POST-TRANSCRIPTIONAL REGULATION OF
SELENOPROTEIN S
by
ERIC MICHAEL COCKMAN
Submitted in partial fulfillment of the requirements for the degree of
Doctor of Philosophy
Department of Molecular Medicine
CASE WESTERN RESERVE UNIVERSITY
August, 2019
CASE WESTERN RESERVE UNIVERSITY
SCHOOL OF GRADUATE STUDIES
We hereby approve the thesis/dissertation of
Eric Cockman
Candidate for the degree of Doctor of Philosophy.*
Donal Luse, Ph.D. Committee Chair
Donna Driscoll, Ph.D. Research Advisor and Committee Member
Richard Padgett, Ph.D. Committee Member
Ofer Reizes, Ph.D. Committee Member
Bela Anand-Apte, M.D., Ph.D. Committee Member
Date of Defense June 28, 2019
*We also certify that written approval has been obtained for any proprietary materials contained therein.
i Table of Contents
List of Tables...... iv
List of Figures...... v
Acknowledgements...... vii
Abstract...... x
CHAPTER I: INTRODUCTION...... 1
Selenium and Selenocysteine...... 1
Synthesis of Sec...... 2
The genetic code...... 5
An overview of general translation elongation and termination...... 7
Insertion of Sec into a growing polypeptide...... 11
Regulation of Sec insertion into selenoproteins...... 16
Selenoproteins in the human body...... 19
Selenoprotein S...... 23
SelS in disease...... 26
CHAPTER II: MATERIALS AND METHODOLOGY...... 30
CHAPTER III: RESULTS...... 40
Recoding efficiency of the SelS 3’ UTR...... 40
V5-surrogate assay for Sec insertion...... 42
Sec is inserted into the SelS-V5 surrogate assay...... 44
ii Efficiency of the SelS-V5 reporter...... 48
Distance restriction of SelS recoding...... 52
The proximal SelS 3’ UTR contains two distinct conserved sequences...... 55
PSL and SPUR mutations do not affect RNA levels or general translation...... 60
PSL and SPUR mutation effects UGA recoding are not cell-type specific...... 62
Point mutations in the SPUR Element inhibit UGA recoding...... 64
The SPUR element does not function with other SECIS elements...... 67
Deletion of the PSL promotes Sec insertion in SelS-V5...... 69
Other sequences can functionally replace the SelS PSL...... 73
The SPUR is required for efficient Sec insertion in the FLAG-SelS...... 79
The effect of the SPUR element is position dependent...... 84
CHAPTER IV: DISCUSSION AND FUTURE DIRECTIONS...... 87
APPENDIX...... 103
REFERENCES CITED...... 104
iii List of Tables
Table 1: List of accession numbers...... 30
Table 2: List of mutagenic primers...... 32
Table 3: Primers used for insertion PCR...... 33
Table 4: Primers used for qRT-PCR...... 36
Table 5: Recoding efficiencies of 3’UTRs...... 41
Table 6: List of peptides queried for SelS-V5 terminal PRM...... 47
Table 7: Selenoprotein S internal peptides...... 72
Table 8: List of peptides queried for FLAG-SelS PRM...... 83
iv List of Figures
Figure 1: Synthesis of Selenocysteine...... 4
Figure 2: The genetic code...... 6
Figure 3: Translational elongation...... 9
Figure 4: Translational termination...... 10
Figure 5: The SECIS element...... 14
Figure 6: Model of UGA recoding...... 15
Figure 7: Recoding efficiencies of SECIS elements from different selenoproteins...... 18
Figure 8: The Human selenoproteome...... 22
Figure 9: Selenoprotein S...... 25
Figure 10: Validation of the SelS-V5 surrogate assay...... 43
Figure 11: MS2 fragmentation of SelS-V5 Sec-containing peptide...... 46
Figure 12: Efficiency of the SelS-V5 reporter...... 50
Figure 13: Efficiency of the SelK-V5 reporter...... 51
Figure 14: Position of the UGA-Sec codon affects UGA recoding in SelS-V5...... 54
Figure 15: The proximal region of the SelS 3’ UTR contains elements that affect
...... V5 expression...... 57
Figure 16: The proximal SelS 3’ UTR contains two conserved regions...... 58
Figure 17: The PSL and SPURdm do not affect mRNA levels or general
...... translation...... 61
v Figure 18: Deletion of the PSL and SPUR element mutation effects are not cell-type
...... specific...... 63
Figure 19: Single point mutations in the SPUR element inhibit UGA recoding...... 66
Figure 20: Mutation of the SPUR element does not affect recoding directed by the
...... SelK or GPx4 SECIS elements...... 68
Figure 21: Deletion of the SelS PSL increases Sec insertion in the SelS-V5
...... construct...... 71
Figure 22: Disruption of the SelS PSL by point mutation and loop deletions has no
...... effect on V5 expression...... 76
Figure 23: Other stem-loops can functionally replace the SelS PSL...... 77
Figure 24: A linear sequence can replace the SelS PSL...... 78
Figure 25: Mutation of the SPUR element effects Sec insertion in FLAG-SelS
...... but PSL deletion has no effect...... 81
Figure 26: Deletion of the PSL does not increase Sec insertion into the FLAG-SelS
...... construct...... 82
Figure 27: Relative position of SPUR element to UGA codon is important ...... f.
...... activity...... 86
Figure 28: Mutations in the SECIS element rescue UGA recoding of the SPURdm..... 92
Figure 29: A protein binds to the SPUR element...... 95
Figure 30: Model for the mechanism of action of the SPUR element...... 97
vi Acknowledgements
It seems like not too long ago I was accepted into the Molecular Medicine program.
At the beginning, I could not wait to start. There were times somewhere in the middle of grad school when it felt like time wasn’t moving at all. And now, at the end, I can’t tell you where all the time went and I cannot believe how fast it was over. Regardless of how long or short it felt, this was one of the best times of my life. I have met countless remarkable individuals, gained life-long friendships, and learned so much. Here I would like to thank the people who have helped me through this journey.
First and foremost, my family. My mom, Kimberly Lopez, and my step-dad, Jason
Lopez, for always encouraging me to continue my education. You both taught me the meaning of hard-work, perseverance, and sacrifice. I can never thank you enough for pushing me to follow my interests in school and life, for letting me take the hard classes in school even if it meant you couldn’t help me with my homework, and for everything you have done for me to help me get this far. I’d like to thank my dad, Thomas Cockman, for instilling a curiosity and love of learning in me from an early age. You taught me how to ask questions and showed me that even if you could not answer it, it didn’t mean it wasn’t worth asking. And the rest of my family: all of my siblings, my grandparents, cousins, aunts, and uncles. They have shown me so much love and support throughout my life and
I would not be the person I am today without all of them. I love you all so much.
Donna Driscoll for allowing me to join her lab and embarking on this journey with me. I have learned and grown so much from her mentorship. She believed in me and pushed me when my first project failed. I truly believe I would not have finished this endeavor if
vii not for her support and encouragement. I will be hard pressed to find another boss or mentor as caring, understanding, and passionate as she is.
All of the current and past Driscoll lab members. From my first day, the lab welcomed me with open arms and treated me like a family. Although members have come and gone, that same comradery and trust has remained. In chronological order as best as I can remember: Angela Minard, Jodi Bubenik, James McConnell, Nisha Tapryal, Tina
Nunn, Alexis Polce, Vivek Narayan, Amanda Vreeland, Hannah Singerline, Maggie
Rybak, Lexi Miller, Ahmad Alhajajreh, Brad Kruithoff, and Kris Skugor. All of you have played a role, however large or small, in getting me through grad school. I am forever grateful for our friendships. I would also like to express extra gratitude toward Angela and
Vivek. Angela’s patience and willingness to teach me a variety of techniques and working with me until they were mastered was instrumental in me becoming a better scientist. As for Vivek, the countless hours we have sat together discussing ideas and working on projects were some of my most well spent. His expertise in mass spectrometry was essential for moving my project forward.
The members of my thesis committee: Don Luse, Rick Padgett, Ofer Reizes, and
Mario Skugor. Your guidance, advice, and suggestions over the years were invaluable. I know it was a little touch and go there for a bit, but I thank you all for seeing it through and believing in me. I would like to especially thank Don for always keeping his door open to talk about data and for being in my corner when things got rough.
Finally, all the friends I have made through grad school. My classmates: Damien
Bellos, Grace Weber, Maha Saber, Hannah Picariello, YeoJung Kim, Matt Hiznay, Emma
Lessiuer, and Jeff Bhasin. We have all grown so much from our first year. Even though
viii many of us have started families and moved away, I know we will all do great things and
I look forward to staying in touch with you over the years. I am also thankful for all the others friends I made within and outside of the Mol Med program. Especially the beautiful and incredibly talented Alyson Wolk. She has encouraged and pushed me to be a better person and scientist.
ix Post-Transcriptional Regulation of Selenoprotein S
Abstract
by
ERIC MICHAEL COCKMAN
Selenoproteins are a unique class of proteins that contain the 21st amino acid, selenocysteine (Sec). Addition of Sec into a protein is achieved by recoding of the UGA stop codon. All 25 mammalian selenoprotein mRNAs possess a 3’ UTR stem-loop structure, the Selenocysteine Insertion Sequence (SECIS), which is required for Sec incorporation. It is widely believed that the SECIS is the major RNA element that controls
Sec insertion, however recent findings in our lab suggest otherwise for Selenoprotein S
(SelS). Here we report that the first 60 nucleotides of the SelS 3’ UTR contains a proximal stem loop (PSL) and a conserved sequence we have named the SelS Positive UGA
Recoding (SPUR) element. We developed a SelS-V5/UGA surrogate assay for UGA recoding, which was validated by mass spectrometry to be an accurate measure of Sec incorporation in cells. Using this assay, we show that point mutations in the SPUR greatly reduce recoding in the reporter; thus, the SPUR is required for readthrough of the UGA-
Sec codon. In contrast, deletion of the PSL increased Sec incorporation. This effect was reversed when the PSL was replaced with other stem-loops or a linear sequence, suggesting that the PSL does not play an active role in Sec insertion. Additional studies revealed that the position of the SPUR relative to the UGA-Sec codon is important for optimal UGA recoding. Our identification of the SPUR element in the SelS 3’ UTR reveals a more complex regulation of Sec incorporation than previously realized.
x Chapter I
INTRODUCTION
Selenium and Selenocysteine
Selenium (Se) is an essential micronutrient that is found in the soil and is involved in multiple facets of human health. Se levels have been linked to the prevention of cancer (1-
3), immunity (4,5), as well as the development of Type II diabetes mellitus (T2DM) (6,7).
The recommended daily allowance of Se is 55 µg/day for adults (8). The benefits of Se appear only to be present at optimal levels. Se levels that are too high or too low are associated with multiple conditions. Interestingly, both high and low Se levels have been associated with increases in T2DM and cancer (6,7). Three specific diseases have been characterized when Se is severely deficient. Keshan Disease, often seen in children from areas of China with poor soil Se levels, is a cardiac disease that can lead to heart failure
(9). Kashin-Beck Disease (10), a bone and joint disease, and Myxedematous Endemic
Cretinism (11), which is characterized by mental retardation are also linked to Se deficiency. While conditions of excessive levels of Se are rare in humans, Selenosis can lead to hair loss, fatigue, and nerve damage (12).
Selenium imparts the majority of its biological activity in the form of selenocysteine
(Sec), the 21st proteogenic amino acid. As the name suggests, Sec has the same structure as cysteine (Cys) but possesses a selenium atom in place of sulfur. The basic chemical properties are similar to Cys, but Sec is much more reactive (13,14). Because of its lower pKa and greater nucleophilicity, Sec carries a negative charge at the physiological pH. Sec can also readily form covalent bonds with other Sec or Cys residues that are similar to disulfide bonds (13). Due to its high electrophobicity, enzymes containing Sec in their
1 active sites are able to easily be reduced back to their active states and resist being irreversibly inactivated by oxidation (15). Multiple studies have shown that Sec is more efficient at catalyzing enzymatic reactions than its sulfur-containing counterpart.
Experiments replacing the Cys residue in peroxidase enzymes with Sec resulted in a protein with a higher catalytic rate (16). In the inverse experiment where Sec was replaced with
Cys, the catalytic activity of the enzyme was greatly diminished (17,18). The chemical advantages of Sec over Cys allow for Sec-containing enzymes to be more active over a broader range of conditions. The advantages of Sec over Cys are great enough to conserve the synthesis and insertion machinery of Sec.
Synthesis of Sec
The mechanism of Sec synthesis is unique among amino acids. Unlike the other 20 standard amino acids, there is no free pool of Sec in the cell. Due to its high reactivity, free
Sec could cause oxidative damage to cells (19). To avoid this, Sec is synthesized on its transfer RNA (tRNA) in a multi-step process (20). As shown in Figure 1, a tRNA[Ser]Sec is charged with a serine residue by the seryl-tRNA synthetase, SerS. The serine is then converted into Sec in a two-step enzymatic process. The serine is phosphorylated by phosphorseryl-tRNA kinase (PSTK). Then Selenocysteine synthetase (SecS) uses
Selenophosphate (SeP), generated by the selenoprotein SPS2, to convert the phosphoserine to Sec. When tRNASer[Sec] has successfully been charged with a Sec residue, it is referred to as Sec-tRNASer[Sec]. Unlike other tRNAs, tRNA[Sec]Ser is larger, possessing a longer variable arm region and a longer acceptor stem (21,22). Furthermore, tRNASer[Sec] has fewer nucleotide modifications than a typical tRNA (21,22). Intriguingly, there are two isoforms of Sec-tRNA[Ser]Sec, one of which contains an additional methyl-group on the uridine
2 nucleotide at position 34 (21,23). The levels of these two isoforms change with Se status of the cell. Under Se deficient conditions, the non-methylated form is the dominant species.
It has been reported that constitutively expressed selenoproteins, such as members of the thioredoxin reductase family, can use either isoform of Sec-tRNASer[Sec} (24,25).
Conversely, selenoproteins that are active during stress responses are synthesized using the methylated form (24).
3
Figure 1: Synthesis of Selenocysteine. Sec is synthesized in a unique, multi-step process where the tRNA[Ser]Sec is charged with serine and the serine is converted to Sec by PSTK and SecS. SPS2 is responsible for converting hydrogen selenite (HSe-) into Selenophosphate (SeP) that is required for the final enzymatic step. Taken from (20).
4
The genetic code
DNA contains the information that is required to produce proteins. The human genome contains an estimated 20,000 protein-coding genes (26). For a cell to access the information stored in DNA to make a protein, it must first transcribe the gene to messenger RNA
(mRNA). The mRNA is heavily processed and exported to the cytoplasm where the genetic code can be translated to a protein. In order to make a protein, the ribosome deciphers the genetic code by reading the mRNA transcript in groups of three nucleotides, known as codons. Different combinations of the four nucleotides found in RNA, Adenosine (A),
Guanine (G), Cytosine (C), and Uracil (U), can be arranged into 64 different codons. Of the 64 possible codons, 61 are deciphered as one of the 20 standard amino acids, and three codons (UAG, UAA, and UGA) are signals for termination of translation. Codon assignment is shown in Figure 2. Within the genetic code, redundancy exists. Multiple codons can be used to specify a single amino acid. For example, tyrosine can be coded for by the UAU or UAC codon (Figure 2). However, there is no ambiguity. A codon can only represent a single amino acid. This property allows for a high degree of translational fidelity. However, Sec is quite extraordinary when compared to the other 20 amino acids.
In what would seem a direct contradiction to the “one codon, one amino acid” rule, Sec is encoded by the UGA stop codon. Because of this, cells require specialized machinery in order to properly recognize an authentic UGA-Sec codon. When the ribosome comes across a UGA codon in a selenoprotein mRNA transcript, the Sec machinery interacts with the ribosome to modify the definition of the UGA codon from termination to Sec in a process known as UGA recoding.
5
Figure 2: The genetic code. Nucleotide triplets known as codons are used to specify specific amino acids to be used in protein synthesis. Each codon denotes a single amino acid and amino acids can be represented by multiple codons. Three of the 64 codons signal termination of protein translation: UAA, UAG, and UGA. Sec is coded for by the UGA codon and requires the existence of a mechanism to distinguish between the dual definitions of this codon. Adapted from
6 An overview of general translation elongation and termination
The dual definition of the UGA codon poses an interesting challenge for protein synthesis. If the UGA codon could be ambiguously decoded as a termination signal or as
Sec, protein translation would be unreliable. Nearly a third of mRNAs use a UGA stop codon (27) however, a third of human proteins are not selenoproteins. In order to maintain translation fidelity, cells use a highly regulated process known as translational recoding
(28).
During normal eukaryotic translation elongation, the 80S ribosome moves from the 5’ to the 3’ end of the mRNA transcript one codon at a time. When the ribosome moves to a new codon, the aminoacyl site (A-site) of the ribosome is empty except for a single codon
(29). In the peptidyl site (P-site), a tRNA rests with the nascent polypeptide attached
(Figure 3A). A charged tRNA that has a cognate anticodon to the codon in the A-site is brought to the ribosome by eukaryotic elongation factor 1A (eEF1A). If the codon and anticodon match, eEF1A hydrolyzes GTP to GDP and allows accommodation of the tRNA into the A-site (Figure 3B). After the tRNA has been accepted into the ribosome, a reaction occurs in the peptidyl transferase center of the ribosome. This enzymatic reaction, catalyzed by the ribosomal RNA, transfers the nascent polypeptide from the tRNA in the
P-site to the tRNA in the A-site (Figure 3C). Hydrolysis of GTP by the elongation factor eEF2 allows the ribosome to move forward by one codon on the mRNA transcript (Figure
3D). This forward movement shifts the tRNA that was in the original A-site into the P-site.
The A-site is empty and ready to accept a new tRNA to repeat the elongation process. The tRNA that was originally in the P-site is ejected from the ribosome and can be re-charged with a new amino acid.
7 When the ribosome encounters a stop codon such as UGA, UAA, or UAG, instead of an aminoacylated tRNA, a ternary complex consisting of release factors eRF1 and eRF3, as well as GTP binds to the A-site of the ribosome (Figure 4A). Hydrolysis of GTP triggers the cleavage of the mature polypeptide from the tRNA in the P-site of the ribosome (Figure
4B). Finally, the 80S ribosome dissociates into the 60S and 40S subunits and can be recycled for new rounds of translation (Figure 4C).
8
Figure 3: Translational elongation. A. The P-site of the 80S ribosome (brown) is occupied by a tRNA (blue) that has the growing polypeptide (purple) attached. The A-site of the ribosome is open and a charged tRNA with the cognate anticodon to the codon in the A-site by eEF1A. Hydrolysis of 2 GTP to GDP by eEF1A allows for accommodation of the tRNA into the A-site. B. The peptidyl-transferase center of the ribosome catalyzes a peptide bond between the growing polypeptide chain in the P-site and the amino acid on the tRNA in the A-site. C. Once the peptide bond is formed, EF2 catalyzes GTP and causes the translocation of the ribosome by one codon. D. The tRNA in the P-site is moved to the E-site and is ejected from the ribosome. The tRNA in the A-site that now has the nascent polypeptide is now in the P-site and the A-site is ready to accept the next charged tRNA in the sequence. Adapted from (30)
9
Figure 4: Translational termination. A. The release factor complex (yellow) consisting of eRF1, eRF3, a GTP binds the A-site of the ribosome (brown) occupied by a stop codon. B. GTP is hydrolyzed to GDP and the mature polypeptide is cleaved from the tRNA in the P-site. C. The 80S ribosome then dissociates into its 60S and 40S subunits. Adapted from (30)
10 Insertion of Sec into a growing polypeptide
The dual definition of the UGA codon requires that cells have the ability to distinguish between a UGA codon that is a stop signal and one that calls for the insertion of the Sec- tRNA[Ser]Sec. This process is carried out by changing a terminating event into a step in the elongation stage of translation. In order to achieve this, cells utilize a host of cis- and trans- acting factors (31). The basal components required for this process are a 3’ untranslated region (3’ UTR) stem-loop RNA structure known as the Sec Insertion Sequence (SECIS)
(28,32), a SECIS binding protein (SBP2) (33), a specialized elongation factor (EFSec)
(28,34), and the ribosomal protein L30 (35).
Within the 3’ UTR of all eukaryotic selenoprotein mRNAs, there is a stem-loop structure known as the SECIS element that is required for UGA recoding (32). The SECIS of each selenoprotein mRNA is unique, but all share a conserved core characterized by a
GA-quartet of non-Watson Crick base pairs (Figure 5) (36,37). The GA-quartet induces a kink-turn motif in the SECIS element and is required for its ability to mediate UGA recoding (36,38). In the apical loop of the SECIS element, there is a conserved AAR motif
(39). This motif is required for Sec incorporation; however, its exact function is unknown.
The kink-turn motif is important for the binding of SBP2 (36). SBP2 is made up of three domains: a Sec incorporation domain (SID), an RNA-binding domain (RBD) found in the C-terminal end, and an N-terminal domain thought to play a regulatory role (33,40).
The interaction between the SECIS and SBP2 is critical for successful UGA recoding, and it has been shown that both the RBD and SID are required for Sec insertion (41,42). The
RBD of SBP2 contains a 50S ribosomal protein L7Ae motif that recognizes and binds to the kink-turn motif of the SECIS element (33). Binding studies revealed the binding site of
11 SBP2 occurs at the GA-quartet and in the 5’ side of the bulge just below the GA-quartet
(Figure 5 red box) (43). Mutations in SBP2 can result in clinical disease (44). One of the first characterized mutations in SBP2 was found in a patient that had deficiencies in the production of thyroid hormones, a process that is controlled by multiple selenoproteins
(45). Another mutation, an RàQ change in the RBD, decreases the ability of SBP2 to bind to the SECIS element of a subset of selenoproteins (41). Interestingly, many of these patients present unique heterozygous mutations in both copies of the SBP2 gene. This observation suggests that a single copy of SBP2 is sufficient and phenotypes are only seen in patients with two deficient SBP2 genes (44).
Due to its non-canonical structure and degree of modification, Sec tRNA[Ser]Sec cannot be recognized by eEF1A (46), the elongation factor that binds and escorts tRNAs to the ribosomal A-site. This prevents insertion of Sec into any UGA codon but also means there must exist an elongation factor for the tRNA[Ser]Sec. In order to facilitate insertion of
Sec into a selenoprotein, cells use a specialized elongation factor known as EFSec. EFSec can recognize and bind tRNA[Ser]Sec (46). EFSec and SBP2 interact in the presence of a
SECIS element and work together to coordinate tRNA[Ser]Sec into the A-site of the translating ribosome (47). Free EFSec is sterically hindered from entering the A-site of the ribosome. However, when in complex with SBP2 and the SECIS element, EFSec undergoes a conformational change and is able to be accommodated by the A-site of the translating ribosome (48).
The ribosomal protein L30 has also been shown to bind to the SECIS element (35).
L30 can exist freely in the cell but the majority is bound to the 60S ribosomal subunit (49).
L30 binds the SECIS through an L7Ae RNA-binding motif with an overlapping site to that
12 of SBP2 (35). L30 has been shown to compete with SBP2 for binding of the SECIS (35).
However, studies have revealed that SBP2 binds preferentially to the open form of the
SECIS element where L30 can bind both the kinked and open forms (35). A model for recoding of the UGA codon is shown in Figure 6. The SECIS element recruits SBP2,
EFSec, and tRNA[Ser]Sec and brings them to the ribosome at the UGA codon. Once at the ribosome, SBP2 is displaced by L30, thereby tethering EFSec and tRNA[Ser]Sec to the ribosome and allowing for completion of UGA recoding.
In addition to the basal Sec incorporation machinery, there are also accessory proteins that can further regulate the recoding of the UGA codon. Nucleolin is one such protein that has been shown to bind to SECIS elements of essential selenoproteins (50).
Nucleolin is an RNA binding protein that is typically found in the nucleolus and plays roles in transcription as well as remodeling of chromatin (51). Interestingly, studies that used siRNA to knock down nucleolin found that it was involved in promoting the synthesis of essential selenoproteins during Se deficiency (50).
The initiation factor eIF4a3, a member of the DEAD-box family of RNA-dependent
ATPases, has also been shown to modulate UGA recoding (52). Canonically, eIF4a3 binds to the exon junction complex and is a part of the nonsense mediated decay surveillance pathway that is responsible for degrading mRNA transcripts containing premature termination codons. However, eIF4a3 has also been shown to bind to the SBP2 binding site in the SECIS of selenoproteins involved in stress responses (53). Binding of eIF4a3 to the SECIS elements under Se deficient conditions is thought to preclude the binding of
SBP2 and decrease the expression of stress-induced, non-essential selenoproteins to allow the available Sec to be used for expression of essential selenoproteins.
13
Figure 5: The SECIS element. The SECIS element is a 3’ UTR stem-loop structure found in all selenoprotein mRNA transcripts. The SECIS core consists of a GA-quartet that is required for SBP2 binding. The SBP2 binding site is represented by the red box. A helix separates the internal loop from the apical loop. The apical loop contains the AAR-motif that is also required for UGA recoding. Adapted from (52)
14
Figure 6: Model of UGA recoding. SBP2 binds to the SECIS element in the 3’ UTR of a selenoprotein transcript (green). EFSec and Sec-tRNASer[Sec] are recruited and are brought to the A-site of the ribosome (grey) at the UGA codon. L30 displaces SBP2 from the SECIS element and tethers EFSec and the Sec-tRNASer[Sec] to the ribosome. The ribosome then catalyzes the peptide bond between the tRNA in the A-site and the growing selenoprotein polypeptide (purple). Nucleolin and eIF4a3 are accessory proteins that can further modulate this process. Taken from (54).
15
Regulation of Sec insertion into selenoproteins
The insertion of Sec into a growing polypeptide is thought to be inefficient. Using in vitro translation assays, successful UGA recoding events were reported to be between
1-5%. These assays require supplementation with SBP2 and may be missing components that are required for high efficiency Sec insertion. Further investigations were performed using modified luciferase assays to measure the efficiency of the 25 human selenoprotein minimal SECIS elements (Figure 7) (55). These investigations into the function of SECIS elements have shown that the Sec incorporation activity of the highest and lowest activity
SECIS elements span at least three orders of magnitude (55). Studies have also shown that the SECIS elements of different selenoproteins have differing affinities for SBP2 (56,57).
Though the SBP2’s binding affinity for the SECIS elements differs, it cannot explain the large disparity in recoding efficiencies. Attempts to quantify the rate of successful Sec insertion in cells have also been made. Many selenoproteins are degraded when termination occurs at the UGA codon; therefore, it is difficult to determine the percentage of full-length protein compared to total synthesized protein. In order to get around this limitation, ribosome profiling of selenoprotein transcripts was used to measure ribosome density upstream and downstream of the UGA codon. These studies found that the ribosome density downstream of the UGA codon was approximately 10% of the ribosome density upstream of the UGA codon (58,59). This closely reflects the efficiencies of UGA recoding determined by reporter assays.
Originally, the efficiency of UGA recoding was thought to be controlled only by the interaction between SBP2 and the SECIS element of a selenoprotein mRNA. A growing interest in the field is the ability of other mRNA sequences to regulate Sec insertion into
16 selenoproteins further. Studies using the protozoan E. crassus have presented evidence that the 3’ UTR context of the SECIS element can play a role in the recoding ability of SECIS elements (60). In this organism, the Thioredoxin reductase 1 (TR1) gene contains seven
UGA codons, but only the final UGA codon is recoded as Sec. The other six UGAs are decoded as cysteine. When the 3’ UTR of TR1 from E. crassus is replaced with the 3’ UTR of another selenoprotein, all the UGA codons are recoded as Sec. This experiment suggests that there are additional elements within the 3’ UTR of TR1, and likely, other selenoproteins that can have profound effects on UGA recoding and possibly the efficiency of Sec insertion. However, the elements that caused this change were not identified and the mechanism by which this action takes place remains elusive.
A series of studies from the Howard group have characterized a stem-loop structure, the Sec Redefinition Element (SRE), in the coding region of Selenoprotein N (SelN) near the UGA-Sec codon (61,62). Single point mutations in the SelN SRE decreased UGA recoding in a dual luciferase reporter assay (63). Furthermore, SelN protein and mRNA levels were greatly reduced in fibroblasts from patients carrying the same point mutations
(63). Similar structures were identified near the UGA codon in SelT and SelP but have not been verified to behave similarly to the SRE in SelN. In addition to sequences found in the coding region, our lab has previously shown that regions outside of the SECIS element in the 3’ UTR of Selenoprotein S (SelS) mRNA can affect UGA recoding (64), but specific elements were not defined.
17
Figure 7: Recoding efficiencies of SECIS elements from different selenoproteins. Minimal SECIS elements consisting of 100-nucleotides were tested for UGA recoding ability using a luciferase assay. A. HEK293 cells were transfected with these constructs and the UGA recoding efficiency was determined by luciferase activity. B. The same experiment was performed using HepG2 cells. The Selenoprotein S (SelS) SECIS has one of the lowest recoding efficiencies in this assay. Adapted from (55)
18 Selenoproteins in the human body
When a protein contains one or more selenocysteine residues, it is classified as a selenoprotein. Selenoproteins are found in all kingdoms of life but are not present in all organisms. Even among organisms that possess the machinery to produce selenoproteins, the number of selenoproteins can vary and not all organisms express the same array of selenoproteins (65). Humans have 25 known selenoproteins (Figure 8). As Sec is more reactive than Cys at the physiological pH (13), many selenoproteins are involved in redox reactions and contain Sec in their active sites. Interestingly, nearly a third of human selenoproteins do not have an assigned function (Figure 8).
Proteins such as those in the glutathione peroxidase and thioredoxin reductase families have well-defined anti-oxidant and reductase activities. The most abundant selenoprotein in humans is Glutathione Peroxidase 1 (GPx1) (66). The main role of GPx1 in cells is to degrade H2O2 and, in turn, help decrease oxidative damage (67). The involvement of GPx1 in the development of diseases such as cancer (68,69) and T2DM (70-72) has been extensively explored. Another member of the glutathione peroxidase family, GPx4, has a wide range of substrate specificity and is highly expressed in testis (73,74). While GPx4 has enzymatic activity, it also serves as a structural component in mature sperm cells (75).
The three members of the Thioredoxin family in humans are also selenoproteins.
Thioredoxin Reductase 1 (TR1) is one of the most important disulfide reductases found in cells. It plays roles in a plethora of biological processes including apoptosis, resolution of antioxidant stress, and regulation of transcription factors (76,77). TR1 has also been implicated in the prevention of cancer (78,79).
19 Selenoproteins are involved in a variety of biological processes other than redox reactions. For example, the thyroid hormone deiodinases are responsible for the production and regulation of thyroid hormones through oxidative deiodination reactions (80). Another particularly interesting selenoprotein is Selenoprotein P (SelP), the protein responsible for transporting selenium throughout the body (81). In humans, SelP contains 10 Sec residues as well as two different SECIS elements in its 3’ UTR (81). SelP is primarily produced in the liver and then secreted into the blood where it is taken up by other tissues (81). SelP is then broken down into its constitutive amino acids. In order to avoid creating a free pool of Sec in cells, Sec is then degraded by an enzyme called Sec lyase into alanine and hydrogen selenide (HSe-) (82). From there, HSe- can be converted into SeP by SPS2 as previously described (20). The SeP is then used to synthesize Sec from phosphoserine by
SecS, thus completing the cycle of Sec metabolism (20).
The endoplasmic reticulum (ER) is important for maintaining many cellular processes from protein translation, folding, and secretion to Calcium ion (Ca2+) signaling and lipid biosynthesis (83-85). Many selenoproteins are found in the ER membrane and help maintain cellular homeostasis. Selenoprotein N (SelN) is an ER-resident membrane protein found in skeletal muscle tissue that plays a critical role in muscular development
(86,87). Mutations found in SelN have been linked to a group of muscle development disorders termed SelN-related myopathies (88). Many of these mutations are predicted to disrupt the process of UGA recoding and decrease the expression of full-length SelN. One such mutation is found in the SECIS of SelN and inhibits the ability of SBP2 to bind and facilitate Sec insertion (89). Another patient mutation is found in the aforementioned SelN
SRE and results in a decrease in total SelN protein levels (63).
20 Other ER-resident selenoproteins have been implicated in resolving ER stress.
SelM is highly expressed in the brain and loss of SelM is associated with an increase in ER stress and alterations to metabolism (90,91). Overexpression of SelM in a mouse model of
Alzheimer’s disease imparted resistance to the development of the disease (92).
Interestingly, Alzheimer’s disease has been linked to metabolic dysfunction (93). These observations may hint at a unique role of SelM in disease progression. Another ER-resident protein, Selenoprotein T, is highly expressed in pancreatic β-cells (94) and has been reported to play a role in Ca2+ regulation (95). In SelT knockout mice, insulin production and secretion were impaired (94), suggesting that SelT is important for maintaining the viability and health of β-cells.
Selenoprotein S (SelS) and Selenoprotein K (SelK) have both been implicated in the resolution of ER stress caused by misfolded proteins and inflammation (96-99). SelK was associated with the formation of atherosclerotic plaques and knockdown of SelK lead to a decrease of foam cell proliferation during plaque formation (100). SelK also plays a role in Ca2+ flux in immune cells such as macrophages and neutrophils (101). Loss of SelK was found to decrease the immune response (101). Recent work has shown that cleavage of SelK by calpain is tightly regulated in macrophages (102). In the resting phase, Sec- containing SelK is not highly expressed due to calpain cleavage of its C-terminal end (102).
Because of this, the majority of SelK in resting macrophages lacks a Sec residue. When the macrophage is activated, calpain cleavage is inhibited and full-length, Sec-containing
SelK is expressed on the ER membrane and able to perform its roles in Ca homeostasis
(102). These findings show that the presence of the Sec-containing form of selenoproteins may be controlled by processes that take place after successful UGA recoding.
21
Figure 8: The Human selenoproteome. There are 25 selenoproteins in humans. Selenocysteine (red line) is required for their functions. Many are involved with anti- oxidant and redox reactions, but overall have a wide range of functions. Nearly a third of human selenoproteins have a putative or unknown function. Figure from (54)
22 Selenoprotein S
SelS is the primary focus of my studies. SelS is an ER and plasma membrane protein that consists of 189 amino acids and contains Sec as its penultimate residue.
Comparative genomic studies have revealed that, among selenoproteins, SelS is one of the most widely expressed across species (96). Nearly all organisms that can synthesize selenoproteins express a homolog of SelS (96). SelS consists of a short N-terminus from residues 1 to 25 (orange) found on the luminal side of the ER, followed by a single-pass transmembrane domain (grey) (Figure 9). The cytoplasmic tail of SelS contains a coiled- coil domain (blue). This domain is important for interactions with other membrane proteins and may allow dimerization of multiple SelS proteins (103,104). After the coiled-coil domain, SelS is intrinsically disordered (green). The Sec residue interacts with an upstream cysteine residue at position 176. The seleno-sulfide bond that forms between these two amino acids is required for the catalytic activity of SelS (105,106).
SelS helps mitigate ER stress through its role in the Unfolded Protein Response
(UPR) (107). During this response, the translation of proteins is diminished (85,108), chaperone protein levels increase to facilitate proper folding (109,110), and misfolded proteins are degraded (99). Unfolded proteins are removed from the ER in a process called
ER-associated degradation (ERAD). In ERAD, the Valosin-containing protein (VCP), which is an ATPase, and other proteins form a retrotranslocation channel in the ER membrane (107). This channel moves unfolded proteins from the ER to the cytoplasm, where they are ubiquitinated and degraded by the proteasome (99). SelS has been shown to associate with the ERAD channel by binding with VCP through a VCP-interacting motif
(VIM) (111) (Figure 9 pink) and interacting with Selenoprotein K (SelK) (98) and Derlin-
23 1 (103). Mutagenic studies have shown that these interactions with SelK and Derlin-1 are dependent on proline residues 178 and 183 found in the disordered region of the C-terminus of SelS (112). Overexpression of SelS has been reported to have protective effects against
ER stress in multiple systems (113-116). Conversely, knockdown of SelS decreases cell survival in conditions of increased ER stress (113,114,117), likely as a result of the toxic accumulation of unfolded proteins in the ER.
24
Figure 9: Selenoprotein S. The N-terminal end of the SelS (orange) is present in the ER lumen. A single-pass transmembrane domain (grey) is followed by a coil-coiled domain present in the cytosol (blue). A VCP interacting motif (pink) is present. The C-terminus of SelS is intrinsically disordered (green). The Sec residue at position 188 interacts with a Cys residue at position 176. Taken from (111).
25 SelS in biology and disease
SelS was first identified as being differentially regulated in the Type II diabetic mouse model, P. obesus (118). In this study, it was shown that SelS levels were elevated in the fasting diabetic state, but remained normal in the control animals (118). This same study showed that SelS could bind the serum amyloid A1b (SAA) protein, an acute-phase inflammatory protein that has been linked with diabetes and inflammation (118-120). Since its discovery, SelS expression has been shown to be inversely regulated by circulating levels of glucose and insulin (97,118). Overexpression of SelS in the rat hepatoma cell- line, H411E, reduced glucose uptake and impaired the cell’s ability to respond to insulin
(121). These studies suggest that SelS may be involved with the control of glucose metabolism and might contribute to insulin resistance. Recently, SelS was shown to protect vascular endothelial cells against apoptosis induced by prolonged exposure to high levels of glucose (122). Another study found that SelS mRNA levels of omental adipose tissue of
T2DM patients were increased in comparison with nondiabetic patients (119). These mRNA levels were also positively correlated with the homeostatic model assessment of insulin resistance (HOMA-IR), an index of insulin resistance, of each patient. It has also been shown that SelS levels increase in human adipocytes after stimulation with insulin
(123). These results suggest that SelS is involved in insulin resistance development in
T2DM. Furthermore, these findings show that regulation of SelS in adipose tissue is different than in other tissues.
A possible role of SelS in the development of T2DM could be mediated through pancreatic β-cells. It is documented that β-cells undergo apoptosis during the progression of T2DM (124,125). As insulin resistance increases in the pre-diabetic state, β-cells need
26 to produce more insulin to compensate. This increase in insulin leads to a greater protein load on the ER and can induce increased ER stress and inflammation (124). However, as insulin resistance increases, the level of glucose increases and in turn, lowers SelS expression (97,118). As β-cells lose their ability to resolve ER stress, the increased need for insulin leads to an accumulation of misfolded proteins that becomes toxic. Indeed, it has been shown that over-expression of SelS in the Min6 pancreatic islet cell-line enhanced cell viability and could protect against oxidative stress (97). Furthermore, suppression of
SelS was recently shown to induce apoptosis in pancreatic β-cells (126). A decrease in SelS expression due to increased glucose levels could deny the β-cells the means to resolve ER stress and eventually lead to apoptosis, further progressing the development of the disease
(121).
SelS is upregulated under conditions that cause ER stress, such as increased pro- inflammatory cytokine levels and nutrient deprivation (97,127,128). Under conditions of
ER stress or inflammation, levels of pro-inflammatory cytokines increase and activate the transcription factor NF-kB. NF-kB then binds to the ER stress element (ERSE), one of which is found in the promoter of SelS, and increases SelS expression at the transcriptional level (127,129). Treatment of HepG2 cells with the acute-phase cytokines tumor necrosis factor α (TNFα), interleukin 6 (IL-6) and interleukin 1β (IL-1β) led to an increase in SelS protein expression (128). Conversely, knockdown of SelS in macrophages resulted in an increase in production of TNFα, IL-6 and IL-1β (113). Thus, there appears to be a negative feedback loop between SelS expression and cytokine production in cells. A proposed model for the importance of SelS is that SelS decreases pro-inflammatory cytokine levels by mitigating ER stress. Ultimately, this would lead to diminished NF-kB levels and protect
27 against cell death by apoptosis. Indeed, it has been shown that siRNA knockdown of SelS promotes apoptosis in murine macrophage cell-lines (113).
Analysis of single nucleotide polymorphisms (SNPs) within the human SelS gene suggests that regulation of SelS expression is important in vivo. The most extensively studied SNP (-105GàA) occurs in the ERSE of the SelS promoter (127). This SNP leads to a decrease in SelS transcription and an increase in levels of the pro-inflammatory cytokines TNFα and IL-6 (127). These acute-phase cytokines have been implicated in the development and rupture of atherosclerotic plaques, which can lead to myocardial infarction (130). Multiple studies have also linked the -105GàA promoter SNP to gastric cancer and preeclampsia (131,132). Other SNPs within SelS have been linked to conditions such as colorectal cancer in females, ischemic stroke, and coronary heart disease (127,133).
All of these diseases are often attributed to an increased inflammatory state, further reinforcing the importance of SelS in controlling inflammation.
As previously mentioned, recoding of the UGA codon as Sec is inefficient and not always successful. Failure to insert Sec into SelS results in a truncated protein that is only two amino acids shorter than full-length SelS. Selenoproteins often require Sec in their active sites to efficiently perform their function. However, it has been reported that the truncated form of SelS is capable of interacting with VCP to form the ERAD channel and can function during ER stress (112,115). In fact, the only unique function of Sec-containing
SelS is an in vitro peroxidase activity (105,106,111), but the substrate for this activity in cells is unknown. The expression of full-length and truncated proteins is further complicated by the fact that there are two SelS mRNA variants (64), only one of which contains a SECIS element and can produce a Sec-containing protein. Unsuccessful
28 recoding events and the presence of an mRNA variant that only encodes a truncated SelS protein suggests that the production of full-length and truncated SelS may be tightly regulated.
We previously reported that the proximal 60 nucleotides of the SelS 3’ UTR were required for efficient UGA readthrough in an in vitro translation assay (64). This result suggests that cis-acting sequences in the SelS 3’ UTR outside of the SECIS element may regulate the production of full-length and truncated SelS. In this study, we establish that our V5-surrogate assay for UGA recoding is a robust and accurate measure of Sec insertion in cells. Furthermore, we show that the first 91 nucleotides of the SelS 3’ UTR contain two elements: a proximal stem loop (PSL), that indirectly affects UGA recoding in a sequence- independent manner, and a conserved sequence we have named the SelS Positive UGA
Recoding (SPUR) element that is required for efficient Sec insertion in cells.
29 CHAPTER II
MATERIALS AND METHODOLOGY
Sequence conservation and secondary structure prediction
Sequences used to perform conservation alignments and structure prediction were obtained using NCBI and Ensemble databases. Accession numbers for all sequences are listed in Table S3. Conservation alignments were performed using the ClustalOmega server
(https://www.ebi.ac.uk/Tools/msa/clustalo/) (134). Structure prediction and structure conservation was performed using the prediction software from RNAVienna websuites
(http://rna.tbi.univie.ac.at/) (135).
Common Name Scientific Name RNA accession Protein accession Cat Felis catus ENSFCAT00000029655 ENSFCAP00000023286 Chimpanzee Pan troglodytes NM_001114756.1 NP_001108228.1 Cow Bos taurus NM_001046114.2 NP_001039579.2 Dolphin Tursiops truncatus ENSTTRT00000011257 ENSTTRP00000010673 Elephant Loxodonta africana ENSLAFT00000004169 ENSLAFP00000003476 Galago Otolemur Garnettii XM_003788628.1 XP_003788676.1 Gorilla Gorilla gorilla ENSGGOT00000009000 ENSGGOP00000008761 Human Homo sapiens NM_018445.6 NP_060915.2 Hyrax Procavia capensis ENSPCAT00000007799 ENSPCAP00000007301 Macaque Macaca mulatta NM_001114755.1 NP_001108227.1 Marmoset Callithrix jacchus NM_001199926.1 NP_001186855.1 Megabat Pteropus vampyrus ENSPVAT00000012871 ENSPVAP00000012138 Mouse Mus musculus NM_024439.3 NP_077759.3 Lemur Microcebus murinus ENSMICT00000012981 ENSMICP00000011829 Orangutan Pongo abelii NM_001200005.1 NP_001186934 Panda Ailluropoda melanoleuca ENSAMET00000007807 ENSAMEP00000007492 Rabbit Oryctolagus cuniculus ENSOCUT00000025451 ENSOCUP00000017852 Rat Rattus norvegicus NM_173120.2 NP_775143 Sloth Choloepus hoffmanni ENSCHOT00000006369 ENSCHOP00000005624 Tarsier Tarsius syrichta ENSTSYT00000010055 ENSTSYP00000009223
Table 1: List of accession numbers for all sequences used for sequence alignment and structure prediction. ENS- Ensembl database, NM,XM,NP,XP- Genbank database.
30
DNA plasmids
The SelS/V5-UGA construct is previously described (64). All mutagenic primers can be found in Table S4. Mutagenic PCR was performed using QuikChange Lightning
Site-Directed Mutagenesis Kit (Agilent). All other PCR was performed using Phusion
Polymerase (New England Biolabs). Primers for insertion PCR can be found in Table S5.
To create GPx4 and SelK chimeric SECIS constructs, mutagenic primers were used to introduce a PacI site upstream and a NotI site downstream of the SelS SECIS in the SelS
3’ UTR (SelS PacI SECIS mutant and SelS NotI SECIS mutant). Primers corresponding to the SECIS elements of SelK (SelK SECIS PacI Fwd and SelK SECIS NotI Rev) or GPx4
(GPx4 SECIS PacI Fwd and GPx4 SECIS NotI Rev) were used to clone the SelK and GPx4 sequences with the PacI and NotI restriction sites. The SelK and GPx4 SECIS elements were digested and cloned into the PacI/NotI sites of the modified SelS 3’ UTR.
31
PCR Primers Sequence SelS V5 UGA->UAA 5'-AGGCCCGTCATCTGGCGGATAAGGCTTCGAAGGTAAGCCTAT-3' SelS V5 UGA->UAG 5'-AGGCCCGTCATCTGGCGGATAGGGCTTCGAAGGTAAGCCTAT-3' SelS V5 UGA->UGU 5'-AGGCCCGTCATCTGGCGGATGTGGCTTCGAAGGTAAGCCTAT-3' SelS V5 SPURdm 5'-TGAACCCTTAACCCTCGATTGAATTCCCTTACGCACGCTTTTCAC-3' FLAG SelS ΔPSL UGA->UGU 5'-GTCATCTGGCGGATGTGGCTAAGAGAACCCT-3' PSL Loop Deletion 5’-GAATCTTGTTAGTGTCATTTTGACATTAGCAAGATGA-3’ PSL Stem mt 5’-GAATCTCGTTAGCGTCACTTTTGACATTAGCAAGATGA-3’ PSL Disruption 5’-CGTACCGGTTAAGAAAGATCTAACTCTCACTTTTGACAT-3’ SelS UGA 178 5’-CTCCTGGAGATGAGGACGCAGAGGC-3’ SelS UGA 168 5'-TATAAGCCGTTGTCTTGAGAAGGAGGCGGA-3' SelS UGA 138 5’-AGCCCCAGGAGTGAGACAGTCCTG-3’ SelS UGA 88 5’-CTGCTCGACTGTGAATGCAAGAAG-3’ SPUR1 5'-GATGAACCCTTATCCCTCGATTC-3' SPUR2 5'-CCCTTAACCCTGGATTCAATTGC-3' SPUR3 5'-TGAACCCTTAACCCTCGATTGAATTGCCTTACGCACGCTTTTCAC-3' SPUR4 5'-TGAACCCTTAACCCTCGATTCAATTCCCTTACGCACGCTTTTCAC-3' SPUR5 5'-CAATTGCCTAACGCACGCTTTTCAC-3' SPUR6 5'-GCCTTACGGACGCTTTTCACAGTGAC-3' SPUR7 5'-CTTACGCACGGTTTTCACAGTGACT-3' SPUR8 5'-CGCTTTTCACAGTCACTAGCCAAG-3' SPUR9 5'-AGTGACTAGCGAAGGGGAGGTGGG-3' SPUR10 5'-TAGCCAAGGGCAGGTGGGGTTG-3' SPUR11 5'-AGGGGAGGTGGCGTTGATTTC-3' AComp 5'-CCCTTACGGACGCTTTTCACAGTGAC-3' BComp 5'-GCCTTACGCAGGGTTTTCACACTGACT-3'
Table 2: List of all mutagenic primers used in this study. Underlined nucleotides denote nucleotides that were changed.
32 PCR Primers Sequence SelS V5 ΔPSL Fwd 5'-CGTACCGGTTAAGAGAACCCTTAACCCTCGATTCAATTG-3' SelS V5 ΔPSL Rev 5'-CGGGTTTAAACATACAGAAACAAAC-3' FLAG SelS ΔPSL 5'-TCTGGCGGATGAGGCTAAGAGAACCCTTAACCCTCGATTC-3' SelS PacI SECIS mutant 5'-GAGGTTAGATTAATTAACTGCTAGGACAGTCTC-3' SelS NotI SECIS mutant 5'-CTGTATATTTATTTTTAGCGGCCGCACTTTGTAAAAACGAAAC-3' GPx4 SECIS PacI Fwd 5'-CTGATTAATTAATGGAGCCTTCCACCGGCACTCATG-3' GPx4 SECIS NotI Rev 5'-CTGAGCGGCCGCGTGCACGCTGGATTTTCGGGTCTG-3' SelK SECIS PacI Fwd 5'-CTGATTAATTAAACAAGGACTGCTCTGTGTCCTCAC-3' SelK SECIS NotI Rev 5'-CTGAAGCGGCCGCTTTATGGAACTGCATGTCCATGTCAGTAGG-3' Lin34 insertion 5'-AAACTGTAATCGTTCTAAAACTGTAATCGTTCTAGAACCCTTAACCCTCGATTCAATTGCCTTACGCACGC-3' Lin34 DS insertion 5'-TTCTAGAAAACTGTAATCGTTCTAAAACTGTAATCGTTCTATGATTAGCAATCTTGATAAAAGAGGCCTAG-3' HSR stem insertion 5'-CGTACCGGTTAAGTATATATATAAAAATAAATATCTCTATTTTATATATATAGAACCCTTAACCCTCGATTC-3' SelK PSL insertion 5'-AAGAAGCAGACAGAACCCTTAACCCTCGATTCAATTGC-3' SelO PSL insertion 5'-CCCCTGGAGTCTCCCGAGGCCGAACCCTTAACCCTCGATTCAATT-3' TR1 PSL insertion 5’-TAAGCTGTGCATATTAGGAGTTGCTAAAATTTAGCCAATACTAATACTGCATGGGAACCCTTAACCCTCG-3’ SelN SRE insertion 5’-ACCGGTTAAGGGGCGGACTCTCCGGGAGACTGTCCTGGAAAGTTCGCCCGAACCCTTAACCCTCGATTC-3’
Table 3: Table of primers used for insertion PCR
33 Cell Culture
McArdle 7777 (rat hepatoma) and HEK 293 (human embryonic kidney) cells were obtained from American Type Culture Collection (ATCC). Cells were cultured in DMEM supplemented with 10% fetal bovine serum (Gibco) and 60 nM sodium selenite in 5% CO2 at 37°C.
V5-surrogate assay
McArdle 7777 cells and HEK 293 cells were plated at 3x105 cells/well in 2 mL of supplemented DMEM in a 6-well plate 24-hours before transfection. A total of 1 µg of
DNA consisting of 600 ng pcDNA 3.1, 200 ng Firefly luciferase, and 200 ng of the appropriate SelS-V5 plasmid DNA were transfected using 8 µL of Lipofectamine reagent
(ThermoScientific) following the manufacturer protocol. Cells were incubated with
DNA:lipofectamine complexes for 24-hours (McArdle 7777 cells) or 48-hours (HEK 293 cells). After transfection, cells were pelleted and proteins extracted using NP40 lysis buffer
(150 mM NaCl; 50 mM Tris Cl, pH 7.0; 1% NP40). Protein concentrations were measured using a pre-diluted BSA standards (ThermoScientific) with Pierce 660nm Protein Assay
Reagent (ThermoScientific) and measuring absorbance at 660nm using a spectrometer
(SpectraMax190, Molecular Devices). To control for transfection efficiency, Firefly luciferase assays were performed by adding 100 µL of Luciferase Assay System Substrate
(Promega) to 1 µg of protein lysate. Luciferase activity was measured in triplicate using a luminometer (Victor Nivo, Perkin Elmer).
Western Blotting
Proteins were separated by SDS-PAGE and transferred to ImmunoBlot polyvinylidene fluoride (PVDF) membrane (Biorad). The primary antibodies used were α-
34 SelS Prestige (Sigma, HPA010025), α-GAPDH (6C5) (Abcam, ab8245), α-V5 (Invitrogen,
R960), α-FLAG (Sigma, A8592), and α-β-Tubulin (Sigma, T0198). The secondary antibodies used were α-mouse-HRP and α-rabbit-HRP (Jackson Immunochemicals, 150-
035-003 and 111-0450144). Proteins were detected using Immobilon Western HRP substrate (Millipore) and imaged by exposure to Biomax MR film (Kodak) or the
Amersham 600 Imager (GE). Analysis and quantification for Western blots were performed using ImageStudioLite (LI-COR Biosciences). qRT-PCR
After transfection, cells were pelleted and RNA was extracted using Trizol
(Invitrogen) according to the manufacturer protocol. To remove contaminating plasmid
DNA, RNA samples were digested with NaeI (New England Biolabs) followed by RQI
DNase (Promega). RNA quantity was then measured using spectrophotometry at 260 and
280 nm and quality was assessed by agarose gel electrophoresis. RNA (2 µg) was used to make cDNA with the SuperScript VILO cDNA Synthesis kit (ThermoScientific). Primer sequences used for qRT-PCR can be found in Table S6. All reactions were performed in triplicate using 2X Fast SYBR Green Master Mix (Applied Biosystems) and set up in
MicroAmp Fast Optical 96-well reaction plates with optical caps (Applied Biosystems).
Reactions lacking cDNA template or reverse transcriptase were used as controls. Reactions were run on a StepOnePlus Real-Time PCR System (Applied Biosystems). Data was analyzed using StepOne Software (Applied Biosystems).
35
RT-qPCR Primers Sequence V5 Fwd 5'-CCTATCCCTAACCCTCTCCTCGGT-3' SelS Rev 5'-GGTTCATCTTGCTAATGTCAA-3' Firefly Luc Fwd 5'-CGGCGCCATTCTATCCTCTA-3' Firely Luc Rev 5'-AGGAACCAGGGCGTATCTCT-3' Renilla Luc Fwd 5'-AAGAGCGAAGAGGGCGAGAA-3' Renilla Luc Rev 5-'TGCGGACAATCTGGACGAC-3' Rat 18s Qiagen-PPR72042A Human 18s Qiagen-PPH05666E
Table 4: List of primers used for qRT-PCR
36 Immunoprecipitation
McArdle 7777 cells were transfected with the SelS-V5/UGA WT, SelS-V5/UGA
DPSL, Flag-SelS/UGA188 WT or Flag-SelS/UGA188 DPSL constructs using Lipofectamine reagent (Invitrogen). Cells were harvested 24-hours after transfection and lysed with NP40 lysis buffer containing 20% glycerol. Protein lysates (3 mg) were diluted 1:1 with radioimmunoprecipitation assay (RIPA) buffer (SelS-V5 lysates) or NP40 buffer w/ glycerol (Flag-SelS lysates), and subjected to immunoprecipitation with 40 µL of α-V5 agarose beads (Abcam; ab1229; 50% slurry; 0.25 mg/mL bound goat polyclonal antibody) or α-Flag M2 affinity gel (Sigma; A2220; 50% slurry), overnight at 4°C. Lysates from untransfected McArdle 7777 cells were subjected to immunoprecipitation under the same conditions, as controls. The beads were centrifuged at 1500 g for 5 min at 4°C, and the unbound fraction was collected for analysis of immunoprecipitation efficiency. The beads were then washed twice with 1 mL (50 volumes) of RIPA buffer for SelS-V5 lysates or phosphate buffered saline with 0.1% tween-20 for Flag-SelS lysates. The immunoprecipitate was eluted off the beads by boiling with 1X Laemmli buffer and separated by SDS-PAGE (15%), followed by Coomassie staining (Gelcode blue safe protein dye; Thermo scientific) to visualize protein bands.
In-gel digestion and liquid chromatography-tandem mass spectrometry
For protein digestion, the bands corresponding to SelS-V5 or Flag-SelS (by size) were cut to minimize excess polyacrylamide, and divided into a number of smaller pieces.
The gel pieces were washed with a solution of 50% ethanol/5% acetic acid, and dehydrated in acetonitrile. The bands were then reduced with DTT followed by alkylation with iodoacetamide prior to in-gel digestion. SelS-V5 bands were digested in-gel by adding 15
37 µL chymotrypsin (Sigma; 11418467001; 25 ng/µl) in 50 mM ammonium bicarbonate, and incubating overnight at room temperature. Flag-SelS bands were digested in-gel by adding
15 µL endoproteinase GluC (Sigma; 11047817001; 50 ng/µl) in 50 mM ammonium bicarbonate, and incubating overnight at room temperature. The resulting peptides were extracted from the polyacrylamide in two aliquots of 30 µL with a solution of 50% acetonitrile/5% formic acid. These extracts were combined and evaporated in a speedvac and then resuspended in 1% acetic acid (HPLC grade) in a final volume of 30 µL for liquid chromatography-tandem mass spectrometry (LC-MS2) analysis.
The LC-MS system was a ThermoScientific Fusion Lumos mass spectrometry system. The HPLC column was a Dionex 15 cm x 75 µm id Acclaim Pepmap C18, 2µm,
100 Å reversed-phase capillary chromatography column. Five µL of the extract were injected and the peptides were eluted from the column by an acetonitrile/0.1% formic acid gradient (2 – 70% over 2 h), at a flow rate of 0.25 µL/min, were introduced into the source of the mass spectrometer on-line. The microelectrospray ion source was operated at 2.5 kV. The digests were analyzed using a data dependent survey analysis acquiring full scan mass spectra to determine peptide molecular weights and product ion spectra to determine amino acid sequence. The data dependent experiments were searched specifically against the sequence of SelS, where the Sec was replaced with a Cys residue, using SequestHT bundled in the Proteome Discoverer 2.2 program (ThermoScientific). These searches used an MS1 mass tolerance of 10 ppm, a MS2 mass tolerance of 0.6 Da, and considered oxidized
Met, carbamidomethylation of Cys, and Cys + 104.96 Da modification (for selenocysteine identification) as a variable modifications. Positive identification of Selenocysteine
38 containing peptides required the presence of several sequence specific ions along with an
MS1 profile consistent with the presence of selenium.
The digests were also analyzed using Parallel Reaction Monitoring (PRM) in which specific m/z ratios were fragmented and analyzed. Quantification of peptide abundances was performed using Xcalibur 4.0 software (ThermoScientific) to plot PRM chromatograms and integrating the peptide peak areas.
Metabolic labeling with 75Se
McArdle 7777 cells were transfected with Lipofectamine as described above. After
24 hours, media was changed to serum-free DMEM that was supplemented with 100nM
75Se (specific activity, 6.29 µCi/µl; Research Reactor Center, University of Missouri,
Columbia, MO). 24 hours later, cells were washed with PBS and lysed with NP40 buffer as described above. Lysates were resolved by SDS-PAGE and imaged by PhosphorImager
(GE Healthcare).
Statistical Analysis
Wherever applicable, data have been represented as mean ± SD. Data were analyzed by unpaired, two-tailed student’s t test, using GraphPad Prism version 8
(GraphPad Software).
39 CHAPTER III
RESULTS
Recoding efficiency of the SelS 3’ UTR
It has been previously reported that the SelS SECIS element has one of the lowest
UGA recoding efficiencies of all human selenoproteins (55). However, that study was performed using a modified luciferase reporter with minimal SECIS elements. The UGU258 codon of the Firefly luciferase coding region was mutated to a UGA-Sec codon and then
100-nucleotides of the SelS SECIS was added as the 3’ UTR. This construct had very low recoding efficiency compared to the constructs with SECIS elements of the other human selenoproteins. One explanation for this low efficiency is that there are other elements in the SelS 3’ UTR that are required for efficient Sec insertion. To test this, the entire 3’ UTR of SelS was added to the modified Firefly luciferase construct (Luc-SelS/UGA). The recoding efficiency was determined by comparing luciferase activity between the Luc-
UGA-SelS construct and a wild-type luciferase construct that had the original UGU-Cys codon at position 258 (Luc-UGU-SelS). Either the Luc-SelS/UGA or Luc-SelS/UGU construct was transfected into McArdle 7777 cells, a rat hepatoma cell line. McArdle 7777 cells are capable of expressing a wide range of selenoproteins and have been used to study selenoprotein synthesis (52,136). Renilla luciferase was co-transfected as a control.
Lysates from cells transfected with either construct were measured for Firefly luciferase activity. Based on these results, the SelS 3’ UTR has a recoding efficiency of 0.02% (Table
5). In order to compare this recoding efficiency to other selenoproteins, the modified luciferase reporter was tested with either the Selenoprotein K (SelK) 3’ UTR (Luc-
SelK/UGA) or the Glutathione Peroxidase 4 (Luc-GPx4/UGA). Schematics for all
40 constructs used can be found in the Appendix. SelK and GPx4 are considered to have efficient SECIS elements based on previous studies. Comparing luciferase activity between the UGA and UGU versions of Luc-SelK and Luc-GPx4, we found that the SelK and GPx4
3’ UTRs have a recoding efficiency of 0.12 % and 1.16% respectively (Table 5). Based on these results, recoding ability of the 3’ UTRs is as follows: SelS < SelK < GPx4.
Interestingly, while this matches the order of relative efficiencies reported by Latreche et al (55), the entire SelS 3’ UTR appears to be more efficient than the SelS SECIS alone.
Comparing SECIS elements alone, SelS is approximately 50-fold worse than SelK. When the recoding efficiencies of the entire 3’ UTRs of SelS and SelK are compared, the SelS 3’
UTR has only a 6-fold lower efficiency compared to the SelK 3’ UTR.
3’ UTR Activity/µg (UGA) Activity/µg (UGU) Efficiency (%) SelS 46.2 ± .0002 225788 ± 4580 0.02 ± .0003 SelK 446.6 ± .001 362736 ± 2386 0.12 ± .003 GPx4 4270.4 ± .011 367415 ± 10845 1.16 ± .006
Table 5: Recoding efficiencies of 3’ UTRs. Relative luciferase activity of Firefly luciferase UGA258 or UGU258 constructs with either the SelS, SelK, or GPx4 3’ UTRs. Activity is expressed as mean relative luciferase counts/ 1 µg of lysate ± standard deviation. Efficiency is expressed as a percentage of UGA counts to UGU counts.
41 V5-surrogate assay for Sec insertion
In order to study UGA recoding in the endogenous context of SelS, we previously developed a V5-reporter construct (64), shown in Figure 10A. The V5-epitope tag was inserted into the human SelS cDNA between the UGA188 codon and UAA190, the natural stop codon, followed by the entire human SelS 3’ UTR (SelS-V5/UGA). The V5-tag can be detected by Western blot when UGA188 is recoded, whereas termination at UGA188 will result in the truncated form of SelS which lacks V5. Originally, the SelS-V5/UGA construct was validated in an in vitro translation assay (64). To test the assay in cells, the
SelS-V5/UGA plasmid or a vector control were transfected into McArdle 7777 cells. After
24 hours, the transfected cells were harvested and lysates were run on SDS-PAGE for analysis by Western blot. As shown in Figure 10B, the V5-tag is robustly expressed in cells transfected with SelS-V5/UGA but not with vector only. Antibodies against SelS and β-
Tubulin were used to control for SelS expression and protein loading, respectively.
Compared to the endogenous SelS levels in the vector-only control, SelS is overexpressed in the SelS-V5/UGA transfected cells. The β-Tubulin signal was consistent between samples, showing that the increased V5 and SelS signals are not due to differences in protein loading.
We next wanted to validate that the V5-reporter assay was UGA codon-specific.
One possibility is that the SelS 3’ UTR allows for readthrough of stop codons in general.
However, when the UGA was mutated to another stop codon, UAA or UAG (Figure 10A),
V5 was not expressed (Figure 10B). The UAA and UAG constructs both overexpressed
SelS, suggesting that SelS is being translated, but termination occurs before the V5-tag can be synthesized (Figure 10B).
42
Figure 10: Validation of the SelS-V5 surrogate assay. A. Schematic representation of the SelS-V5/UGA construct. The V5-epitope tag was placed between the UGA-Sec and UAA-stop codons of the human SelS coding region followed by the entire SelS 3’ UTR. The UGA-Sec codon was mutated to UAG or UAA as indicated. B. Representative Western blot from three separate experiments of cells transfected with the SelS-V5/UGA and stop codon mutants as well as a vector control. Samples were immunoblotted with α- V5 antibody. The same blot was reprobed for SelS and β-Tubulin.
43 Sec is inserted into the SelS-V5 surrogate assay
A limitation of UGA recoding reporter assays is that they only measure readthrough and cannot distinguish whether Sec or another amino acid has been inserted at the UGA codon. Metabolic labeling of cells with [75Se] in the form of selenous acid has been used to detect proteins that contain selenium. However, this technique cannot determine whether an amino acid other than Sec is incorporated into the nascent selenoprotein. Therefore, we decided to use a mass spectrometric approach to identify the amino acid that is inserted at
UGA codon in our V5-surrogate assay. V5-tagged SelS was immunoprecipitated from
McArdle 7777 cells that were transfected with the SelS-V5/UGA construct. The immunoprecipitation efficiency was robust, with more than 90% depletion of the target.
The immunoprecipitated proteins were analyzed by SDS-PAGE. The band corresponding to V5-tagged SelS was cut out of a Coomassie-stained gel, and the protein was digested in- gel with chymotrypsin. The digested sample was analyzed by LC-MS2 as described in the methods. The spectra generated by MS2 fragmentation were searched specifically against the human SelS protein sequence using Proteome Discoverer 2.2. The analysis revealed 26 unique peptides, covering 64% of the protein sequence. To detect the presence of Sec (U), the survey data were queried for the presence of a Cys residue (C) with an addition of
104.96 Da (C + 104.96). This modification accounts for the mass difference between U and C (41.9 Da), and the mass of the alkylation (57.02 Da). It also considers the isotopic distribution of selenium, which results in the most prominent peak being the M+6 peak
(5.99 Da). The C-terminal Sec-containing peptide, RPGRRGPSSGGUGF, was identified.
The MS2 spectra of this peptide are shown in Figure 11. Also included is the MS1 profile
44 of this peptide which has an isotope pattern consistent with the presence of selenium
(Figure 11, inset).
The digest was further analyzed by parallel reaction monitoring (PRM) to search for the presence of the other 20 standard amino acid residues in place of Sec. The peptide sequences that were queried are shown in Table 6. No other amino acids were detected in the PRM analysis. Therefore, the SelS-V5 surrogate assay is a reliable method for measuring recoding of UGA as Sec in cells.
45
Figure 11: MS2 fragmentation of SelS-V5 Sec-containing peptide. SelS-V5/UGA was transfected into McArdle 7777 cells, immunoprecipitated with α-V5 beads, digested with chymotrypsin, and analyzed by LC/MS2. Data represents analysis from three separate transfections. The most abundant isotope of the Sec-containing peptide was analyzed by MS2 fragmentation. Sequence: RPGRRGPSSGGUG, charge: +3, monoisotopic m/z: 499.2173 Da, [M+H] = 1495.6375. Fragment mass tolerance used for search = 0.6 Da, precursor mass tolerance 10 ppm. Fragments used for search – b; b-NH3 (red); y; y-NH3 (blue). Inset: Isotopic distribution of triply charged Sec-containing peptide.
46
[M+H] m/z z Sequence 1447.7 483.2372 3 RPGRRGPSSGGCGF 1447.7 724.3522 2 RPGRRGPSSGGCGF 1495.6 499.2173 3 RPGRRGPSSGGUGF 1344.7 448.901 3 RPGRRGPSSGGGGF 1358.7 453.573 3 RPGRRGPSSGGAGF 1374.7 458.904 3 RPGRRGPSSGGSGF 1384.7 462.245 3 RPGRRGPSSGGPGF 1386.7 462.917 3 RPGRRGPSSGGVGF 1388.7 463.576 3 RPGRRGPSSGGTGF 1400.8 467.588 3 RPGRRGPSSGGLGF 1400.8 467.588 3 RPGRRGPSSGGIGF Amino acid 1401.7 467.908 3 RPGRRGPSSGGNGF substitutions 1402.7 468.236 3 RPGRRGPSSGGDGF 1415.7 472.58 3 RPGRRGPSSGGQGF 1415.7 472.58 3 RPGRRGPSSGGKGF 1416.7 472.9079 3 RPGRRGPSSGGEGF 1424.7 475.58 3 RPGRRGPSSGGHGF 1434.7 478.906 3 RPGRRGPSSGGMGF 1230.6 410.887 3 RPGRRGPSSGGF 1443.7 481.927 3 RPGRRGPSSGGRGF 1246.6 416.218 3 RPGRRGPSSGGY 1269.6 423.89 3 RPGRRGPSSGGW
Table 6: List of peptides queried to check for amino acid substitutions, instead of Sec, during parallel reaction monitoring experiment
47 Efficiency of the SelS-V5 reporter
In order to analyze the relative UGA recoding efficiency of the SelS-V5/UGA construct, the UGA188 was mutated to UGU, the codon for cysteine (Figure 12A). Insertion of cysteine at UGU188 will result in all translated SelS protein containing the V5-tag. The
SelS-V5/UGA lysate (10 µg) and increasing amounts of the SelS-V5/UGU lysate (0.125
µg, 0.25 µg, 0.5 µg, and 1.0 µg) were run on SDS-PAGE and analyzed by Western blot
(Figure 12B). The V5 expression from increasing amounts of SelS-V5/UGU lysates was quantified to generate a standard curve, which was linear (Figure 12C). The SelS-V5/UGA lysate (10 µg) gave a V5 signal which was between what was observed for 0.125 and 0.25
µg of the SelS-V5/UGU lysate (Figure 12C). Using the equation generated by the standard curve in Figure 12D, the level of V5 expression from SelS-V5/UGA is approximately 2.0% compared to V5 expression from SelS-V5/UGU (100%). The recoding efficiency is 2.5- fold higher than what was previously reported for a luciferase-based recoding assay using the Glutathione Peroxidase 4 (GPx4) SECIS in the same cell-type (137).
As previously mentioned, when using minimal SECIS elements in a Firefly luciferase reporter, the SelS SECIS is approximately 50-fold weaker than the SelK SECIS
(55). Using the full 3’ UTRs in the same luciferase assay, SelS recoding is only 6-fold weaker than that of SelK. To determine the relative recoding ability of SelS and SelK with the entire 3’ UTRs as well as the appropriate coding region, a SelK-V5 construct was generated (Figure 13A). Like the SelS-V5 construct, the SelK-V5 construct contains the entire SelK coding region and the SelK 3’ UTR. Between the UGA-Sec codon and the natural UAA stop codon, a V5-epitope tag was inserted (SelK-V5/UGA). The UGA-Sec codon was mutated to UGU-Cys (SelK-V5/UGU) (Figure 13A). Using these SelK-V5
48 constructs, the same approach taken to determine the SelS-V5/UGA recoding efficiency was used to determine the recoding efficiency of the SelK-V5/UGA construct. After transfection of SelK-V5/UGA or UGU into McArdle 7777 cells, lysates were run on SDS-
PAGE and probed for V5 expression. V5 expression from 10 µg of SelK-V5/UGA was compared to increasing amounts of SelK-V5/UGU (0.125 µg, 0.25 µg, 0.5 µg, and 1.0 µg)
(Figure 13B). The V5 signal from the SelK-V5 constructs was quantified (Figure 13C) and a curve was generated (Figure 13D). Based on the equation from the curve in Figure 13D, the SelK-V5/UGA construct has a recoding efficiency of 5%, only a two-fold increase in recoding efficiency over the SelS-V5/UGA construct.
Taken together, these results suggest that context affects recoding efficiency of the
SelS 3’ UTR. In the luciferase reporter assay, the minimal SECIS element is highly inefficient. By simply replacing the minimal SECIS with the entire SelS 3’ UTR, recoding ability greatly improves. This observation suggests that the SelS 3’ UTR may contain additional cis-elements besides the SECIS element that can affect Sec incorporation.
Furthermore, when the coding region was changed from Firefly luciferase to SelS, the recoding ability of the SelS 3’ UTR was much more comparable to the efficiency of the
SelK-V5/UGA construct.
49
Figure 12: Efficiency of the SelS-V5 reporter. A. The UGA codon in the SelS-V5/UGA construct was mutated to UGU B. Western blot of 10 µg of lysate from McArdle 7777 cells transfected with SelS-V5/UGA or decreasing amounts (1 µg, 0.5 µg, 0.25 µg, and 0.125 µg) of lysate from SelS-V5/UGU transfected cells. Lysates were resolved on SDS-PAGE, transferred to PVDF membrane, and immunoblotted for V5. The V5 signals from SelS- V5/UGA and UGU constructs were quantified and graphed. D. Figure 12C was analyzed using linear regression to determine the slope of the line.
50
Figure 13: Efficiency of the SelK-V5 reporter. A. The UGA codon in the SelK-V5/UGA construct was mutated to UGU. B. Western blot of 10 µg of lysate from McArdle 7777 cells transfected with SelK-V5/UGA or decreasing amounts (1 µg, 0.5 µg, 0.25 µg, and 0.125 µg) of lysate from SelK-V5/UGU transfected cells. Lysates were resolved on SDS- PAGE, transferred to PVDF membrane, and immunoblotted for V5. C. The V5 signals from SelK-V5/UGA and UGU constructs were quantified and graphed. D. Figure 12C was analyzed using linear regression to determine the slope of the line.
51 Distance restriction of SelS UGA recoding
One possible explanation for the context dependence of UGA recoding in SelS is the position of the SelS SECIS element in relation to the UGA-Sec codon. UGA258 in the
Firefly luciferase ORF is nearly 1500 nucleotides away from the SelS SECIS, while the
SECIS is only 250 nucleotides away in the endogenous SelS context. Indeed, it has been shown that the position of the UGA-Sec codon is important and recoding appears to be distance restricted in Thioredoxin reductase 1 (TR1), another selenoprotein with Sec as its penultimate amino acid residue (138). When the UGA-Sec codon in TR1 was moved upstream of its natural position, Sec insertion was drastically decreased (138).
To test if changing the distance between the SelS SECIS and the UGA-Sec codon has an effect on recoding, a series of mutations were generated that moved the UGA codon upstream. The UGA188 codon in the SelS-V5 construct was mutated to UGU-Cys and the codons at positions 178 (UGA178), 138 (UGA138), and 88 (UGA88) were mutated to UGA
(Figure 14A). Constructs were transfected into McArdle 7777 cells and lysates were analyzed by SDS-PAGE and Western blot, shown in Figure 14B. V5 expression for the
SelS-V5/UGA188 was robust. However, when the UGA-Sec codon was moved upstream by ten amino acids (UGA178), V5 expression was greatly reduced. Codon positions UGA138 and UGA88 had no detectable V5 signal. Truncated SelS protein could be detected in all samples suggesting that, while SelS is expressed, UGA recoding was not able to take place
(Figure 14B). Based on these results, moving the UGA-Sec codon of SelS upstream greatly decreases UGA recoding efficiency similarly to what was reported for TR1. This result further supports the idea that the context of the UGA codon is important for recoding by
52 the SelS 3’ UTR. Additionally, these results helps explain the poor recoding efficiency of the SelS 3’ UTR in the Firefly luciferase reporters.
53
Figure 14: Position of the UGA-Sec codon affects UGA recoding in SelS-V5. A. Representative schematic of constructs used. The UGA-Sec (UGA188) codon in the SelS-V5 construct was moved to codon position 178 (UGA178), 138 (UGA138), and 88 (UGA88). The original UGA-Sec codon was mutated to UGU-Cys. B. Representative Western blot. Lysates were run on SDS-PAGE and probed for V5, SelS, and GAPDH. Truncated SelS bands are due to termination at the UGA codon.
54 The proximal SelS 3’ UTR contains two distinct conserved sequences
We previously reported that deletion of the first 60 nucleotides of the SelS 3’ UTR decreased V5 expression in a cell-free translation assay (64). To investigate whether this deletion had the same effect in cells, the D60 construct, which lacks the first proximal 60 nucleotides of the SelS 3’ UTR (Figure 15A), was transfected into McArdle 7777 cells. V5 expression for the D60 construct was greatly reduced compared to the wild-type 3’ UTR
(Figure 15B).
In our previous study, we proposed that a stem-loop structure in the proximal 60 nucleotides of the SelS 3’ UTR is responsible for this effect (64). This proximal stem loop
(PSL) spans nucleotides 3-36 of the SelS 3’ UTR and is conserved in sequence and predicted structure across species (64). To test if the PSL alone is responsible for the decrease in UGA recoding, we deleted nucleotides 3-36 of the SelS 3’ UTR (DPSL) (Figure
15A). Unexpectedly, the V5 signal from the DPSL construct increased by approximately
2.5-fold when compared to the wild-type 3’ UTR construct (Figure 15B and C).
Since the PSL is not responsible for the effect of the 60 nucleotide deletion, we further analyzed the proximal SelS 3’ UTR. Sequences of the SelS 3’ UTR from various species were collected from the NCBI database and analyzed for conservation using the
ClustalOmega server (https://www.ebi.ac.uk/Tools/msa/clustalo/) (134). In addition to the
PSL, we identified a non-conserved region (nucleotides 37-54), as well as a downstream sequence (nucleotides 55-91) that is 76% conserved, which we have named the SelS
Positive UGA Recoding (SPUR) element (Figure 16A). For comparison, nucleotides 93-
335 (from the SPUR element to the beginning of the SECIS) are only 15% conserved. The consensus secondary structure of the proximal 3’ UTR from across species was determined
55 using the prediction software Vienna RNA Websuite (http://rna.tbi.univie.ac.at/) (135).
Our analysis revealed a secondary structure for nucleotides 55-92 consisting of two small stem-loops denoted A and B (Figure 16B and 7C). These secondary structures are strongly conserved across primates, but begin to drop off as the diversity of species increases. The
D60 deletion removes nucleotides 55-60 that are a part of the SPUR element (Figure 16A).
We mutated two strongly conserved nucleotides in this region, C56àG and G61àC, referred to as SPURdm (Figure 15A). This double point mutation greatly decreased V5 expression but did not affect the overexpression of SelS (Fig. 15B and C). There were no differences in loading based on GAPDH levels. Furthermore, deletion of the PSL in the context of the SPURdm was not able to rescue the V5 expression to wild-type levels
(Figure 15B).
56
Figure 15: The proximal region of the SelS 3’ UTR contains elements that affect V5 expression. A. Schematic representation of the SelS-V5/UGA construct and the different 3’ UTR mutants (D60, DPSL, and SPURdm). B. Representative Western blot of lysates from McArdle 7777 cells transfected with SelS-V5 constructs that contained different mutant 3’ UTRs. Lysates were resolved on SDS-PAGE, transferred to PVDF membrane, and immunoblotted for V5, followed by stripping and reprobing for SelS and GAPDH. C. Quantification of the relative V5 expression from the wild-type and DPSL constructs. V5 signals were normalized to SelS and GAPDH signals. V5 levels are expressed relative to wild-type. ***; p < 0.001. Data represents results from three independent experiments (n=3).
57
Figure 16: The proximal SelS 3’ UTR contains two conserved regions. A. The sequences of the first 91 nucleotides of SelS 3’ UTR from different mammals were analyzed for nucleotide conservation by the ClustalOmega alignment program. Nucleotide positions with ‘*’ underneath have complete conservation across all species. Positions with ‘:’ have conservation between groups of strongly similar properties and positions marked with a ‘.’ have conservation between groups with weakly similar properties. The green box shows the first 60 nucleotides of the SelS 3’ UTR. The orange box shows the nucleotides that make up the PSL and the blue box encompasses the SPUR element. Nucleotides C56 and G61 were mutated to form the SPURdm. B. SelS 3’ UTR sequences were analyzed for
58 predicted structure using the RNAlifold program. The color code indicates the number of base pair types found at each position: ochre-2, green-3, turquoise-4, blue-5, violet-6. Less saturated colors indicate that a base pair cannot be formed in some of the sequences. The orange box denotes the PSL and the blue box shows the SPUR element. C. The predicted consensus secondary structure of the first 90 nucleotides of the SelS 3’ UTR. Nucleotides shown in black circles indicate compensatory mutations within the sequences. The probability of a base-pair is indicated on a scale from 0 (blue) to red (1) as shown by the legend. The PSL in orange has a highly conserved structure while the SPUR element (blue box) is made up of two smaller stem-loops.
59 PSL and SPUR mutations do not affect RNA levels or general translation
3’ UTRs have been reported to have a wide variety of functions, including the regulation of mRNA stability and translation (139). Thus, the effect of mutations in the
PSL and SPUR element on V5 expression could be due to mechanisms unrelated to UGA recoding. While the DPSL and the SPURdm did not affect SelS overexpression (Figure
15B), we wanted to confirm that these mutations did not alter mRNA levels or general translation. To test if either the PSL or the SPUR element affects mRNA levels, the SelS-
V5/UGA DPSL and SelS-V5/UGA SPURdm (Figure 17A) constructs were transfected into
McArdle 7777 cells along with a Renilla luciferase plasmid. Total RNA was extracted from each sample and cDNAs were generated. The relative transcript levels were measured by
RT-qPCR using a forward primer in the nucleotide sequence corresponding to the V5- epitope tag and a reverse primer downstream in the SelS 3’ UTR. V5 RNA levels were normalized to Renilla and 18s RNA levels to control for transfection efficiency and loading, respectively. As shown in Figure 17B, the deletion of the PSL or mutation of the
SPUR element had no effect on transfected SelS-V5 mRNA levels when compared to the wild-type 3’ UTR. When the UGA codon in the WT, DPSL, and SPURdm SelS-V5 constructs was converted to a UGU-Cys codon, the mutations in the PSL and SPUR element no longer had an effect on V5 expression (Figure 17C). Taken together, these results show that the differences observed in V5 expression with the DPSL and SPURdm mutants are due to changes in UGA recoding.
60
Figure 17: The PSL and SPURdm do not affect mRNA levels or general translation. A. Schematic representation showing the SelS-V5/UGU construct and the mutant SelS 3’ UTRs. B. qRT-PCR results showing the effects of deletion of the PSL and mutation of the SPUR element on SelS-V5 mRNA transcript levels. RNA levels were normalized to Firefly luciferase and 18s RNA levels and are compared to the wild-type levels, which is taken as 1.0. Data represents results from two independent experiments analyzed in triplicate (n=6). There were no statistically significant differences between groups. C. Representative Western blot from three separate transfections. The UGA-Sec codon was mutated to UGU- Cys and each mutant 3’ UTR was tested to measure its effect on protein expression. Lysates were resolved on SDS-PAGE, transferred to PVDF membrane, and immunoblotted for V5, followed by stripping and reprobing for SelS and GAPDH..
61 PSL and SPUR mutation effects on UGA recoding are not cell-type specific
The experiments described above were all performed in the McArdle 7777 rat hepatoma line. To test whether these effects are cell-line or species specific, we transfected the SelS-V5/UGA constructs containing the DPSL and SPURdm mutations into the Human
Embryonic Kidney cell line, HEK 293. V5 expression increased when the PSL was deleted but it decreased when the SPUR element was mutated (Figure 18). Based on the above results, the SelS 3’ UTR contains two elements that affect UGA recoding in more than one cell-type.
62
Figure 18: Deletion of the PSL and SPUR element mutation effects are not cell-type specific. A. Representative Western blot from HEK293 lysates. Lysates were analyzed by SDS-PAGE and transferred to PVDF membrane. Westerns were immunoblotted for V5, SelS, and GAPDH.
63 Point mutations in the SPUR element inhibit UGA recoding
We first analyzed the SPUR element because it is required for efficient V5 expression. No homology was found between the SPUR sequence and the rest of the human genome. Furthermore, there were no robust hits when we searched RNA binding protein database (RNPDB) (140) to identify RNA-binding proteins that have been shown to bind or crosslink to the region encompassing the SPUR element. Because these searches were not fruitful, we employed a scanning point mutation approach to identify nucleotides that are required for function of the SPUR element. Nucleotides 55-92 of the SelS 3’ UTR are highly conserved across mammalian species and the SPURdm only takes into account a small portion of this conserved region (Figure 19A). Eleven transversion point mutations
(A ßà T or C ßà G) were generated from nucleotides 46 and 104 (SPUR1-11) (Figure
19B). SPUR mutants 1, 2, 10, and 11 flank the highly-conserved region whereas, SPUR3 through SPUR9 focus on some of the most highly conserved nucleotides within the SPUR element. These point mutation constructs were transfected into McArdle 7777 cells and analyzed by Western blot (Figure 19C). V5 expression was quantified and normalized to
SelS and GAPDH (Figure 19D). None of the point mutations had an effect on SelS overexpression. SPUR mutants 1, 10, and 11 had V5 signals similar to the wild-type 3’
UTR. Interestingly, SPUR5 did not show a statistically significant difference in V5 expression compared to the wild-type construct. In contrast, all other point mutations had a negative effect on V5 levels, reducing the signal by 60% or more (Figure 19D). SPUR3 and 4, the two point mutations that make up the original SPUR double mutation, were exceptionally deleterious to UGA recoding, each reducing V5 expression to less than 10%
64 of wild-type levels. Thus, multiple nucleotides in the SPUR element are required for activity.
As described above, the human SPUR element is predicted to form two small stem- loop structures, Stem Loop A and Stem Loop B (Figure 19A). Interestingly, the SPUR4 and SPUR7 mutations are predicted to abolish the structure potential of Stem Loop A and
Stem Loop B, respectively (Figure 19E). To test if either stem-loop is responsible for the activity of the SPUR element, we generated compensatory mutations in SPUR4 and
SPUR7, called AComp and BComp, which are predicted to reform Stem Loops A and B.
However, compensatory mutations restoring the stem-loops did not rescue V5 expression
(Figure 19F). These results suggest that either predicted Stem Loops A and B do not form in cells or that the structures are not important for SPUR activity.
65
Figure 19: Single point mutations in the SPUR element inhibit UGA recoding. A. Schematic showing the predicted structure of the human SPUR element. Nucleotide colors show probability of base pairing from 0 (blue) to 1 (red) as shown in the legend. The two nucleotides mutated in the SPURdm are indicated by arrows. B. Schematic of human SPUR element sequence. The blue box indicates the conserved nucleotides of the SPUR element. Stem Loops A and B are denoted by brackets. Red nucleotides in the sequence show the original nucleotide (WT) and what it was mutated to (MT) C. Representative Western blots showing the effect of single point mutations on V5 expression. Westerns were performed as described in Figure 15B. D. Quantification of Western blots. V5 expression was quantified and normalized to SelS overexpression and GAPDH loading. Normalized V5 levels for each point mutant were compared to wild-type. Data represents results from two independent experiments analyzed in duplicate (n=4). *; p < 0.05, **; p < 0.01, ***; p < 0.001, ****; p < 0.0001, and ns - not significant) E. Predicted structure of mutations made in the human SPUR element. SPUR4 and SPUR7 disrupt Stem Loop A and B, respectively. Compensatory point mutations (Acomp and Bcomp) are predicted to restore the stem-loop structures. F. Representative Western blot of compensatory point mutations from two transfections analyzed in duplicate. Western blots were performed as described above.
66 The SPUR element does not function with other SECIS elements
SECIS elements vary in sequence and structure across selenoproteins. Therefore, we investigated whether the SPURdm would inhibit UGA recoding activity of other SECIS elements. Chimeric 3’ UTRs were created in which the SelS SECIS was replaced with either the SelK or GPx4 SECIS elements (Figure 20A). These were tested in both the wild- type SelS 3’ UTR and SPURdm contexts. As shown in Figure 20B, the level of V5 expression from constructs with the wild-type 3’ UTR varied with the SECIS element.
Compared to the SelS SECIS, V5 expression was lower with the SelK SECIS but higher with the GPx4 SECIS. This may be due to differences in recoding efficiencies when different SECIS elements are placed in the SelS 3’ UTR. As reported above, mutation of the SPUR element in the wild-type SelS 3’ UTR inhibited V5 expression. However, the
SPURdm mutation had little effect when the 3’ UTR contained either the SelK or GPx4
SECIS elements, instead of the SelS SECIS (Figure 20B). These observations cannot be explained by differences in overexpression (SelS) or loading (GAPDH). These data suggest that the SPUR element cannot function with the SelK or GPx4 SECIS elements and that its activity may be specific to the SelS SECIS.
67
Figure 20: Mutation of the SPUR element does not affect recoding directed by the SelK or GPx4 SECIS elements. A. Schematic representation of the SelS-V5/UGA construct. The SelS SECIS element was replaced with the SECIS element from either SelK or GPx4. These SECIS elements were tested in both the wild-type SPUR and the SPURdm contexts. B. Representative Western blot from two independent experiments. WT = wild- type 3’ UTR, dm = SPURdm, and V = vector only. Lysates were resolved on SDS-PAGE, transferred to PVDF membrane, and immunoblotted for V5, followed by stripping and reprobing for SelS and GAPDH.
68 Deletion of the PSL promotes Sec insertion in SelS-V5
Unlike the SPUR element, the SelS PSL inhibits UGA recoding since its deletion increased V5 expression in our surrogate assay. In contrast to the PSL in SelS,
Selenoprotein N (SelN) has a stem-loop structure in its coding region directly downstream of the UGA codon, known as the selenocysteine redefinition element (SRE), which is required for maximal UGA recoding (61). Mutations in the SelN SRE inhibited Sec insertion in an in vitro translation system and cells from patients with the same mutations had reduced SelN protein levels (63). Interestingly, the SelN SRE also promoted readthrough of other stop codons in cells (61). Because of this finding, we were interested in whether deletion of the SelS PSL would also allow for readthrough of other stop codons.
However, when the UGA codon in the SelS-V5/UGA DPSL construct was mutated to either the UAA or UAG stop codon (Figure 21A), SelS was overexpressed, but there was no expression of the V5-tag (Figure 21B).
We next wanted to determine whether deletion of the PSL promoted Sec incorporation in cells. V5-tagged SelS was immunoprecipitated from McArdle 7777 cells that were transfected with either the SelS-V5/UGA WT or the SelS-V5/UGA DPSL construct. The bands corresponding to V5-tagged SelS were processed for LC-MS2 analysis as described in detail in the materials and methods (Chapter II). The immunoprecipitated proteins from the wild-type and DPSL samples were positively identified as human SelS, with 25 and 34 unique peptides, covering 64% and 81% of the protein sequence respectively. Peptides from human SelS were not detected in immunoprecipitations from untransfected cell lysates.
69 A PRM analysis was performed on three independent immunoprecipitations to quantify the recoded SelS protein in the DPSL sample, relative to the wild-type sample.
The experiment was set up to detect the Sec-containing peptide, RPGRRGPSSGGUGF, as well as two abundant peptides from different regions of the SelS sequence (Table 7). The internal peptides (Table 7) of the DPSL samples were increased by 2-fold compared to the wild-type sample. This result is congruent with the increase of V5-containing protein seen with deletion of the PSL when analyzed by Western blot. The peak areas of the
RPGRRGPSSGGUGF peptide were then compared between the wild-type and DPSL samples. The relative abundance of the Sec-containing peptide was 2-fold higher in the
DPSL samples when compared to the wild-type samples (Figure 21C) suggesting that the increase in V5-containing peptide is due to Sec insertion. The ratios of the Sec peptide to the average internal peptide abundance in the wild-type and DPSL samples were similar
(Figure 21D), suggesting that Sec is inserted into all V5-containing protein. To confirm this, the DPSL digest was analyzed by PRM for the presence of the other 20 amino acid residues in place of Sec (Table 6). Based on the list of peptides queried, no amino acid substitutions were detected. Taken together, our results show that deletion of the PSL promotes Sec incorporation into the SelS-V5/UGA construct.
70
Figure 21: Deletion of the SelS PSL increases Sec insertion in the SelS-V5 construct. A. Schematic of the SelS-V5/UGA DPSL construct. The UGA Sec codon was mutated to the UAG or UAA stop codon. B. Representative Western blot of SelS-V5 DPSL constructs that contain either UGA, UAG, or UAA at codon position 188. Lysates were resolved on SDS-PAGE, transferred to PVDF membrane, and immunoblotted for V5, followed by stripping and reprobing for SelS and GAPDH. C. Abundance of Sec-containing peptide from SelS-V5/UGA DPSL sample relative to SelS-V5/UGA WT sample. Data represents three independent experiments. **; p < 0.01. D. Comparison of the ratio of the Sec- containing peptide to the average SelS internal peptide, from SelS-V5/UGA DPSL sample, relative to the ratio of the same from SelS-V5/UGA WT sample. No statistically significant changes were observed (ns). Data represents three independent experiments.
71
[M+H] m/z z Sequence 967.3 484.1823 2 SGEGGGACSWa Internal 2037.1 679.7078 3 DRAAAAVEPDVVVKRQEALa peptides
Table 7: Selenoprotein S internal peptides used for normalization of Sec peptide abundance relative to SelS-V5 protein
72 Other sequences can functionally replace the SelS PSL
The SelS PSL is a 34-nucleotide stem-loop with a 14-base paired stem and a six- nucleotide loop. When the PSL is deleted in the SelS-V5/UGA construct, V5 expression increases suggesting that the PSL is inhibiting UGA recoding. In order to identify the sequences or structures that were important for PSL function, we made mutations that removed the loop (Loop Deletion) or introduced bulges in the stem (Disrupted PSL and
Stem mt) (Figure 22A). If the structure of the SelS PSL was important for its inhibitory effect, these mutations would be expected to behave similarly to the PSL deletion mutation.
However, none of these mutations increased V5 expression compared to wild-type levels
(Figure 22B) suggesting that the exact sequence and/or structure may not be important for the inhibitory activity of the PSL. We then investigated whether the SelS PSL could be functionally replaced by other stem-loops from selenoprotein mRNAs. In addition to SelS, there are seven other mammalian selenoproteins that contain C-terminal Sec residues
(141). Of these, six are predicted to possess a stem-loop near the beginning of their 3’ UTR based on RNA structure prediction software. We tested the proximal stem-loops from
SelK, Selenoprotein O (SelO), and TR1. The SelK PSL is smaller than the SelS PSL with an 8-base pair stem and 6-nucleotide loop, while the SelO and TR1 PSLs are larger and contain multiple bulges in their stem as well as an internal loop (Figure 23A and 23C). The
SelK, SelO, and TR1 PSLs were cloned into the SelS-V5/UGA ∆PSL construct. As reported above, V5 expression increased when the SelS PSL was deleted. However, insertion of either the SelK, SelO, or TR1 PSL into the DPSL construct reduced V5 expression back to wild-type 3’ UTR levels (Figure 23B and 23D).
73 We next tested whether a non-selenoprotein stem-loop could take the place of the
SelS PSL. The SelS PSL was replaced with a 36-nucleotide stem-loop structure in the
Hypoxia Stability Region (HSR) found in the VEGF-A 3’ UTR (142) (Figure 23C). When the HSR was inserted into the DPSL construct, V5 expression was reduced compared to the DPSL construct (Figure 23D). Furthermore, this expression was lower than what was observed with the wild-type 3’ UTR.
Finally, the SRE from SelN was put in place of the SelS PSL (Figure 23C). In SelN, the SRE is found directly downstream of the UGA-Sec codon and promotes UGA recoding
(61,62). While we have shown that the PSL has an opposite effect on recoding than the
SRE, we wondered if the SRE could functionally replace the PSL and inhibit V5 expression. Alternatively, it is possible that the SelN SRE would have the same stimulatory effect on the SelS-V5/UGA construct as it has on recoding of the SelN UGA and increases
V5 expression. However, replacing the SelS PSL with the SelN SRE resulted in reduced
V5 signal when compared to the SelS-V5/UGA DPSL construct (Figure 23D). Like the
HSR, expression was lower than was observed with the construct containing the wild-type
3’ UTR.
Since stem-loops of different sequences and structures can functionally replace the
SelS PSL, we wondered if a structure was required at all. It could be that the PSL is acting as a type of spacer and any sequence could replace it. To test this, we cloned a 34- nucleotide sequence into the DPSL construct (Lin34, Figure 24A). This sequence is of similar GC content as the SelS PSL and is not predicted to form any structure. As shown in Figure 24B, the V5 signal from Lin34 was much lower than what was observed with the
74 DPSL construct and, in fact, was only 25% of what we observed with the wild-type 3’ UTR.
These results suggest that a linear sequence can functionally replace the SelS PSL.
One explanation for the above results is that deletion of PSL increased Sec insertion because the position of the SECIS element relative to the UGA-Sec codon has changed, making the SECIS more efficient. If this is the case, inserting the 34-nucleotide linear sequence in another part of the DPSL 3’ UTR before the SECIS element should also decrease V5 expression. We cloned the linear sequence immediately downstream of the
SPUR element into the non-conserved portion of the SelS 3’ UTR in the DPSL 3’ UTR context (DPSL DS 34; Figure 24A). As shown in the graph in Figure 24B, there was no significant difference in V5 expression between DPSL and DPSL DS 34. Therefore, deletion of the PSL does not increase UGA recoding due to the fact the SECIS element has been moved upstream. Taken together, these results suggest that the SelS PSL is affecting
UGA recoding in the SelS-V5/UGA construct in a sequence and structure independent manner. Furthermore, it appears that the increase in V5 expression seen when the PSL is deleted may be due to changes in spacing and distance of elements in the SelS 3’ UTR other than the SECIS such as the SPUR element.
75
Figure 22: Disruption of the SelS PSL by point mutation and loop deletions has no effect on V5 expression. A. Schematic representation of constructs. The SelS PSL was mutated. Multiple point mutations to disrupt structure (disrupted PSL), single point mutations to introduce bulges into the stem (stem mt), and a deletion in the loop (loop deletion) were tested. Base-pair probabilities are indicated by color 0 (blue) being the lowest probability and 1 (red) being the highest. B. Representative Western blot. Lysates were run on SDS-PAGE, transferred to PVDF membrane and probed for V5, SelS, and GAPDH.
76
Figure 23: Other stem-loops can functionally replace the SelS PSL. A and C. Schematic representation of the SelS-V5/UGA DPSL construct. The SelS PSL was replaced with the proximal stem-loop from either the SelK or SelO 3’ UTRs or the HSR found in the VEGF- A 3’ UTR, the TR1 PSL, or SelN SRE. Base-pair probabilities are indicated by color 0 (blue) being the lowest probability and 1 (red) being the highest. B and D. Representative Western blots. Lysates were resolved on SDS-PAGE, transferred to PVDF membrane, and immunoblotted for V5, followed by stripping and reprobing for SelS and GAPDH.
77
Figure 24: A linear sequence can replace the SelS PSL. A. Schematic representation of constructs used to test the linear sequence. The SelS PSL was replaced with an unstructured 34-nucleotide sequence (Lin34). The linear sequence was also placed downstream of the SPUR element in the DPSL context (DPSL DS 34). B. Quantification of Western blots. V5 expression was quantified and normalized to SelS overexpression and GAPDH loading. Data represents three independent experiments (n=3). Relative V5 levels are expressed relative to WT. No statistically significant changes were observed between DPSL and DPSL DS 34 (ns).
78 The SPUR is required for efficient Sec insertion in the FLAG-SelS
All of the above experiments were performed using the SelS-V5 construct. In the
SelS-V5 construct, the V5-tag is separating the coding region of SelS from the 3’ UTR.
The above studies focusing on the SelS PSL suggest that the position of the SPUR element in relation to the coding region is important for its function. If this is true, the presence of the V5-tag between the SelS coding region and the SPUR element could be having unintended effects on our experiments. To test whether deleting the PSL or mutating the
SPUR element would affect UGA recoding in the absence of the V5-tag, we generated a
SelS construct that had a FLAG epitope tag at the N-terminus. In the FLAG-SelS construct, the UGA codon is in its natural position relative to the SPUR element. FLAG-SelS constructs were made with the wild-type, DPSL, and SPURdm 3’ UTRs (Figure 25A) and transfected into McArdle 7777 cells. After 24-hours of transfection, cells were incubated with [75Se] selenous acid for an additional 24-hours. Lysates were analyzed by SDS-PAGE.
A prominent band corresponding to the FLAG-SelS protein can be seen (Figure 25B).
Unlike with the SelS-V5 construct, there was no change in FLAG-SelS band intensity when the PSL is deleted from the 3’ UTR. However, mutation of the SPUR element still reduces
Sec insertion into FLAG-SelS resulting in decreased signal of the FLAG-SelS band (Figure
25B).
Metabolic labeling of the FLAG-SelS constructs confirmed our result with the
SPUR element mutations in the SelS-V5/UGA construct, but suggested that deletion of the
PSL was not increasing Sec insertion. One possibility is that the increase in V5 signal observed with deletion of the PSL is not due to an increase of Sec insertion, but instead insertion of another amino acid. Insertion of a non-Sec amino acid would be undetectable
79 by metabolic labeling with [75Se] selenous acid. To test this, we moved to a mass spectrometric approach. The FLAG-SelS/UGA constructs containing the wild-type and
DPSL 3’ UTRs (Figure 25A) were transfected into McArdle 7777 cells, the FLAG-SelS was immunoprecipitated and digested with endoproteinase GluC. Mass spectrometry was performed to detect the Sec-containing peptide, GGGACSWRPGRRGPSSGGUG (Figure
26A). We found that deletion of the PSL in the FLAG-SelS/UGA188 context did not increase Sec insertion (Figure 26B). The digest was further analyzed by parallel reaction monitoring (PRM) to search for the presence of the other 20 standard amino acid residues in place of Sec. The peptides that were queried are shown in Table 8. No other amino acids were detected in the PRM analysis. The mass spectrometry supports the metabolic labeling results showing that deletion of the PSL does not change Sec insertion in the FLAG-SelS construct. These results suggest that in the endogenous context the SelS PSL has no effect on UGA recoding. Deletion of the PSL only affects recoding when the V5-tag is present and adds additional space between the SPUR element and the UGA-Sec codon.
80
Figure 25: Mutation of the SPUR element effects Sec insertion in FLAG-SelS but PSL deletion has no effect. A. Schematic representation of the FLAG-SelS construct. A FLAG tag was placed upstream of the SelS coding region. The SPUR element double mutant (SPURdm) and the deletion of the PSL (DPSL) were tested in this construct. B. Metabolic labeling of McArdle 7777 cells transfected with FLAG-SelS constructs. McArdle 7777 cells were transfected with the FLAG-SelS constructs and treated with 75Se. Lysates were resolved by SDS-PAGE and exposed to a phosphorscreen.
81 3+ y18 637.68 A) 100 B) 95 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 90 b-ions
85 G G G A C S W R P G R R G G S S G G U G 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 y-ions 80 1.5 ns 75 506.71 70 506.96 3+ 65 b18 -H2O 586.99 60 506.21 507.21 1.0 55 506.46 50 507.46 3+ b19 505.96 45 650.71 505.71 507.71 40 505.46 507.96 0.5 491.95 Relative Abundance Relative 35 3+ y16 30 581.29 594.96 Sec Peptide Abundance 25 0.0 3+ 20 y17 467.76 618.37 WT ΔPSL 15 604.28 2+ y12 -H2O 2+ 2+ y13 10 3+ 432.91 4+ b13 -NH3 y11 -NH3 y17 669.92 674.90 b + 360.16 463.48 5 4 497.62 548.91 243.16 267.07 377.20 500.38 718.37 751.32 813.38 864.48 0 306.07 783.36 880.42 912.39 968.71 997.66 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 m/z
Figure 26: Deletion of the PSL does not increase Sec insertion into the FLAG-SelS construct. A. Representative analysis of Flag-SelS by mass spectrometry. FLAG-SelS was transfected into McArdle 7777 cells, immunoprecipitated using α-FLAG beads, digested with GluC, and analyzed by mass spectrometry. Data represents analysis from two independent transfections. The most abundant isotope of the Sec-containing peptide was analyzed by MS2 fragmentation. Sequence: GGGACSWRPGRRGPSSGGUG, charge: +4, monoisotopic m/z: 506.7096 Da, [M+H] – 2023.8167. Fragment mass tolerance used for search = 0.6 Da. Fragments used for search – b; b-NH3; b-H2O (red); y; y-NH3; y-H2O (blue). Inset: Isotopic distribution of triply charged Sec-containing peptide. B. Quantification of FLAG-SelS Sec-containing peptide by mass spectrometry
82
[M+H] m/z z Sequence
1977.1018 494.7235 4 GGGACSWRPGRRGPSSGGCG 2025.0463 506.7096 4 GGGACSWRPGRRGPSSGGUG 1873.9889 468.9712 4 GGGACSWRPGRRGPSSGGGG 1888.0160 472.4751 4 GGGACSWRPGRRGPSSGGAG 1904.0154 476.4739 4 GGGACSWRPGRRGPSSGGSG 1914.0542 478.9790 4 GGGACSWRPGRRGPSSGGPG 1916.0701 479.4830 4 GGGACSWRPGRRGPSSGGVG 1918.0424 479.9778 4 GGGACSWRPGRRGPSSGGTG 1930.0972 482.9869 4 GGGACSWRPGRRGPSSGGLG 1931.0412 483.2266 4 GGGACSWRPGRRGPSSGGNG 1932.0258 483.4726 4 GGGACSWRPGRRGPSSGGDG Amino acid 1873.8470 469.2172 4 GGGACSWRPGRRGPSSGGD substitutions 1945.0682 486.7305 4 GGGACSWRPGRRGPSSGGQG 1945.1119 486.7396 4 GGGACSWRPGRRGPSSGGKG 1946.0529 486.9765 4 GGGACSWRPGRRGPSSGGEG 1889.0007 472.7211 4 GGGACSWRPGRRGPSSGGE 1954.0787 488.9806 4 GGGACSWRPGRRGPSSGGHG 1964.1294 491.4747 4 GGGACSWRPGRRGPSSGGMG 1964.1145 491.4830 4 GGGACSWRPGRRGPSSGGFG 1973.1253 493.7411 4 GGGACSWRPGRRGPSSGGRG 1980.1139 495.4817 4 GGGACSWRPGRRGPSSGGYG 2003.1515 501.2357 4 GGGACSWRPGRRGPSSGGWG 1758.8201 586.9449 3 GGGACSWRPGRRGPSSGG 1758.8201 440.4605 4 GGGACSWRPGRRGPSSGG
Table 8: List of peptides queried to check for amino acid substitutions, instead of Sec, during parallel reaction monitoring experiment
83
The effect of the SPUR element is position dependent
These FLAG-SelS results, in conjunction with our data from Figure 24, suggest that distance between the UGA-Sec codon and the SPUR element is important. We hypothesized that the effect of the DPSL mutation may be due to the fact that the position of the SPUR element has changed when the PSL is deleted. In the SelS-V5/UGA construct, the SPUR element is 118 nucleotides downstream from the UGA-Sec codon because of the V5-tag, compared to 61 nucleotides in endogenous SelS. Deletion of the PSL would move the SPUR element 34 nucleotides closer to UGA-Sec. If the distance between the
UGA-Sec and the SPUR element is important, we predict that deletion of the PSL in the
FLAG-SelS context would increase UGA recoding if the UGA was moved upstream of its natural position. To test this, the UGA-Sec codon was moved upstream by 60 nucleotides to codon 168 and the natural UGA-Sec codon at position 188 was mutated to UGU-Cys
(FLAG-SelS/UGA168; Figure 27A bottom two). This FLAG-SelS/UGA168 construct is analogous to the SelS-V5/UGA construct in that the UGA codon is 121 nucleotides upstream of the SPUR element. The UGA168 mutation was also made in the DPSL 3’ UTR context, which decreases the distance between the UGA-Sec and SPUR element to 87 nucleotides and is comparable to the SelS-V5/UGA DPSL construct. The FLAG-
SelS/UGA188 and UGA168 constructs with either the wild-type or DPSL 3’ UTRs were transfected into McArdle 7777 cells and analyzed by Western blot with the α-FLAG antibody. Because there is only a two amino acid difference between full-length and truncated SelS when the UGA codon is at the natural position, there is no differentiation between the two forms (Figure 27B, 188 WT and DPSL). As shown in Figure 27B, most of the FLAG-SelS/UGA168 protein is truncated due to premature termination at UGA168
84 but full-length FLAG-SelS was also detected. As predicted, deletion of the PSL in the
FLAG-SelS/UGA168 construct increased UGA recoding approximately 2-fold compared to the wild-type 3’ UTR (Figure 27C). This is similar to the increase in Sec insertion seen when the PSL is deleted in the SelS-V5/UGA context. These results suggest that position of the SPUR element relative to the UGA-Sec codon is important for its function.
85
Figure 27: Relative position of SPUR element to UGA codon is important for activity. A. Schematic of FLAG-SelS/UGA188 (top two) and UGA168 (bottom two) constructs with either the wild-type or DPSL 3’ UTR. B. Representative Western blot. Vector only (V) FLAG-SelS/UGA188 (188) and UGA168 (168) with either the wild-type (WT) or DPSL 3’ UTR were transfected into McArdle 7777 cells. Lysates were resolved on SDS-PAGE, transferred to PVDF membrane, and immunoblotted for V5, followed by stripping and reprobing for SelS and GAPDH. Full-length and truncated forms of SelS are indicated. Possible SelS degradation bands are marked by a bracket. C. Quantification of Western blots. Expression of the full-length FLAG-SelS protein from the UGA168 lanes were quantified and normalized to GAPDH. Data represents three independent experiments (n=3). Relative FLAG levels are expressed relative to WT. **; p < 0.01.
86 CHAPTER IV
DISCUSSION AND FUTURE DIRECTIONS
Recoding efficiency of the SelS 3’ UTR
SelS is a widely conserved selenoprotein that has two known activities (96). The most well-defined function is its role in the ERAD retrotranslocation channel, which removes unfolded proteins from the ER during ER stress (98,103). SelS also has a peroxidase activity which has only been defined in vitro (106,111). Interestingly, there is the potential to produce two different forms: a full-length, Sec-containing protein and a truncated protein resulting from premature termination at the UGA-Sec codon. The enzymatic functions of many selenoproteins require the Sec residue, and indeed, this is true of the reductase function of SelS (105). Whether the Sec residue plays a role in the ERAD function of SelS has not been directly tested, but several lines of evidence suggest that the truncated protein is sufficient (112,115). Given its important role in ERAD, it is not surprising that expression of SelS is increased at the transcriptional level by ER stress and pro-inflammatory cytokines (97,127,128). However, it remains unknown whether the incorporation of Sec into SelS is regulated.
Initially SelS was considered to have one of the worst SECIS elements in regards to recoding efficiency. However, this conclusion was based on results from a modified luciferase reporter using minimal SECIS elements (55). When the entire 3’ UTR of SelS was tested using the Firefly luciferase reporter, recoding efficiency was greatly improved in comparison to the SelK and GPx4 SECIS elements. In the minimal SECIS experiments,
87 SelS recoding efficiency was approximately 50-fold lower than that of SelK. When the full
3’ UTRs of either SelS or SelK were tested in the reporter, SelS recoding was only 6-fold lower than that of SelK. When the Firefly luciferase ORF was replaced with the SelS-V5
ORF, recoding efficiency of the SelS 3’ UTR was nearly 2.5% compared to the 5% recoding efficiency of the SelK-V5 construct containing the SelK 3’ UTR. Based on these results, the recoding ability of the SelS SECIS is much stronger than initially reported.
Furthermore, recoding efficiency was highest when the natural 3’ UTR and ORF of SelS were used, suggesting that there may be additional elements in the 3’ UTR required for recoding and that the context of the UGA-Sec codon is important. The requirement of context for recoding of the UGA-Sec codon was further illustrated by experiments that moved the position of the UGA codon. When the UGA codon was moved just ten codon positions (30 nucleotides) upstream in the SelS-V5 ORF, V5 expression was greatly reduced. This result is likely due to increasing the distance between the SelS SECIS element and the UGA-Sec codon. Together, these results suggest that insertion of Sec into
SelS is distance restricted. This could explain why the SelS 3’ UTR has poor recoding efficiency in the Firefly luciferase reporter where the UGA codon is outside of the optimal distance. A similar distance restriction was reported for TR1, another selenoprotein containing Sec as near its C-terminus (138). In contrast, the SECIS of TR3 was able to successfully recode UGA independent of the codon position (138).
88 The SelS Positive UGA Recoding (SPUR) element
In this study, we identified the SPUR element, this is the first example of a defined
3’ UTR sequence, beyond the SECIS, that regulates Sec incorporation in cells. The nucleotide sequence of the SPUR element is conserved in the SelS genes of mammals, but it is not found in other genes and is not a known functional motif. Single point mutations across this element decreased UGA recoding, often by 60% or more. Intriguingly, mutation at nucleotide 65 (SPUR5) has little to no effect on UGA recoding, whereas nucleotides on either side are required. This observation raises the possibility that the SPUR element may be bipartite. Furthermore, while the SPUR element is predicted to form two small stem- loops, these structures do not appear to play a role in function based on our mutagenesis studies.
The effect of the SPUR element mutation on Sec insertion was also confirmed in the FLAG-SelS construct. In the FLAG construct, there is no V5-tag between the SPUR element and the UGA-Sec codon. Metabolic labeling of McArdle 7777 cells with [75Se] selenous acid showed that mutation of the SPUR element decreased Sec insertion compared to the wild-type SPUR element. We were also interested in whether the PSL deletion and SPUR mutation would have the same effects in a heterologous gene such as the modified Firefly luciferase assay. However, because the recoding efficiency of the SelS
3’ UTR is so low in this reporter (Table 5), we were unable to confidently answer this question. Furthermore, because the amount of full-length luciferase protein is so low, we are unable to verify that Sec is being inserted into the Firefly luciferase reporter by metabolic labeling or immunoprecipitation and mass spectrometry.
89 We have also shown that the SPUR element mutations are only deleterious to UGA recoding in the presence of the SelS SECIS, but not when the SelS SECIS is replaced with either the SelK or GPx4 SECIS elements. One possible explanation for this is that the
SPUR element has specific long-range interactions with the SelS SECIS. Indeed, there are short sequences in the SPUR element that have the potential to base-pair with different regions in the SelS SECIS (Figure 28A). Intriguingly, the base-pairing occurs in a region of the SelS SECIS element that is important for SBP2 binding and is predicted to be loosely structured (Figure 28A and B, black box). While attempting to create the SelS SECIS cassette used in Figure 19 to replace the SelS SECIS with the SelK or GPx4 SECIS elements, a mutation was made that is predicted to change the structure of the SelS SECIS
(Figure 28B). When this SECIS mutation was tested with the wild-type SPUR element, the recoding ability of the SelS SECIS was not affected and V5 expression was similar between the wild-type and mutant SECIS constructs (Figure 28C). Unexpectedly, the SECIS mutation was able to rescue V5 expression in the context of the SPURdm. These results suggest that the SelS SECIS and the SPUR element may be interacting. One possible mechanism to explain this result is that the SPUR element base-pairs with the SelS SECIS element, stabilizes it, and allows for efficient recoding. When the SECIS element is mutated, the structure becomes more stable, and the SPUR element is no longer required for the SelS SECIS to efficiently insert Sec at the UGA codon. An experiment to determine if the SPUR element and the SelS SECIS base-pair is by an RNA electromobility shift assay (REMSA). The RNA sequence of the SelS proximal 3’ UTR would be labeled using
32P-UTP. This probe could be incubated with the SelS SECIS in trans. If interactions are
90 present between the two RNAs, the complex will move slower through the gel and cause a shift in the probe.
Understanding this observation may be key to understanding the mechanism of action of the SPUR element; however, there are many challenges. Perhaps the most important consideration is that generating mutations in the SelS SECIS, especially near the
SPB2 binding site, could lead to a non-functional SECIS element. Indeed, initial efforts to create single point mutations in the SECIS that could rescue recoding in the SPURdm 3’
UTR were encumbered by this issue. While structure and base-pair prediction give clues into what portion of the SelS SECIS may be of interest for interaction with the SPUR element, the nucleotides that are responsible still need to be experimentally determined.
The potential base-pairing between the SPUR element and the SECIS encompasses SPUR2 and SPUR3 which had inhibitory effects on V5 expression (Figure 19C). Mutating these two nucleotides may disrupt the base-pair potential between the SPUR and the SECIS. One interesting experiment would be making compensatory point mutations in the SECIS element to restore base-pairing. If this model is correct, compensatory mutations should rescue V5 expression. However, a negative result for this experiment is not conclusive. It is also possible that any interactions between the SPUR element and the SECIS are much more complex than simple base-pair interactions. If this SECIS is mediated by RNA- binding proteins, compensatory mutations in the SECIS element may not be enough to rescue V5 expression.
91
Figure 28: Mutations in the SECIS element rescue UGA recoding of the SPUR mutation. A. Schematic of possible base pairing between the SPUR element (grey) and the SelS SECIS (red). Asterisks above base-pairs indicate SPUR3 and SPUR4, the two mutations that make up SPURdm. B. Predicted structure for the wild-type (WT) and mutated (mt) SelS SECIS. Bold nucleotides show the conserved AUGA core required for function. The box on the wild-type SECIS shows where SBP2 would bind. C. Representative Western blot of the effect of the SPURdm on the wild-type and mutant SelS SECIS elements. Lysates were resolved on SDS-PAGE, transferred to PVDF membrane, and immunoblotted for V5, followed by stripping and reprobing for SelS and GAPDH.
92 RNA-binding proteins
Our study of the SPUR element to this point has focused on sequence and structure requirements for function. Understanding the mechanism of action likely hinges on the identification of a binding protein. Preliminary data from our lab using REMSA with the radiolabeled proximal SelS 3’ UTR sequence and lysates from McArdle 7777 cells suggests that a protein binds to the proximal 3’ UTR of SelS (Figure 29) (Narayan, unpublished). This shift is absent when the SPUR element is mutated. Notably, the
REMSA shift does not require the SelS SECIS to occur. Efforts are being made to identify the protein or proteins responsible for this shift. Previously in the lab, we have used an
RNase-assisted affinity purification approach to identify proteins that bind to the 3’ UTR of selenoproteins mRNA transcripts adapted from Michlewski and Caceres (143). A full explanation and protocol for this technique can be found in Cockman et al (144). Briefly, this purification process aims to identify proteins that bind differentially between the wild- type SPUR element and mutant SPUR elements. To begin, either the wild-type or mutant
RNA is bound to agarose beads and then incubated with lysates from McArdle 7777 cells.
After incubation and multiple washes to remove any unbound protein, RNase A and T1 are added to the beads. The RNase cleaves the RNA, thereby releasing RNA-binding proteins and leaving behind proteins that bound non-specifically to the agarose beads. The eluates are then run on SDS-PAGE and stained with Coomassie blue. Differential protein bands between the wild-type and SPURdm samples are then excised and analyzed by mass spectrometry. Potential protein candidates would then be verified by siRNA knockdown in cells. Lysates from the knockdown cells would be used in the REMSA to determine whether the protein of interest was responsible for the shift.
93 Identification of a protein that binds to the SPUR element would accelerate understanding of the biological importance and mechanism of action. If the activity of the
SPUR element is regulated, identifying proteins that bind would provide insight into what processes or stress conditions require full-length, Sec-containing SelS. Furthermore, the exact binding site of the protein to the SPUR element could be determined by footprinting and RNase-protection assays. Knowing where these proteins are binding may also be able to explain why changes in the SelS SECIS structure can rescue UGA recoding when the
SPUR element is mutated.
One intriguing possibility is that the proteins of the Sec insertion machinery bind to the SPUR element. The SelS SECIS has been shown to have a weak affinity for SBP2
(57). The SPUR element may act by binding SBP2 and transferring it to the SelS SECIS.
When the SECIS structure is changed due to mutations near the SECIS element, the SelS
SECIS’ affinity for SBP2 may increase and the SPUR element is no longer required to facilitate SBP2 binding. Similar possibilities exist with the other required factors of UGA recoding. Testing whether or not these proteins are binding to the SPURdm can be performed by siRNA knockdown followed by an analysis of lysates by REMSA. These experiments can be done alongside the affinity purification and may be able to quickly identify proteins binding to the SPUR element.
94
Figure 29: A protein binds to the SPUR element. Representative REMSA using 32P- UTP labelled wild-type and SPURdm RNA probes incubated with lysates from McArdle 7777 lysates. Increasing amounts of lysates were incubated with either wild-type of SPURdm probe. Complexes were run on a native gel along with a probe-only control (probe). Gels were dried and exposed on Biomax MR film. Image courtesy of Vivek Narayan.
95 Mechanistic model of the SPUR element
It is likely the role the SPUR element plays in promoting Sec insertion into SelS is through its interactions with the SelS SECIS element. A possible role for the SPUR element is recruitment of the SelS SECIS and the Sec recoding machinery and bringing it into proximity of the ribosome at the UGA codon. (Figure 30). When the SPUR element is mutated, this interaction cannot take place and the recoding efficiency is greatly impaired.
Whether the interaction between the SelS SECIS and the SPUR element is mediated by
RNA binding proteins is yet to be determined.
This model may also explain why deletion of the PSL only increases recoding in the SelS-V5 construct and not in the FLAG-SelS construct. If ribosome proximity is important, then addition of the V5-tag between the UGA codon and the SPUR element can sterically hinder SECIS:ribosome interactions that are needed for Sec insertion. Deletion of the PSL in SelS-V5 could relieve this steric hinderance and allow the SelS SECIS to successfully meditate Sec insertion. This same reasoning can be used to also explain the distance restriction exhibited by the SelS SECIS (Figure 14). As the UGA-Sec codon is moved further upstream, recruitment of the SECIS element by the SPUR element would not position the SECIS near the upstream ribosome and recoding efficiency becomes lower the further the UGA codon is moved upstream from the SPUR element.
96
Figure 30: Model for the mechanism of action of the SPUR element. The SelS SECIS (red stem-loop) associates with SBP2 (green), EFSec (red), and the Sec tRNA (black). The interaction of the SPUR element (grey) with the SECIS brings the SECIS into position to interact with the ribosome at the UGA-Sec codon and facilitate Sec insertion.
97 The proximal stem loop
In contrast to the SPUR element, deletion of the PSL stimulated Sec incorporation in the V5-surrogate assay. We found that the SelS PSL could be replaced with other stem- loops or even a linear sequence. These results suggest that removing the PSL indirectly affects Sec incorporation. We excluded the possibility that changing the position of the
SelS SECIS was responsible for the increase in UGA recoding observed with the deletion of the PSL. Our experiments using the FLAG-SelS constructs support a model in which the efficiency of Sec incorporation is dependent on the distance between the SPUR element and the UGA-Sec codon. One could envision that the SPUR element may recruit the SelS
SECIS and facilitate the delivery of the tRNA[Ser]Sec to the ribosome at the UGA-Sec codon.
By increasing the distance between the SPUR and the UGA-codon, the interaction with the ribosome becomes less sterically favorable and Sec insertion is impaired.
Our results raise an intriguing question: why is the SelS PSL conserved if it does not play a role in UGA recoding? It is possible that the PSL is important for Sec incorporation in other tissues, developmental stages, or under specific stress conditions.
Alternatively, the PSL may play a role in processes other than UGA recoding. We have shown that the PSL has no effect on mRNA stability or general translation but did not test if has a role in splicing. We previously reported that there are two SelS mRNA variants produced through alternative splicing (64), only one of which contains a SECIS element.
Both of these SelS transcripts are expressed in cell lines (64) and human tissues (Cockman
EM, unpublished), with the SECIS-containing transcript representing 85-90% of the total
SelS mRNA. The splice site for the SelS mRNA variant lacking the SECIS is between nucleotides 13 and 14 of the 3’ UTR and is predicted to be sequestered in the 5’ stem of
98 the PSL (64). The structure of the PSL may be conserved across species due to evolutionary pressure to prevent splicing between nucleotides 13 and 14 and promote expression of the
SECIS-containing SelS mRNA transcript.
There are eight human selenoproteins that contain C-terminal Sec residues. Our analysis revealed that six of these eight selenoproteins (including SelS and SelK) have the potential to form stem-loops in their 3’ UTR immediately downstream of the canonical stop codon. The PSLs from TR1 and SelK do not encompass a splice site and their function is unknown. We generated a SelK-V5/UGA construct in which the UGA-Sec codon is moved upstream from its natural position due to the V5-tag. Unlike our results with SelS, deletion of the SelK PSL had no effect on UGA recoding when compared to the wild-type
3’ UTR. This result could be explained by the fact that SelK lacks a conserved sequence immediately downstream of the PSL that would be analogous to the SPUR element, based on our computational analysis. TR1 is another selenoprotein with a C-terminal Sec residue.
Like SelK, we have not identified a SPUR-like element in the proximal region of the TR1
3’ UTR. Interestingly, Turanov et al reported that efficient Sec incorporation into TR1 only occurred when the UGA-Sec codon was in its natural C-terminal position (138). Much like our SelS-V5 and FLAG-SelS distance studies, when the UGA codon of TR1 was moved upstream of its natural position, UGA recoding was greatly diminished. Unlike SelS,
Turanov et al found that this restriction of Sec incorporation appears to be defined by the
TR1 SECIS element and its distance to the UGA codon. Deletion of the TR1 PSL in the wild-type construct did not increase Sec insertion, but this was not tested in the context of the upstream UGA-Sec codons.
99
Tools for future studies
In this study, we have developed important new tools for analyzing Sec incorporation in mammalian cells. Standard approaches have used either modified reporter constructs which measure UGA recoding or metabolic labeling with [75Se] selenous acid.
However, neither of these approaches specifically identify Sec as the incorporated amino acid nor do they exclude the possibility of the insertion of other amino acids. Using mass spectrometry, we have explicitly shown that Sec is incorporated into the SelS-V5 protein.
Furthermore, none of the other 20 standard amino acids were detected in the peptide, suggesting that only Sec is inserted at the UGA codon. To our knowledge, this study is the first report that a cell-based reporter assay for UGA recoding assay is a direct measure of
Sec incorporation. The success of the SelS-V5 construct in identifying the SPUR element is likely to lead to a similar exploration of other C-terminal selenoproteins. Our lab has already generated a SelK-V5/UGA construct and similar constructs can be made for TR1 and other C-terminal Sec-containing selenoproteins. Using these V5 reporters, mutagenesis of conserved regions in the 3’ UTRs of different selenoproteins may lead to the discovery of more potential regulatory elements for UGA recoding. While we were unable to identify conserved, SPUR-like elements in the proximal 3’ UTRs of SelK and TR1, our computational analyses do not exclude the existence of a SPUR-like element in more distal regions of these 3’ UTRs. This is an interesting possibility for future projects.
In addition to studying the mechanism of Sec insertion, the mass spectrometry approaches we developed could be used to study expression and regulation of endogenous
SelS. The efficiency of Sec insertion into endogenous selenoproteins is an open and
100 important area of interest in the field. Reporter constructs and selenium labeling would suggest that the rate of Sec insertion is near 5%. Indeed, we calculate the recoding efficiency of our SelS-V5/UGA construct to be 2.5%. UGA recoding efficiency has been estimated using ribosome profiling by comparing reads from before and after the UGA-
Sec codon. While this approach works for most selenoproteins, the selenoproteins with Sec near the C-terminal end do not have ribosome reads after the Sec residue due proximity to the stop codon. With the described mass spectrometry techniques, we are now in a position to determine the abundance of endogenous full-length and truncated SelS and other C- terminal selenoproteins in cells. Furthermore, we can explore how Sec-status in these proteins differ between cell-types, disease states, stress conditions, and developmental stages. Of particular interest would be the Sec-status of SelS during ER stress. During ER stress, SelS is transcriptionally upregulated, but it remains unknown if the amount of full- length SelS changes. It would also be interesting to study the expression of full-length and truncated SelS in animal models of T2DM, where SelS was first discovered, and other inflammatory diseases. Many of these experiments are currently underway and are likely to yield intriguing results.
In order to study the potential role of regulation of Sec insertion into SelS, new approaches will need to be developed. Generation of cell-lines expressing mutations in the
SelS ORF and SPUR element would be powerful tools to answer these questions. Cells expressing the mutant SPURdm can be subjected to a large variety of stresses including
ER stress, hypoxia, and glucose deprivation to study the importance of the SPUR element in SelS expression and response to stress. Additional experiments can also be performed to create mutant cell-lines that express the truncated form of SelS or a variant that contains
101 Cys instead of Sec. These cells will be instrumental in understanding the role of the Sec residue in SelS function and biology.
Conclusion
Finally, our findings have implications for future studies on elucidating the mechanism and regulation of Sec incorporation. Very few studies have investigated whether 3’ UTR sequences outside of the SECIS element contribute to the efficiency of
Sec insertion. Our discovery of the SPUR element in SelS highlights the need to consider this possibility when analyzing other selenoprotein mRNAs. The SelS SECIS has been reported to have weak activity but this conclusion was based on the analysis of a minimal
SECIS of ~100 nucleotides (55). Under basal conditions, the SPUR element is required for efficient UGA recoding. It is tempting to speculate that the activity of the SPUR may be regulated, which could affect the production of the Sec-containing and truncated SelS proteins. Future studies on SelS will focus on understanding the mechanism by which the
SPUR element effects UGA recoding and further elucidating how SelS is regulated post- transcriptionally.
102 Appendix
103 REFERENCES CITED
1. Yu, S. Y., Zhu, Y. J., and Li, W. G. (1997) Protective role of selenium against hepatitis B virus and primary liver cancer in Qidong. Biol Trace Elem Res 56, 117- 124 2. Qiao, Y. L., Dawsey, S. M., Kamangar, F., Fan, J. H., Abnet, C. C., Sun, X. D., Johnson, L. L., Gail, M. H., Dong, Z. W., Yu, B., Mark, S. D., and Taylor, P. R. (2009) Total and cancer mortality after supplementation with vitamins and minerals: follow-up of the Linxian General Population Nutrition Intervention Trial. J Natl Cancer Inst 101, 507-518 3. Meyer, F., Galan, P., Douville, P., Bairati, I., Kegle, P., Bertrais, S., Estaquio, C., and Hercberg, S. (2005) Antioxidant vitamin and mineral supplementation and prostate cancer prevention in the SU.VI.MAX trial. Int J Cancer 116, 182-186 4. Beck, M. A., Levander, O. A., and Handy, J. (2003) Selenium deficiency and viral infection. J Nutr 133, 1463s-1467s 5. Avery, J. C., and Hoffmann, P. R. (2018) Selenium, Selenoproteins, and Immunity. Nutrients 10, 1203 6. Rayman MP. (2012) Selenium and human health. Lancet 379, 1256-1268 7. Sarkozy, M., Szucs, G., Pipicz, M., Zvara, A., Eder, K., Fekete, V., Szucs, C., Barkanyi, J., Csonka, C., Puskas, L. G., Konya, C., Ferdinandy, P., and Csont, T. (2015) The effect of a preparation of minerals, vitamins and trace elements on the cardiac gene expression pattern in male diabetic rats. Cardiovasc Diabetol 14, 85 8. Prabhu, K. S., and Lei, X. G. (2016) Selenium12. in Adv Nutr. pp 415-417 9. Coppinger RJ, and Diamond AM. (2001) Selenium deficiency and human disease. in Selenium: Its Molecular Biology and Role in Human Health (Hatfield, D. ed.), Kluwer Academic Publishers, Norwell, Massachusetts. pp 219-234 10. Zhang, R., Guo, H., Yang, X., Zhang, D., Li, B., Li, Z., and Xiong, Y. (2019) Pathway- based network analyses and candidate genes associated with Kashin-Beck disease. Medicine (Baltimore) 98, e15498 11. Bellis, G., Roux, F., and Chaventre, A. (1998) Endemic cretinism in a traditional society in Mali: from the collectivity to the individual. Coll Antropol 22, 23-30 12. Goldhaber, S. B. (2003) Trace element risk assessment: essentiality vs. toxicity. Regul Toxicol Pharmacol 38, 232-242 13. Byun, B., and Kang, Y. (2011) Conformational Preferences and pKa Value of Selenocysteine Residue. Biopolymers 95, 345-353 14. Arner, E. S. (2010) Selenoproteins-What unique properties can arise with selenocysteine in place of cysteine? Exp Cell Res 316, 1296-1303 15. Snider, G. W., Ruggles, E., Khan, N., and Hondal, R. J. (2013) Selenocysteine Confers Resistance to Inactivation by Oxidation in Thioredoxin Reductase: Comparison of Selenium and Sulfur Enzymes†. Biochemistry 52, 5472-5481 16. Hazebrouck, S., Camoin, L., Faltin, Z., Strosberg, A. D., and Eshdat, Y. (2000) Substituting selenocysteine for catalytic cysteine 41 enhances enzymatic activity
104 of plant phospholipid hydroperoxide glutathione peroxidase expressed in Escherichia coli. J Biol Chem 275, 28715-28721 17. Berry, M. J., Maia, A. L., Kieffer, J. D., Harney, J. W., and Larsen, P. R. (1992) Substitution of cysteine for selenocysteine in type I iodothyronine deiodinase reduces the catalytic efficiency of the protein but enhances its translation. Endocrinology 131, 1848-1852 18. Zhong, L., and Holmgren, A. (2000) Essential role of selenium in the catalytic activities of mammalian thioredoxin reductase revealed by characterization of recombinant enzymes with selenocysteine mutations. J Biol Chem 275, 18121- 18128 19. Fan, C. D., Fu, X. Y., Zhang, Z. Y., Cao, M. Z., Sun, J. Y., Yang, M. F., Fu, X. T., Zhao, S. J., Shao, L. R., Zhang, H. F., Yang, X. Y., and Sun, B. L. (2017) Selenocysteine induces apoptosis in human glioma cells: evidence for TrxR1-targeted inhibition and signaling crosstalk. Sci Rep 7, 6465 20. Xu, X., Carlson, B., Mix, H., Zhang, Y., Saira, K., Glass, R., Berry, M., Gladyshev, V., and Hatfield, D. (2007) Biosynthesis of selenocysteine on its tRNA in eukaryotes. PLoS Biology 5, e4 21. Diamond, A. M., Choi, I. S., Crain, P. F., Hashizume, T., Pomerantz, S. C., Cruz, R., Steer, C. J., Hill, K. E., Burk, R. F., McCloskey, J. A., and Hatfield, D. L. (1993) Dietary selenium affects methylation of the wobble nucleoside in the anticodon of selenocysteine tRNA([Ser]Sec). J Biol Chem 268, 14215-14223 22. Diamond, A., Dudock, B., and Hatfield, D. (1981) Structure and properties of a bovine liver UGA suppressor serine tRNA with a tryptophan anticodon. Cell 25, 497-506 23. Hatfield, D., Diamond, A., and Dudock, B. (1982) Opal suppressor serine tRNAs from bovine liver form phosphoseryl-tRNA. Proc Natl Acad Sci U S A 79, 6215- 6219 24. Carlson, B. A., Moustafa, M. E., Sengupta, A., Schweizer, U., Shrimali, R., Rao, M., Zhong, N., Wang, S., Feigenbaum, L., Lee, B. J., Gladyshev, V. N., and Hatfield, D. L. (2007) Selective restoration of the selenoprotein population in a mouse hepatocyte selenoproteinless background with different mutant selenocysteine tRNAs lacking Um34. J Biol Chem 282, 32591-32602 25. Carlson, B. A., Xu, X. M., Gladyshev, V. N., and Hatfield, D. L. (2005) Selective rescue of selenoprotein expression in mice lacking a highly specialized methyl group in selenocysteine tRNA. J Biol Chem 280, 5542-5548 26. (2004) Finishing the euchromatic sequence of the human genome. Nature 431, 931-945 27. Sun, J., Chen, M., Xu, J., and Luo, J. (2005) Relationships among stop codon usage bias, its context, isochores, and gene expression level in various eukaryotes. J Mol Evol 61, 437-444 28. Tujebajeva, R. M., Copeland, P. R., Xu, X. M., Carlson, B. A., Harney, J. W., Driscoll, D. M., Hatfield, D. L., and Berry, M. J. (2000) Decoding apparatus for eukaryotic selenocysteine insertion. EMBO Rep 1, 158-163
105 29. Dever, T. E., and Green, R. (2012) The elongation, termination, and recycling phases of translation in eukaryotes. Cold Spring Harb Perspect Biol 4, a013706 30. Translation. Protein from RNA. http://www.sliderbase.com/spitem-695-1.html 31. Driscoll, D. M., and Copeland, P. R. (2003) Mechanism and regulation of selenoprotein synthesis. Annu Rev Nutr 23, 17-40 32. Berry, M., Banu, L., Chen, Y., Mandel, S., and Kieffer, J. (1991) Recognition of UGA as a selenocysteine codon in type I deiodinase requires sequences in the 3' untranslated region. Nature 353, 273-276 33. Copeland, P., Fletcher, J., Carlson, B., Hatfield, D., and Driscoll, D. (2000) A novel RNA binding protein, SBP2, is required for the translation of mammalian selenoprotein mRNAs. EMBO J 19, 306-314 34. Fagegaltier, D., Hubert, N., Yamada, K., Mizutani, T., Carbon, P., and Krol, A. (2000) Characterization of mSelB, a novel mammalian elongation factor for selenoprotein translation. Embo j 19, 4796-4805 35. Chavette L, Brown BA, and Driscoll DM. (2005) Ribosomal protein L30 is a component of the UGA-selenocysteine recoding machinery in eukaryotes. Nat Struct Mol Biol. 12, 408-416 36. Walczak, R., Westhof, E., Carbon, P., and Krol, A. (1996) A novel RNA structural motif in the selenocysteine insertion element of eukaryotic selenoprotein mRNAs. Rna 2, 367-379 37. Rother, M., Resch, A., Gardner, W. L., Whitman, W. B., and Bock, A. (2001) Heterologous expression of archaeal selenoprotein genes directed by the SECIS element located in the 3' non-translated region. Mol Microbiol 40, 900-908 38. Goody, T. A., Melcher, S. E., Norman, D. G., and Lilley, D. M. (2004) The kink-turn motif in RNA is dimorphic, and metal ion-dependent. Rna 10, 254-264 39. Martin, G. W., 3rd, Harney, J. W., and Berry, M. J. (1998) Functionality of mutations at conserved nucleotides in eukaryotic SECIS elements is determined by the identity of a single nonconserved nucleotide. Rna 4, 65-73 40. Copeland, P. R., Stepanik, V. A., and Driscoll, D. M. (2001) Insight into mammalian selenocysteine insertion: domain structure and ribosome binding properties of Sec insertion sequence binding protein 2. Mol Cell Biol 21, 1491- 1498 41. Bubenik, J. L., and Driscoll, D. M. (2007) Altered RNA binding activity underlies abnormal thyroid hormone metabolism linked to a mutation in selenocysteine insertion sequence-binding protein 2. J Biol Chem 282, 34653-34662 42. Takeuchi, A., Schmitt, D., Chapple, C., Babaylova, E., Karpova, G., Guigo, R., Krol, A., and Allmang, C. (2009) A short motif in Drosophila SECIS Binding Protein 2 provides differential binding affinity to SECIS RNA hairpins. Nucleic Acids Res 37, 2126-2141 43. Fletcher JE, Copeland PR, Driscoll DM, and Krol A. (2001) The selenocysteine incorporation machinery: Interactions between the SECIS RNA and the SECIS- binding protein SBP2. Rna-a Publication of the Rna Society 7, 1442-1453 44. Duntas, L. H. (2010) On the trail of the SBP2-syndrome: clues in a Daedalean maze. in J Clin Endocrinol Metab, United States. pp 3618-3621
106 45. Dumitrescu, A. M., Liao, X. H., Abdullah, M. S., Lado-Abeal, J., Majed, F. A., Moeller, L. C., Boran, G., Schomburg, L., Weiss, R. E., and Refetoff, S. (2005) Mutations in SECISBP2 result in abnormal thyroid hormone metabolism. Nat Genet 37, 1247-1252 46. Carlson, B. A., Xu, X. M., Kryukov, G. V., Rao, M., Berry, M. J., Gladyshev, V. N., and Hatfield, D. L. (2004) Identification and characterization of phosphoseryl- tRNA[Ser]Sec kinase. Proc Natl Acad Sci U S A 101, 12848-12853 47. Donovan, J., Caban, K., Ranaweera, R., Gonzalez-Flores, J. N., and Copeland, P. R. (2008) A novel protein domain induces high affinity selenocysteine insertion sequence binding and elongation factor recruitment. J Biol Chem 283, 35129- 35139 48. Huttenhofer, A., and Bock, A. (1998) Selenocysteine inserting RNA elements modulate GTP hydrolysis of elongation factor SelB. Biochemistry 37, 885-890 49. Dabeva, M. D., and Warner, J. R. (1993) Ribosomal protein L32 of Saccharomyces cerevisiae regulates both splicing and translation of its own transcript. J Biol Chem 268, 19669-19674 50. Miniard, A., Middleton, L., Budiman, M., Gerber, C., and Driscoll, D. (2010) Nucleolin binds to a subset of selenoprotein mRNAs and regulates their expression. Nucleic Acids Research 38, 4807-4820 51. Tajrishi, M. M., Tuteja, R., and Tuteja, N. (2011) Nucleolin: The most abundant multifunctional phosphoprotein of nucleolus. Commun Integr Biol 4, 267-275 52. Budiman ME, Bubenik J, Miniard A, Middleton LM, Gerber CA, and Driscoll DM. (2009) Eukaryotic initiation factor 4a3 is a selenium-regulated RNA -binding protein that selectively inhibits selenocysteine incorporation. Molecular Cell 35, 479-489 53. Budiman M, Bubenik JL, and Driscoll DM. (2011) Identification of signature motif for the eIF4a3-SECIS interaction. Nucleic Acids Research 39, 7730-7739 54. Labunskyy, V. M., Hatfield, D. L., and Gladyshev, V. N. (2014) Selenoproteins: molecular pathways and physiological roles. Physiol Rev 94, 739-777 55. Latreche L, Jean-Jean O, Driscoll DM, and L, C. (2009) Novel structural determinants in human SECIS elements modulate the translational recoding of UGA as selenocysteine. Nucleic Acids Research 37, 5868-5880 56. Low, S. C., Grundner-Culemann, E., Harney, J. W., and Berry, M. J. (2000) SECIS- SBP2 interactions dictate selenocysteine incorporation efficiency and selenoprotein hierarchy. Embo j 19, 6882-6890 57. Squires, J. E., Stoytchev, I., Forry, E. P., and Berry, M. J. (2007) SBP2 binding affinity is a major determinant in differential selenoprotein mRNA translation and sensitivity to nonsense-mediated decay. Mol Cell Biol 27, 7848-7855 58. Fradejas-Villar, N., Seeher, S., Anderson, C. B., Doengi, M., Carlson, B. A., Hatfield, D. L., Schweizer, U., and Howard, M. T. (2017) The RNA-binding protein Secisbp2 differentially modulates UGA codon reassignment and RNA decay. Nucleic Acids Res 45, 4094-4107 59. Mariotti, M., Shetty, S., Baird, L., Wu, S., Loughran, G., Copeland, P. R., Atkins, J. F., and Howard, M. T. (2017) Multiple RNA structures affect translation initiation
107 and UGA redefinition efficiency during synthesis of selenoprotein P. Nucleic Acids Res 45, 13004-13015 60. AA, T., AV, L., DE, F., HG, M., ML, S., LA, K., DL, H., and VN, G. (2009) Genetic code supports targeted insertion of two amino acids by one codon. Science 323, 259 61. Howard, M., Aggarwal, G., Anderson, A., Kharti, S., Flanigan, K., and Atkins, J. (2005) Recoding elements located adjacent to a subset of eukaryal selenocysteine-specifying UGA codons. EMBO 24, 1596-1697 62. Howard, M., Moyle, M., Aggarwal, G., Calson, B., and Anderson, A. (2007) A recoding element that stimulates decoding of the UGA codons by Sec tRNA [Ser]Sec. RNA, 912-920 63. Maiti, B., Arbogast, S., Allamand, V., Moyle, M., Anderson, C., Richard, P., Cuicheney, P., Ferreiro, A., Flanigan, K., and Howard, M. (2010) A mutation in the SEPN1 SRE reduces selenocysteine incorporation and leads to SEPN1-related myopathy. Human Mutation 30, 411-416 64. Bubenik J, Miniard A, and Driscoll DM. (2013) Alternative Transcripts and 3'UTR Elements Govern the Incorporation of Selenocysteine into Selenoprotein S. PLOS One 8, e62102 65. Mariotti, M., Ridge, P., Zhang, Y., Lobanov, A., and Pringle, T. (2012) Composition and evolution of the vertebrate and mammalian selenoproteomes. PLoS One 7, e33066 66. Flohe, L. (2009) The labour pains of biochemical selenology: the history of selenoprotein biosynthesis. Biochim Biophys Acta 1790, 1389-1403 67. Lubos, E., Loscalzo, J., and Handy, D. E. (2011) Glutathione peroxidase-1 in health and disease: from molecular mechanisms to therapeutic opportunities. Antioxid Redox Signal 15, 1957-1997 68. Zhang, Q., Xu, H., You, Y., Zhang, J., and Chen, R. (2018) High Gpx1 expression predicts poor survival in laryngeal squamous cell carcinoma. Auris Nasus Larynx 45, 13-19 69. Kim, U., Kim, C. Y., Lee, J. M., Oh, H., Ryu, B., Kim, J., and Park, J. H. (2019) Phloretin Inhibits the Human Prostate Cancer Cells Through the Generation of Reactive Oxygen Species. Pathol Oncol Res 70. Lei, X. G., Wang X. . (2012) Glutathioned peroxidase 1, diabetes. in Selenium: Its Molecular Biology and Role in Human Health (Hatfield DL, B. M., Gladyshev VN ed.), Springer, New York. pp 261-270 71. McClung, J. P., Roneker, C. A., Mu, W., Lisk, D. J., Langlais, P., Liu, F., and Lei, X. G. (2004) Development of insulin resistance and obesity in mice overexpressing cellular glutathione peroxidase. Proc Natl Acad Sci U S A 101, 8852-8857 72. Pepper, M. P., Vatamaniuk, M. Z., Yan, X., Roneker, C. A., and Lei, X. G. (2011) Impacts of dietary selenium deficiency on metabolic phenotypes of diet- restricted GPX1-overexpressing mice. Antioxid Redox Signal 14, 383-390 73. Ursini, F., Maiorino, M., Brigelius-Flohe, R., Aumann, K. D., Roveri, A., Schomburg, D., and Flohe, L. (1995) Diversity of glutathione peroxidases. Methods Enzymol 252, 38-53
108 74. Ursini, F., Heim, S., Kiess, M., Maiorino, M., Roveri, A., Wissing, J., and Flohe, L. (1999) Dual function of the selenoprotein PHGPx during sperm maturation. Science 285, 1393-1396 75. Maiorino, M., Roveri, A., Benazzi, L., Bosello, V., Mauri, P., Toppo, S., Tosatto, S. C., and Ursini, F. (2005) Functional interaction of phospholipid hydroperoxide glutathione peroxidase with sperm mitochondrion-associated cysteine-rich protein discloses the adjacent cysteine motif as a new substrate of the selenoperoxidase. J Biol Chem 280, 38395-38402 76. Arner, E. S., and Holmgren, A. (2000) Physiological functions of thioredoxin and thioredoxin reductase. Eur J Biochem 267, 6102-6109 77. Nordberg, J., and Arner, E. S. (2001) Reactive oxygen species, antioxidants, and the mammalian thioredoxin system. Free Radic Biol Med 31, 1287-1312 78. Merrill, G. F., Dowell, P., and Pearson, G. D. (1999) The human p53 negative regulatory domain mediates inhibition of reporter gene transactivation in yeast lacking thioredoxin reductase. Cancer Res 59, 3175-3179 79. Hatfield, D. L., Yoo, M. H., Carlson, B. A., and Gladyshev, V. N. (2009) Selenoproteins that function in cancer prevention and promotion. Biochim Biophys Acta 1790, 1541-1545 80. Bianco, A. C., Salvatore, D., Gereben, B., Berry, M. J., and Larsen, P. R. (2002) Biochemistry, cellular and molecular biology, and physiological roles of the iodothyronine selenodeiodinases. Endocr Rev 23, 38-89 81. Saito, Y., and Takahashi, K. (2002) Characterization of selenoprotein P as a selenium supply protein. Eur J Biochem 269, 5746-5751 82. Mihara, H., Kurihara, T., Watanabe, T., Yoshimura, T., and Esaki, N. (2000) cDNA cloning, purification, and characterization of mouse liver selenocysteine lyase. Candidate for selenium delivery protein in selenoprotein synthesis. J Biol Chem 275, 6195-6200 83. Kim, I., Xu, W., and Reed, J. C. (2008) Cell death and endoplasmic reticulum stress: disease relevance and therapeutic opportunities. Nat Rev Drug Discov 7, 1013-1030 84. Fagone, P., and Jackowski, S. (2009) Membrane phospholipid synthesis and endoplasmic reticulum function. J Lipid Res 50 Suppl, S311-316 85. Harding, H. P., Zhang, Y., and Ron, D. (1999) Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase. Nature 397, 271-274 86. Deniziak, M., Thisse, C., Rederstorff, M., Hindelang, C., Thisse, B., and Lescure, A. (2007) Loss of selenoprotein N function causes disruption of muscle architecture in the zebrafish embryo. Exp Cell Res 313, 156-167 87. Petit, N., Lescure, A., Rederstorff, M., Krol, A., Moghadaszadeh, B., Wewer, U. M., and Guicheney, P. (2003) Selenoprotein N: an endoplasmic reticulum glycoprotein with an early developmental expression pattern. Hum Mol Genet 12, 1045-1053 88. Arbogast, S., and Ferreiro, A. (2010) Selenoproteins and protection against oxidative stress: selenoprotein N as a novel player at the crossroads of redox signaling and calcium homeostasis. Antioxid Redox Signal 12, 893-904
109 89. Allamand, V., Richard, P., Lescure, A., Ledeuil, C., Desjardin, D., Petit, N., Gartioux, C., Ferreiro, A., Krol, A., Pellegrini, N., Urtizberea, J. A., and Guicheney, P. (2006) A single homozygous point mutation in a 3'untranslated region motif of selenoprotein N mRNA causes SEPN1-related myopathy. EMBO Rep 7, 450-454 90. Gromer, S., Eubel, J. K., Lee, B. L., and Jacob, J. (2005) Human selenoproteins at a glance. Cell Mol Life Sci 62, 2414-2437 91. Addinsall, A. B., Wright, C. R., Andrikopoulos, S., van der Poel, C., and Stupka, N. (2018) Emerging roles of endoplasmic reticulum-resident selenoproteins in the regulation of cellular stress responses and the implications for metabolic disease. Biochem J 475, 1037-1057 92. Hwang, D. Y., Cho, J. S., Oh, J. H., Shim, S. B., Jee, S. W., Lee, S. H., Seo, S. J., Lee, S. K., and Kim, Y. K. (2005) Differentially expressed genes in transgenic mice carrying human mutant presenilin-2 (N141I): correlation of selenoprotein M with Alzheimer's disease. Neurochem Res 30, 1009-1019 93. Cai, H., Cong, W. N., Ji, S., Rothman, S., Maudsley, S., and Martin, B. (2012) Metabolic dysfunction in Alzheimer's disease and related neurodegenerative disorders. Curr Alzheimer Res 9, 5-17 94. Prevost, G., Arabo, A., Jian, L., Quelennec, E., Cartier, D., Hassan, S., Falluel- Morel, A., Tanguy, Y., Gargani, S., Lihrmann, I., Kerr-Conte, J., Lefebvre, H., Pattou, F., and Anouar, Y. (2013) The PACAP-regulated gene selenoprotein T is abundantly expressed in mouse and human beta-cells and its targeted inactivation impairs glucose tolerance. Endocrinology 154, 3796-3806 95. Grumolato, L., Ghzili, H., Montero-Hadjadje, M., Gasman, S., Lesage, J., Tanguy, Y., Galas, L., Ait-Ali, D., Leprince, J., Guerineau, N. C., Elkahloun, A. G., Fournier, A., Vieau, D., Vaudry, H., and Anouar, Y. (2008) Selenoprotein T is a PACAP- regulated gene involved in intracellular Ca2+ mobilization and neuroendocrine secretion. Faseb j 22, 1756-1768 96. Shchedrina, V., Everley, R., Zhang, Y., Gygi, S., and Hatfield, D. (2011) Selenoprotein K binds multiprotein complexes and is involved in the regulation of endoplasmic reticulum homeostasis. Journal of Biological Chemistry 286, 42937-42948 97. Gao Y, Feng H, Walder K, Bolton K, Sunderland T, Bishara N, Quick M, Kantham L, and Collier GR. (2004) Regulation of the selenoproteins SelS by glucose deprivation and endoplasmic reticulum stress- SelS is a novel glucose-regulated protein. FEBS Letters 563, 185-190 98. Lee, J. H., Park, K. J., Jang, J. K., Jeon, Y. H., Ko, K. Y., Kwon, J. H., Lee, S. R., and Kim, I. Y. (2015) Selenoprotein S-dependent Selenoprotein K Binding to p97(VCP) Protein Is Essential for Endoplasmic Reticulum-associated Degradation. J Biol Chem 290, 29941-29952 99. Smith, M. H., Ploegh, H. L., and Weissman, J. S. (2011) Road to ruin: targeting proteins for degradation in the endoplasmic reticulum. Science 334, 1086-1090 100. Meiler, S., Baumer, Y., Huang, Z., Hoffmann, F. W., Fredericks, G. J., Rose, A. H., Norton, R. L., Hoffmann, P. R., and Boisvert, W. A. (2013) Selenoprotein K is
110 required for palmitoylation of CD36 in macrophages: implications in foam cell formation and atherogenesis. J Leukoc Biol 93, 771-780 101. Verma, S., Hoffmann, F. W., Kumar, M., Huang, Z., Roe, K., Nguyen-Wu, E., Hashimoto, A. S., and Hoffmann, P. R. (2011) Selenoprotein K knockout mice exhibit deficient calcium flux in immune cells and impaired immune responses. J Immunol 186, 2127-2137 102. Huang, Z., Hoffmann, F. W., Norton, R. L., Hashimoto, A. C., and Hoffmann, P. R. (2011) Selenoprotein K is a novel target of m-calpain, and cleavage is regulated by Toll-like receptor-induced calpastatin in macrophages. J Biol Chem 286, 34830-34838 103. Ye Y, Shibata Y, Yun C, Ron D, and Rapoport TA. (2004) A membrane protein complex mediates retro-translocation from the ER lumen into the cytosol. Nature Genetics 429, 841-847 104. Turanov, A. A., Shchedrina, V. A., Everley, R. A., Lobanov, A. V., Yim, S. H., Marino, S. M., Gygi, S. P., Hatfield, D. L., and Gladyshev, V. N. (2014) Selenoprotein S is involved in maintenance and transport of multiprotein complexes. Biochem J 462, 555-565 105. Liu, J., and Rozovsky, S. (2013) Contribution of selenocysteine to the peroxidase activity of selenoprotein S. Biochemistry 52, 5514-5516 106. Liu, J., Li, F., and Rozovsky, S. (2013) The intrinsically disordered membrane protein selenoprotein S is a reductase in vitro. Biochemistry 52, 3051-3061 107. Ye Y, Shibata Y, Kikkert M, Voorden S, Wiertz E, and Rapoport TA. (2005) Recruitment of the p97 ATPase and ubiquitin ligases to the site of retrotranslocation at the endoplasmic reticulum membrane. PNAS 102, 14132- 14138 108. Raven, J. F., Baltzis, D., Wang, S., Mounir, Z., Papadakis, A. I., Gao, H. Q., and Koromilas, A. E. (2008) PKR and PKR-like endoplasmic reticulum kinase induce the proteasome-dependent degradation of cyclin D1 via a mechanism requiring eukaryotic initiation factor 2alpha phosphorylation. J Biol Chem 283, 3097-3108 109. Wu, J., Rutkowski, D. T., Dubois, M., Swathirajan, J., Saunders, T., Wang, J., Song, B., Yau, G. D., and Kaufman, R. J. (2007) ATF6alpha optimizes long-term endoplasmic reticulum function to protect cells from chronic stress. Dev Cell 13, 351-364 110. Yamamoto, K., Sato, T., Matsui, T., Sato, M., Okada, T., Yoshida, H., Harada, A., and Mori, K. (2007) Transcriptional induction of mammalian ER quality control proteins is mediated by single or combined action of ATF6alpha and XBP1. Dev Cell 13, 365-376 111. Christensen LC, Jensen NW, Vala A, Kamarauskaite J, Johansson L, Winther JR, Hofmann K, Teilum K, and Ellgaard L. (2012) The human selenoprotein VCP- interacting membrane protein (VIMP) is non-globular and harbors reductase function in an intrinsically disordered region. Journal of Biological Chemistry 287, 26388-26399 112. Lee, J. H., Kwon, J. H., Jeon, Y. H., Ko, K. Y., Lee, S. R., and Kim, I. Y. (2014) Pro178 and Pro183 of selenoprotein S are essential residues for interaction with
111 p97(VCP) during endoplasmic reticulum-associated degradation. J Biol Chem 289, 13758-13768 113. Kim KH, Gao Y, Walder K, Collier GR, Skelton J, and Kissebah AH. (2007) SEPS1 protects RAW264.7 cells from pharmacological ER stress agent-induced apoptosis. Biochemical and Biophysical Research Communications 354, 127-132 114. Fradejas N, P. M., Mora-Lee S, Tranque P, Calvo S. (2008) SEPS1 gene is activated during astrocyte ischemia and shows prominent antiapoptotic effects. J Mol Neurosci 35, 259-265 115. Kelly, E., Greene, C., Carroll, T., McElvaney, N., and O'Neill, S. (2009) Selenoprotein S/SEPS1 modifies endoplasmic reticulum stress in Z variant alpha 1-antitrypsin deficiency. Journal of Biological Chemistry 284, 16891-16897 116. Zheng HT, Z. L., Huang CJ, Hua X, Jian R, Su BH, Fang F. (2008) Selenium inhibits high glucoseand high insulin induced adhesion molecule expression in vascular endothelial cells. Archives of Medical Research 39, 373-379 117. Zhao, Y., Li, H., Men, L. L., Huang, R. C., Zhou, H. C., Xing, Q., Yao, J. J., Shi, C. H., and Du, J. L. (2013) Effects of selenoprotein S on oxidative injury in human endothelial cells. J Transl Med 11, 287 118. Walder, K., Kantham, L., McMillan, J., Trevaskis, J., and Kerr, L. (2002) Tanis: a link between type 2 diabetes and inflammation? Diabetes 51, 1859-1866 119. Jian-ling, D., Chang-kai, S., Bo, L., Li-li, M., Jun-jie, Y., Li-jia, A., and Gui-rong, S. (2008) Association of SelS mRNA expression in omental adipose tissue with Homa-IR and serum amyloid A in patients with type 2 diabetes mellitus. Chinese Medical Journal 121, 1165-1168 120. Karisson, H., Tsuchida, H., Lake, S., Koistinen, H., and Krook, A. (2004) Relationship between Serum Amyloid A level and Tanis/SelS mRNA expression in skeletal muscle and adipose tissue from healthy and type 2 diabetic subjects. Diabetes 53, 1424-1428 121. Gao Y, Walder K, Sunderland T, Kantham L, Feng HC, Quick M, Bishara N, de Silva A, Augert G, Tenne-Brown J, and Collier G. R. (2003) Elevation in Tanis expression alters glucose metabolism and insulin sensitivity in H4IIE cells. Diabetes 52, 929- 934 122. Yu, S., Liu, X., Men, L., Yao, J., Xing, Q., and Du, J. (2018) Selenoprotein S protects against high glucose-induced vascular endothelial apoptosis through the PKCbetaII/JNK/Bcl-2 pathway. J Cell Biochem 123. Olsson, M., Olsson, B., Jacobsen, P., Thelle, D., Bjorkegren, J., Walley, A., Froguel, P., Carlsson, L., and Sjoholm, K. (2011) Expression of the selenoprotein S (SELS) gene in subcutaneous adipose tissue and SELS genotype are associated with metabolic risk factors. Metabolism 60, 114-120 124. Butler, A., Janson, J., Bonner-Weir, S., Ritzel, R., Rizza, R., and Butler, P. (2003) B- cell apoptosis in human with type 2 diabetes. Diabetes 52, 102-110 125. Yoon, K., Ko, S., and Cho, J. (2003) B-cell loss and a-cell xpansion in patients with type 2 diabetes mellitus in Korea. J Clin Endocrnol Metab 88, 2300-2308
112 126. Men, L., Sun, J., and Ren, D. (2018) Deficiency of VCP-Interacting Membrane Selenoprotein (VIMP) Leads to G1 Cell Cycle Arrest and Cell Death in MIN6 Insulinoma Cells. Cell Physiol Biochem 51, 2185-2197 127. Curran J, Jowett JBM, Elliott K, Gao Y, Gluschenko K, Wang J, Azim D, Cai G, Collier GR, Kissebah AH, and Blangero J. (2005) Genetic variation in selenoprotein S influences inflammatory response. Nature Genetics 37 128. Hannan, N., Wanyonyi, S., Konstantopolous, N., Pagnon, J., Feng, H., Jowett, J., Kim, K., Walder, K., and Collier, G. (2006) Activation of the selenoprotein SEPS1 gene expression by pro-inflammatory cytokines in HepG2 cells. Cytokine 33 129. Alanne M, Kristiansson K, Auro K, Silander K, Kuulasmaa K, Peltonen L, Salomaa V, and Perola M. (2007) Variation in selenoprotein S gene locus is associated with coronary heart disease and ischemic stroke in two independent Finnish cohorts. Human Genetics 122, 355-365 130. Libby P. (2002) Inflammation in atherosclerosis. Nature 420, 868-874 131. Moses, E., Johnson, M., Tommerdal, L., Forsmo, S., and Curran, J. (2008) Genetic association of preeclampsia to the inflammatory response of SEPS1. Am J Obstet Gynecol 198, 336 132. Shibata, T., Arisawa, T., Tahara, T., Ohkubo, M., and Yoshioka, D. (2009) SelenoproteinS (SEPS1) gene -105G>A promoter polymorphism influences the susceptibility to gastric cancer in the Japanese population. BMC Gastroenterol 9 133. Meplan, C., Hughes, D., Pardini, B., Naccarati, A., and Soucek, P. (2010) Genetic variants in selenoprotein genes increases risk of colorectal cancer. Carcinogenesis 31, 1074-1079 134. Sievers, F., and Higgins, D. G. (2014) Clustal Omega, accurate alignment of very large numbers of sequences. Methods Mol Biol 1079, 105-116 135. Gruber, A. R., Lorenz, R., Bernhart, S. H., Neubock, R., and Hofacker, I. L. (2008) The Vienna RNA websuite. Nucleic Acids Res 36, W70-74 136. Bubenik, J. L., Ladd, A. N., Gerber, C. A., Budiman, M. E., and Driscoll, D. M. (2009) Known turnover and translation regulatory RNA-binding proteins interact with the 3' UTR of SECIS-binding protein 2. RNA Biol 6, 73-83 137. Mehta, A., Rebsch, C., Kinzy, S., Fletcher, J., and Copeland, P. (2004) Efficiency of mammalian selenocysteine incorporation. Journal of Biological Chemistry 279, 37852-37859 138. Turanov, A. A., Lobanov, A. V., Hatfield, D. L., and Gladyshev, V. N. (2013) UGA codon position-dependent incorporation of selenocysteine into mammalian selenoproteins. Nucleic Acids Res 41, 6952-6959 139. Mayya, V. K., and Duchaine, T. F. (2019) Ciphers and Executioners: How 3'- Untranslated Regions Determine the Fate of Messenger RNAs. Front Genet 10, 6 140. Berglund, A. C., Sjölund, E., Östlund, G., and Sonnhammer, E. L. L. (2008) InParanoid 6: eukaryotic ortholog clusters with inparalogs. Nucleic Acids Res 36, D263-266 141. Driscoll, D. M., and Chavatte, L. (2004) Finding needles in a haystack. In silico identification of eukaryotic selenoprotein genes. EMBO Rep 5, 140-141
113 142. Ray, P. S., Jia, J., Yao, P., Majumder, M., Hatzoglou, M., and Fox, P. L. (2009) A stress-responsive RNA switch regulates VEGFA expression. Nature 457, 915-919 143. Michlewski, G., and Caceres, J. F. (2010) RNase-assisted RNA chromatography. Rna 16, 1673-1678 144. Cockman, E. M., and Driscoll, D. M. (2018) Identification and Characterization of Proteins that Bind to Selenoprotein 3' UTRs. Methods Mol Biol 1661, 61-71
114