
Livers were rapidly excised, and frozen in liquid nitrogen. Approximately 100 mg of tissue were pulverized under liquid nitrogen by mortar and pestle. While still frozen, 2 mL of TRIzol were added and total RNA isolated according to the manufacturer’s instructions. Library construction was performed using the Illumina TruSeq Stranded Total RNA Sample Preparation Kit with Ribo-Zero Human/Mouse/Rat (Illumina, San Diego, CA, USA). Briefly, ribosomal RNA was removed from total RNA samples using biotinylated Ribo-Zero oligos attached to magnetic beads. Following purification, the rRNA-depleted sample was fragmented and primed with random hexamers. First strand reverse transcription was accomplished using Superscript II Reverse Transcriptase (Invitrogen/Thermo Fisher Scientific, Grand Island, NY, USA). Second strand cDNA synthesis was accomplished using DNA polymerase I and RNase H under conditions in which dUTP is substituted for dTTP. An A-base was added to the blunt ends and ligated to adapters containing a T-base overhang. Ligated fragments were PCR-amplified (12–15 cycles) under conditions in which the PCR reaction enables amplification of the first strand cDNA product only. Libraries were subjected to 50-cycle single-end sequencing on an Illumina HiSeq 2000 Instrument.
2.8. Bioinformatic Analysis of RNA-Seq and Ribosome Profiling
Adapter sequences were removed using the Hannon laboratory FastX toolkit  rRNA sequences were removed using Bowtie (Johns Hopkins University, Baltimore, MD, USA) . Sequences were aligned against mouse rRNA, and all unaligned sequences were retained for further processing. RefSeq FASTA sequences were obtained from the UCSC genome browser (mm9). These FASTA files were reduced to a single entry for each mRNA corresponding to the longest isoform. For coding sequence alignments, the first 15 and last 3 codons were excluded to avoid bias at the initiation and termination codons. Uniquely aligning sequences, allowing for 2 mismatches, were identified using the Bowtie sequence aligner .
Ribosome profiling and total RNA sequences were aligned separately to selenoprotein mRNAs using the Bowtie alignment parameters described above against the following RefSeq entries: NM_027652.2 Seli, NM_027905 Selo, NM_007860.3 Dio1, NM_013759.2 Sepx1, NM_024439.3 Sels, NM_015762.2 Txnrd1, NM_008160.6 Gpx1, NM_008162.2 Gpx4, NM_009155.3 Sepp1, NM_053102.2 Sep15, NM_013711.3 Txnrd2, NM_019979.2 Selk, NM_001040396.2 Selt, NM_009156.2 Sepw1, NM_009266.3 Sephs2, NM_030677.2 Gpx2, NM_001178058.1 Txnrd3, NM_029100.2 Sepn1, NM_053267.2 Selm, NM_008161.3 Gpx3, NM_001033166.2 Selh, NM_172119.2 Dio3, NM_010050.2 Dio2, NM_175033.3 Selv, NM_008084.2 Gapdh.
For quantitative analysis, the 5′ end of ribosome profiling and RNA reads were offset to the predicted A-site, as determined previously [17