The mRNA Structure Has Potent Regulatory Effects on Type 2 Iodothyronine Deiodinase Expression
Balázs Gereben,
Anna Kollár,
John W. Harney and
P. Reed Larsen
Institute of Experimental Medicine (B.G.), Department of Neurobiology, Budapest H-1083, and University of Pécs, Faculty of Sciences, Institute of Biology, Pécs H-7624, Hungary; Szent István University (A.K.), Faculty of Veterinary Science, Department of Physiology and Biochemistry, Budapest H-1078, Hungary; Thyroid Division (B.G., J.W.H., P.R.L.), Department of Medicine, Brigham and Womens Hospital, Harvard Medical School, Boston, Massachusetts 02115
Address all correspondence and requests for reprints to: Dr. P. Reed Larsen, Thyroid Division, Department of Medicine, Brigham and Womens Hospital, Harvard Medical School, Room 560, Harvard Institutes of Medicine, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115. E-mail: larsen{at}rascal.med.harvard.edu.
 |
ABSTRACT
|
---|
Type 2 deiodinase (D2) is a selenoenzyme catalyzing the activation of T4 to T3. D2 activity/mRNA ratios are often low, suggesting that there is significant posttranscriptional regulation. The D2 mRNA in higher vertebrates is more than 6 kb, containing long 5' and 3' untranslated regions (UTRs). The D2 5'UTRs are greater than 600 nucleotides and contain 35 short open reading frames. These full-length 5'UTRs reduce the D2 translation efficiency approximately 5-fold. The inhibition by human D2 5'UTR is localized to a region containing the first short open reading frame encoding a tripeptideMKG. This inhibition was abolished by mutating the AUG start codon and weakened by modification of the essential purine of the Kozak consensus. Deletion of the 3.7-kb 3'UTR of the chicken D2 mRNA increased D2 activity approximately 3.8-fold due to an increase in D2 mRNA half-life. In addition, alternatively spliced D2 mRNA transcripts similar in size to the major 6- to 7-kb D2 mRNAs but not encoding an active enzyme are present in both human and chicken tissues. Our results indicate that a number of factors reduce the D2 protein levels. These mechanisms, together with the short half-life of the protein, ensure limited expression of this key regulator of T4 activation.
 |
INTRODUCTION
|
---|
TYPE 2 DEIODINASE (D2) catalyzes the 5' monodeiodination of T4 to T3, the first step found in the process by which T4 produces its effects. It is widely expressed in humans being in brain, pituitary, brown adipose tissue, placenta, skeletal, and cardiac muscle and skin (1, 2). Because D2 contains the rare amino acid selenocysteine encoded by UGA in its active center, its successful translation requires specific sequences, the selenocysteine insertion sequence (SECIS) element, in its 3' untranslated region (UTR) (3). Even so, its translational efficiency, like that of other selenoproteins, is much less than that of typical proteins (4). The process can also be influenced by selenium and by the components of the selenocysteine insertion machinery (5, 6, 7).
Interestingly, D2 activity/mRNA ratios show marked variations. In tissues such as the normal human thyroid, D2 activity is lower than would be expected from the amount of D2 mRNA expressed. For example, in the thyroid tissue of a Graves patient, the D2 activity/mRNA ratio was 25-fold that in normal human thyroid (8, 9). In human thyroid adenomas, the D2 activity/mRNA ratio is also higher than in normal thyroid, indicating that there are variations in posttranslational processing (10).
It is not understood which components of the highly complex D2 regulation might be involved in the generation of these variations in mRNA/activity ratios between tissues. The dio2 (type 2 iodothyronine deiodinase) gene has multiple transcription start sites (TSSs), and the transcriptional regulatory component involves cAMP, thyroid transcription factor-1, T3, and activator protein-1 regulated pathways (11, 12, 13, 14, 15). In addition, T4 or 3,3'5' triiodothyronine (reverse T3, rT3) cause significant posttranslational down-regulation. The latter effect involves substrate-accelerated selective proteolysis via the ubiquitin/26 S proteasome pathway (16, 17, 18).
The D2 mRNAs of vertebrates are unusually long (
67 kb) compared with the 2- to 2.5-kb length of the other two members of the selenodeiodinase family. Despite this large size, the open reading frame (ORF) encoding the active enzyme is only approximately 800 bp. The 3'UTR is extremely long (>
4.7 kb) and contains a SECIS element at its 3' end. This is the greatest distance yet recognized between a SECIS element and the UGA codon and could theoretically attenuate successful readthrough of the two UGA codons in the mRNA (19, 20, 21). The 5'UTR of the D2 mRNAs is at least approximately 600-bp long and the mRNAs of the four species cloned all contain 35 short ORFs (sORF), which may also modulate translation. The goal of our study was to elucidate the role of mRNA structure in the posttranscriptional regulation of this key rate- limiting gene in thyroid hormone activation.
 |
RESULTS
|
---|
The Translation of D2 mRNA Is Regulated by Its 5'UTR
Comparison of the 600- to 700-bp 5'UTR of the dio2 genes of higher vertebrates shows a number of common features. The human D2 (hD2) 5'UTR has 73, 70, and 46% identity to the rat, mouse, and chicken sequences, respectively. Virtual identity exists in the most 5' approximately 20 bases, which form putative stem loop structures by GCG FOLDRNA analysis (Fig. 1A
). The more 3' portions of these 5'UTR contain 35 putative sORFs (Fig. 1B
). The sORFs were defined considering the unique feature of selenoprotein mRNAs in which SECIS elements in the 3'UTR suppress the stop codon function of UGA codons. Unambiguous stop codons (UAA and UAG) and in-frame UGA codons followed by purines were considered as translational terminators, whereas in-frame UGAs followed by pyrimidines favor readthrough and are indicated by an asterisk (22). The deduced peptide sequences of these ORFs have low similarity between species with the exception of rat and mouse (Fig. 1B
).

View larger version (52K):
[in this window]
[in a new window]
|
Figure 1. Alignment of D2 5'UTR Portions
A, Alignment of the 5' portion of the human, chicken, rat, and mouse D2 mRNAs. The region just 3' to the transcription start site is highly conserved. Alignments were performed by GCG PILEUP. B, Deduced amino acid sequences of the putative amino acid sequence of the sORFs in the human, chicken, rat, and mouse D2 5'UTR. Unambiguous stop codons and in frame UGAs followed by purines were considered as translational terminators. In-frame UGAs in possible readthrough position (codon followed by a pyrimidine) are indicated by an asterisk (22 ). The presence of a strong translational initiation sequence (-3 purine where position 1 is the A of the start codon) is indicated by a plus sign (39 ).
|
|
We developed a chicken D2 (cD2)-containing reporter system to explore the potential role of these 5'UTR sequences in modulating the translation efficiency of this selenoprotein. The human and chicken cD2 5'UTR caused a 5-fold reduction in transient cD2 expression in HEK-293 cells (Fig. 2
). This was not due to the conserved sequences in the 5' region (Fig. 1A
) because deletion of these had no effect. The 5'UTR of cD2 was as effective as that of the human mRNA in decreasing D2 activity. Northern blots showed no inhibitory effect of the sORFs on the transient expression of the cD2 mRNA (data not shown). In fact, the D2/actin mRNA/human GH (hGH) ratio of full-length cD2 was about 3-fold higher than that of the
5'UTRcD2 construct, whereas the activity in the same experiments was 2- to 3-fold lower. The effect on D2 activity of 5'UTR fragments containing the hORF-A was not different from that of the hD25'UT
containing the sORFs. However, constructs lacking the sORF-A were significantly less effective in suppressing D2 activity than those containing sORF-B or C (Fig. 3
). Thus, the sORF-A encoding the tripeptide MKG was the predominant translation inhibitory region in the hD2 5'UTR.

View larger version (15K):
[in this window]
[in a new window]
|
Figure 2. Effect of the Human and Chicken 5'UTR on D2 Expression
The hD2 or cD2 5'UTR were inserted between SacII and EcorI of the cD2 reporter containing the cD2 coding region followed by the rat D1 minimal SECIS element. Plasmids were transiently transfected into HEK-293 cells as described in Materials and Methods. Data are the mean ± SEM of relative D2 activities of duplicate plates in at least three separate experiments as a percentage of the activity of the cD2 reporter. All results are corrected for transfection efficiency. *, P < 0.001 vs. cD2 by ANOVA followed by Newman-Keuls.
|
|

View larger version (15K):
[in this window]
[in a new window]
|
Figure 3. Structure/Functional Analysis of the Human 5'UTR
The hD2 5'UTR fragments were inserted into the cD2 reporter (see Fig. 2 ) and transiently transfected into HEK-293 cells as described in Materials and Methods. Data are the mean ± SEM of the relative D2 activities of duplicate plates in at least three separate experiments as a percent of the cD2 reporter control. *, P < 0.05; **, P < 0.01 vs. hD25'UT by ANOVA followed by Newman-Keuls.
|
|
Mutagenesis was used to inactivate the AUG codon of the human sORF-A and the shortest chicken ORF-B to determine the structure-activity relationships (Fig. 1B
). Replacement of ATG by TTG completely abolished the inhibitory effect of the sORFs on D2 translation (Fig. 4
). The suppressive activity of human sORF-A was also decreased by changing the Kozak consensus replacing the -3A with C. These data indicate that translational initiation of the sORF is required to decrease the translation of the 3' major coding cistron. The hORF-A(Wt)-cD2 and hORF-A(Mut-ATG)-cD2 were also transfected into GH4C1 and Met5A (which express endogenous D2) and COS-7 and JEG cells (no endogenous D2) to analyze for possible cell type specific effects. In all cell lines tested, the relative D2 activity (calculated as percentage of the cD2 reporter) produced by the hORF-A(Wt)-cD2 was significantly lower than that of produced by hORF-A(Mut-ATG)-cD2 constructs (Table 1
). This indicates that the sORF based translational inhibition of D2 activity is not restricted to cell types not expressing endogenous D2.

View larger version (20K):
[in this window]
[in a new window]
|
Figure 4. Effect of Site-Directed Mutagenesis on the Inhibitory Function of sORFs of the D2 5'UTR
Mutagenized nucleotides are shown in bold and underlined. Fragments were inserted into the cD2 reporter (see Fig. 2 ) and transiently transfected into HEK-293 cells as described in Materials and Methods. Data are the mean ± SEM of relative D2 activities of duplicate plates in at least three separate experiments. *, P < 0.05; **, P < 0.001 vs. hORF-A(Wt)-cD2 by ANOVA followed by Newman-Keuls. ***, P < 0.05 vs. cORF-B(Wt)-cD2 by unpaired t test.
|
|
Wild-type (wt) or mutant (mut) ATG-containing sORF-A containing cDNAs were also inserted in separate eukaryotic expression plasmids and cotransfected with the cD2 reporter. The cotransfection of the wt, MKG or mut LKG, encoding expression plasmids did not affect cD2 expression from the cD2 plasmid, indicating neither the peptide nor the ORF-A mRNA is inhibitory (not shown). When the wt or TTG-containing human sORF-A was inserted 5' to the rat D1 coding sequence, the wt sORF-A, but not the mut, suppressed D1 activity, indicating that the suppressive effect of this sORF is not D2 specific (Fig. 5
).

View larger version (15K):
[in this window]
[in a new window]
|
Figure 5. Effect of the wt or mut ATG Containing hD2 5'UTR sORF-A on D1 Activity
The fragments were inserted into a rD1 reporter element and the plasmid transiently transfected into HEK-293 cells as described in Materials and Methods. The mutagenized nucleotide is shown in bold and underlined. Data are the mean ± SEM of relative D1 activities of duplicate plates in at least three separate experiments as percent of the rD1 reporter control. *, P < 0.05 vs hORF-A(Wt)-rD1 by unpaired t test.
|
|
The D2 3'UTR Affects the D2 mRNA Half-Life and Translation Efficacy
The 3'UTR of the vertebrate D2 mRNAs is at least 4.7 kb, and each contains the required SECIS element at its 3' terminus (19, 20, 21). The human, chicken, and mouse D2 3'UTR (GenBank accession no. AF096875) also contain 11, 9, and 7 AUUUA putative mRNA instability motifs, respectively. These sequences are surrounded by adenine/uracil (A/U) rich regions as is characteristic of instability motifs (19, 21, 23).
The cD2 3'UTR was restricted to remove 3710 nucleotides (nt) containing 8 of the 9 AUUUA motifs between the coding region and the SECIS element (cD23.7
, in Fig. 6A
). To keep the distance constant between the active center and the SECIS element, exogenous DNA from Neurospora crassa or from the mouse thyroid receptor
, without AUUUA motifs, was inserted (cD23.7exo). Replacement of the 3'UTR increased D2 activity by approximately 50%, whereas elimination of the 3'UTR increased D2 activity approximately 3.8-fold relative to the cD2wt (Fig. 6A
). Transfections were performed with 1, as opposed to 10, µg D2-expression plasmid with the same result, indicating that the selenoprotein synthesis was not saturated.
The D2 mRNA was quantified by Northern blots in two separate experiments using a coding region probe. The mRNA of the cD23.7exo and cD23.7
were 0.6- and 4-fold that of cells transfected with cD2wt, respectively (data not shown).
To evaluate possible differences in mRNA stability, similarly transfected cells were exposed to tetracycline to stop transcription from the tetracycline-repressible promoter (see Materials and Methods). After 13 h of tetracycline exposure, the amount of cD23.7exo mRNA was approximately 2.5-fold higher than in the cells transfected with the cD2wt (Fig. 6B
). This suggested that elimination of the 8 AUUUAs contained in the 3'UTR increased the mRNA half-life.
As is apparent in Fig. 6B
, deletion of most of the 3'UT (cD23.7
) leads to the presence of large quantities of two D2 transcripts. The larger transcript is of the expected size (approximately 2.1 kb). The blot was reprobed using a cD2 SECIS region probe, and the same doublet was observed indicating that both mRNAs are potentially functional. The densities of the bands were 15-fold (larger) and 40-fold higher than the wt, respectively.
Cloning and Functional Testing of Alternatively Spliced D2 Coding Region Transcripts in Chicken and Human Tissues
Despite similar D2 mRNA levels in the telencephalon and liver of the adult chicken (21), the maximum velocity for D2 is 2.6-fold higher in brain tissue (408 vs. 156 fmol T4/h·mg protein). While only a single 6.1-kb D2 was observed in both tissues, a potential explanation for this discrepancy is the presence of a second mRNA in liver similar in size but not encoding an active protein. This proved to be the case with cDNAs cloned from two animals demonstrating a 77-bp deletion just 5' to the region corresponding to the exon/intron junction in the human D2 coding region (Fig. 7A
). The absence of these nucleotides resets the reading frame and the deduced amino acid sequence of the
77cD2 protein is terminated short of the active center (Fig. 7B
). The coexistence of the normal and
77cD2 mRNA was shown in the telencephalon and liver by PCR (Fig. 7C
). As predicted, the
77cD2 mRNA did not encode an active D2 in transiently transfected HEK-293 cells.
Alternatively spliced human D2 mRNAs involving the same intron/exon junction were also found in human thyroid tissue samples (see Materials and Methods). Three samples had two transcripts (see Fig. 8A
). The cDNAs of the three abnormal (longer) transcripts were cloned and sequenced. All had an identical 108-bp in-frame insertion that also contained an in frame TGA followed by a C, a context that can support UGA translation (Fig. 8B
). To see if this mRNA could encode a functional D2, the hD2+108 coding region was placed 5' to a SelP SECIS element with a FLAG epitope fused to the NH2 terminus. The protein was transiently expressed in HEK-293 cells in Na2(75Se)O3 containing media and immunoprecipitated by anti-FLAG antibody. Autoradiography showed that the hD2+108 coding region encoded a protein of approximately 35 kDa (Fig. 8C
). This corresponds to the calculated size based on the deduced amino acid sequence of the putative full-length D2+108 protein indicating that the UGA is translated. No band of this size was visible in the control lane. The hD2+108 transfected HEK-293 cell sonicates contained no D2 activity. Sequence analysis showed that the 108 bp is derived from the midportion of the approximately 8-kb intron separating hdio2 exon
and ß (Fig. 8D
).

View larger version (38K):
[in this window]
[in a new window]
|
Figure 8. Sequence and Expression of the hD2+108 Transcript
A, PCR of an alternatively spliced hD2+108 transcript from human thyroid. The RNA was isolated from thyroid tissue of a patient with a thyrotropin-producing pituitary adenoma (lane 1) or Graves disease (lane 2) using the intron spanning Bp97-Bp41 oligos (sequence indicated in Table 2 ). Both the expected 491-bp amplicon and an approximately 600-bp product were generated. The sequences are shown in panel B. B, Nucleic acid and deduced amino acid sequence of the cloned thyroidal hD2+108 cDNA. The 108-bp intronic sequence inserted in frame between the exon/intron junction of the hD2 coding region are bold and underlined. The inserted sequence contains an in-frame UGA (boxed). A HindIII site was used for assembling the cDNA as described in Materials and Methods. Position 1 is the start of the coding region. C, Immunoprecipitation of Na2(75Se)O3 labeled HEK-293 cells transfected with hD2+108-SelP SECIS (lane 1) or N-FLAG hD2+108-SelP SECIS (lane 2) using an anti-FLAG antibody. Immunoprecipitate was resolved by SDS-PAGE followed by autoradiography for 12 d. N-FLAG hD2+108-SelP SECIS expresses a D2 protein of approximately 35 kDa showing that the inserted UGA is translated. No 35-kDa band was present in lane 1, indicating that the FLAG IP is specific. D, Genomic organization of the alternatively spliced hdio2 gene. The 108-bp fragment from the intron is inserted between codons 74 and 75 (see panel B).
|
|
 |
DISCUSSION
|
---|
Our results document several previously unrecognized characteristics common to the D2 mRNAs of four different species which can explain discrepancies between D2 mRNA content and D2 activity in different tissues. Quantitatively most important is the effect of the 5'UTR, which causes a 5-fold reduction in D2 expression in HEK-293 cells (Fig. 2
). While there are 35 sORFs in the 5'UTRs of the human, chicken, rat, and mouse D2 mRNAs (Fig. 1B
), in the human D2 it is the most 5' sORF (hORF-A), which is primarily responsible for this effect (Fig. 3
). These sORFs encode a tri- (human, rat, mouse) or di-peptide (chicken). The hORF-A operates only in cis and requires an AUG codon. All of the shortest D2 5'UT ORFs have Kozak consensus sequences with purines at -3 and +4 relative to the ATG, and an A to C substitution at -3 of the hORF-A reduces the translational inhibition by about 2-fold (Fig. 4
). The inhibitory effect was not D2-specific (Fig. 5
). Our results suggest there is no tissue specificity to the inhibitory function of hORF-A (Table 1
).
The best characterized examples of sORF-based translational regulation occur in yeast and viruses. The translation of the yeast GCN4 transcription factor is increased by amino acid starvation. This process is regulated by the 4 sORFs in the 5'UTR in a manner independent of the amino acid sequence of the putative peptides. The phosphorylation state of eIF-2 controls the re-initiation frequency at ORF4 (see Ref. 24 for review). In contrast, the yeast CP1 leader expresses a 25 amino acid leader peptide that suppresses the translation of the glutaminase subunit of the carbamoyl-phosphate synthetase in a sequence-specific manner (25). Another well-documented mechanism for bypassing sORFs occurs in Picornaviruses where specific cis acting RNA sequences, the internal ribosome entry segment, allow translation of major 3'ORFs (26). On the other hand, the Cauliflower mosaic virus 35S RNA utilizes nonlinear ribosome migration, ribosome shunting, to bypass the inhibitory sORFs (for review see Ref. 27).
The translational mechanism for higher vertebrates containing a 5'UTR with sORFs upstream of the major coding sequence is poorly understood. Only 10% of 5'UTR vertebrate mRNAs contain such sequences (28). This group of genes includes transcription and growth factors, proto-oncogenes, receptors of the G protein-coupled receptor superfamily, and signal transduction components (29). Examples include Angiotensin II type IA receptor (30), CCAAT/enhancer-binding (31), ornithine decarboxylase (32), fibroblast growth factor-5 (33), the alcohol dehydrogenase-5/formaldehyde dehydrogenase and Myf6 genes (34), serine hydroxymethyltransferase (35), the HER-2 receptor (36), and the ß2 adrenergic receptor (37). The human ß2 adrenergic receptor 5'UTR contains an sORF encoding a 19-amino acid peptide that inhibits the translation of the protein. As in the case of D2 mRNA, the effect is not specific to the homologous coding sequence and it functions only in cis. Similar to human D2, a CTT for ATG substitution in the sORF increases the receptor protein translation approximately 2-fold. There is a single nucleotide polymorphism in this mRNA altering amino acid 19 from ARG to CYS. This leads to a 2-fold increase in the translation efficiency of the receptor in a cell-line endogenously expressing the protein indicating that the suppressive effect of the ORF is influenced by the peptide (38). The number and sequence of the sORFs in the D2 5'UTRs are not conserved between species, not even between the most potent sORF-A, indicating that the suppressive effect is independent of peptide sequence but common to all D2 mRNAs. Interestingly, the human D2 sORF-B also has a -3 purine (G), but this sORF does not suppress D2 translation (Figs. 1B
and 3
). This, taken together with the successful initiation from the weak translation initiation sequence of the ß2 adrenergic receptor sORF, suggests in accordance with the findings of Kozak (39) that other factors, such as secondary structure, should be taken into consideration when assessing the translational inhibitory potential of a 5'UT sORF. The 5' terminal end of the D2 5'UTRs have high sequence similarity (Fig. 1A
) and computer-assisted methods predict secondary structures for this region. However, we found no suppressive effect of this portion of the human D2 5'UTR on D2 translation.
It is not yet clear whether the translational suppression caused by the D2 5'UTR is constitutive or inducible. Within a given cell type or tissue, increases in D2 mRNA, such as those induced by adrenergic mechanisms in pineal gland or during development, increase D2 activity more or less in parallel (40, 41, 42). However, recent analyses of D2 activities in hyperfunctioning human thyroid adenomas show that the ratios between adenoma and normal are greater for D2 activity (approximately 5-fold) than for D2 mRNA (approximately 3-fold), suggesting there may be more efficient translation as well as increased mRNA as an explanation for increased D2 (10). Further studies will be required to explore these possibilities.
The long 3'UTR of the D2 mRNA has many AUUUA instability motifs and the D2 mRNA has an approximately 2-h half-life (14, 19, 21). The study of the functional role of these structures is complicated by the fact that reducing the length of the 3'UTR simultaneously alters the relative proximity of the SECIS element to the UGA in the catalytic center of the coding region. Increasing the distance between the SECIS element and the coding region of the rat type 1 deiodinase by 1.5 kb does not decrease D1 expression (43). However, the larger change of 3.7 kb could have such effects. Substitution of Neurospora and mouse thyroid receptor
DNA, which have no AUUUA elements, for the chicken D2 3'UT cause a 50% increase in transient D2 expression (Fig. 6A
). However, the amount of D2 mRNA 13 h after inhibition of D2 transcription was 2.5-fold higher (Fig. 6A
). Because both the cD2wt, cD23.7exo, and cD23.7
mRNAs were expressed using the same promoter contained in the same nucleotide context, this finding suggests that the half-life of the chimeric mRNA was prolonged. Deletion of the 3.7-kb fragment increases D2 activity 3.5-fold, but there is a much greater increase in the mRNA after blocking transcription, again suggesting that the truncated mRNA is more stable.
Even though the structures of the transcripts in cD23.7
transfected cells are not defined (Fig. 6B
), both the shorter and longer mRNAs hybridize with both coding sequence and the SECIS element containing probes indicating that both can potentially be translated. The differences in D2 expression are not changed by a 10-fold reduction in transfected plasmid making it highly unlikely that saturation of one or more of the components required for selenoprotein synthesis limits translation. Thus, we are unable to address definitively the role of the 3'UTR in D2 translation efficiency.
Alternative Splicing in the Coding Region of the D2 mRNA Gives Rise to Inactive D2 Proteins
At least three 6- to 7-kb D2 mRNA transcripts differing by 500700 nt have been identified in human thyroid, and two in brain and other tissues (1, 9, 11). However, differences of 50100 nt are impossible to recognize by size alone in such a long mRNA. We have identified two D2 transcripts in chicken brain and liver, one of which encodes an inactive protein. The cDNA encoding the inactive D2 lacks 77 nt in the coding region, apparently due to the use of an alternative splice site. The genomic organization of the cdio2 gene is not known but the 3' end of the missing cD2 77 bps maps exactly to the position of the exon/intron junction of the mouse and human dio2 genes, suggesting the alternative processing of an intron in the same position (Fig. 7A
) (11, 20, 44). PCR studies show that both wt and
77cD2 transcripts are expressed in both telencephalon and liver and different ratios could account for different D2 activity/mRNA ratios.
Similarly, an alternatively spliced hD2 mRNA encoding an inactive protein is expressed in human thyroid tissue. This transcript is readily seen on RT-PCR using oligonucleotides spanning the exon-intron junction between the two coding exons
and ß (Fig. 8
, A and D). It derives from an alternative 5' exon-intron junction leading to insertion of 108 intronic nucleotides after codon 74 (Fig. 8
, B and D). The insertion does not change the reading frame and is translated even though it contains an additional UGA codon (Fig. 8
, B and C). It has no catalytic activity. Parallel with the cloning of the thyroidal hD2+108 coding region described here an hDII-b splice variant of the same sequence (GenBank accession no. AB041843) was cloned from a human umbilical vein endothelial cell line ECV304 by Ohba et al. (45). They also demonstrated the presence of the hDII-b transcript by RT-PCR in the human brain, lung, kidney, heart, and trachea. Another hD2 mRNA with a 242-bp-long genomic insertion (hDII-c) has also been cloned from ECV304 cells (GenBank accession no. AB041844) (45). We could not detect this transcript in human thyroid by RT-PCR.
Two other D2 mRNA variants have been cloned. In a mouse cochlear cDNA library, a clone was identified containing a truncated D2 coding region followed by the 3'UTR (GenBank AF177197) (42). Interestingly, in this case the divergence point was 35 bp 3' to the conserved exon/intron junction. The transcript abundance and its functional properties were not reported. The hD2 5'UTR contains an alternatively spliced intron in human thyroid (11). The last four nucleotides of hORF-B and the whole region encoding hORF-C are missing from this D2 mRNA but the potent hORF-A is not affected.
In summary, a number of posttranslational mechanisms can regulate the D2 content of a given tissue. Selenium deficiency reduces D2 activity approximately 30-fold in the mesothelioma cell line MSTO-211 (6). Substrate-induced acceleration of D2 ubiquitination leads to rapid proteasomal degradation, reducing the D2 protein half-life 2- to 3-fold (18). The present studies show that sORFs in the 5'UTR reduce D2 expression by as much as 5-fold. Alternative splicing is another mechanism that dictates the D2 concentration in specific tissues, such as the brain, liver, and thyroid of chickens or humans. While the long, approximately 5-kb 3'UT separating the UGA in the D2 active center from the SECIS element might have been expected to reduce the D2 activity/mRNA ratio by reducing translation, our studies suggest that the AUUUA instability motifs and, more importantly, reducing the distance between the SECIS and coding region may decrease D2 expression by destabilizing the D2 mRNA. However, more direct studies of translational efficiency will be required to confirm this.
The multiplicity of potential regulatory mechanisms for this protein argues that there is meticulous control of the deiodinative activation of T4 to T3. The demonstration that much of this occurs at a posttranscriptional level indicates that D2 activity in different tissues cannot be inferred simply by quantitating its mRNA.
 |
MATERIALS AND METHODS
|
---|
DNA Transfection
Cells were transfected as previously described using calcium phosphate precipitation (46). GH4C1 cells were transfected using Lipofectamine Plus (Life Technologies, Inc., Gaithersburg, MD). Ten micrograms of pUHD10-3 based vector encoding the deiodinase was transiently transfected together with 4 µg pUHD-15, which is required for the transcriptional activation of the pUHD10-3 promoter. This activation can be suppressed by tetracycline that inhibits the binding of the activator expressed by pUHD-15 to the pUHD10-3 promoter (47). The transfection efficiency was monitored by the cotransfection of 3 µg TKGH and hGH was assayed in the media and used to correct D2 or D1 activities (48). Results are given as the mean ± SEM of D2 or D1 activities of duplicate plates of at least three separate experiments as the percentage of the cD2 or rD1 control, respectively.
Expression Constructs
The cD2 reporter construct contained a 33-bp cD2 5'UTR fragment as spacer between the cloning site and the initiator ATG followed by the cD2 coding region between EcorI-HindIII and a rD1 minimal SECIS element between HindIII-NotI (21). In the constructs described below, different UTR fragments were cloned between the SacII site of the D10 vector and RI if not indicated otherwise. The human 5'UTR Vent PCR fragments were generated from the hdio2 clone 2 (11). The name of the construct is followed by the oligos in parentheses used to prepare it: hdio25'UT (Bp35-Bp37); hdio25'UT
(Bp36-Bp37); hORF-A,B-cD2 (Bp36-Bp38); hORF-A-cD2 (SacII-ApoI fragment of the Bp36-Bp38 PCR); hORF-B-cD2 (ApoI-EcoRI fragment of the Bp36-Bp38 PCR with the ApoI at 5'); hORF-C-cD2 (Bp39-Bp37); hdio25'UT(41/240)-cD2 (Bp36-Bp60); hORF-A(Wt)-cD2 (Bp52 was ligated to the corresponding antisense, latter was not indicated in Table 2
); hORF-A(Mut-ATG)-cD2 (Bp54 and corresponding antisense); hORF-A(Mut-Koz)-cD2 (Bp62 and corresponding antisense); cORF-B(Wt)-cD2 (Bp68 and corresponding antisense); cORF-B(Mut-ATG)-cD2 (Bp70 and corresponding antisense).
The hORF-A(Wt)-cD2 and hORF-A(Mut-ATG)-cD2 were cut by EcorI-NotI and religated, generating in this way hORF-A(Wt)-D10 and hORF-A(Mut-ATG)-D10, respectively. As the rD1 reporter a construct containing a rD1 coding sequence followed by rat D1 minimal SECIS element was used (49). The hORF-A(Wt)-cD2 and hORF-A(Mut-ATG)-cD2 were cut by EcorI-HindIII and the EcorI-HindIII fragment of the rD1 reporter (the rD1 coding region) was ligated to generate the hORF-A(Wt)-rD1 and the hORF-A(Mut-ATG)-rD1, respectively. The cD25'UT was produced by inserting the SacII-RI fragment of the full-length cD2 cDNA between these sites of the cD2 reporter.
The cD23.7
was generated by cutting the intact cD2 cDNA (21) by Bsu36I and BsrGI. The ends were filled in and religated. This removed 3710 bp from the 3'UTR (from 16005310 of GenBank accession no. AF125575) between the coding region and SECIS. In the cD23.7exo the same length of exogenous cDNA containing no AUUUA motifs was inserted. In brief, the intact cD2 cDNA was cut by NcoI and BsrG1 (BsrGI blunted) and the NcoI-XbaI (blunt) fragment of the Pho2 gene of Neurospora crassa (kindly provided by Dr. Dorsey Stuart (Neugenesis Corp., San Carlos, CA) was ligated between these sites. The resulting construct was cut by Bsu36I-NcoI (NcoI blunted) and the Bsu36I cut of Bp107-Bp108 Vent PCR fragment (approximately 980 bp) generated from mouse TR
-CDM13 template (50) was inserted. The constructs were confirmed by automated sequencing. Oligonucleotide sequences are indicated in Table 2
.
The
77cD2 Coding Region
Total telencephalic RNA of an adult chicken was reverse transcribed and amplified using the Expand Long Template PCR System (Roche Molecular Biochemicals, Indianapolis, IN) as described earlier (21). The fragment was ligated into pGEM-T and sequenced. The clone was completed with the missing portion of the 3'UTR and subcloned into D10 after the strategy used earlier for the assembling of the cD2 cDNA (21). The mRNA isolated from the telencephalon and liver from another adult chicken was reverse transcribed using Bp11 and amplified by Taq polymerase (Sigma, St. Louis, MO) by Bp3-Bp90 primers. The bands generated on the brain cDNA were cloned into pGEM-T and sequenced. The amplifications were performed in two separate reactions.
The hD2+108 Coding Region
The mRNA from thyroid of two patients, one with a thyrotropin-producing pituitary adenoma or Graves disease and a normal thyroid obtained postmortem were isolated by Trizol (Life Technologies, Inc.). Reverse transcription was started by Bp112 and amplified by the intron spanning Bp97-Bp41 primers using the GIBCO Supermix. The approximately 600-bp long upper band was cloned from all three samples. They were identical by sequencing. A D2+108 PCR fragment obtained by Bp113111 was also cloned from the Graves sample, ligated into pGEM-T (Promega Corp.) (5' at T7). A SacII-HindIII fragment (the latter from the insert) was placed between these sites of the Bp97-Bp41 insert producing the hD2+108 coding in pGEM-T (hD2+108pGEM-T). To place the insert 5' to a SECIS element in the D10 vector the SphI-PstI fragment of the hD2+108pGEM-T was inserted between these sites of hD2-D10-SelP or N-FLAGhD2-D10-SelP producing D2+108-SelP and FLAG D2+108-SelP, respectively. The constructs were sequenced in the ABI Prism 377 automated sequencer using dye terminators.
Deiodinase Assays
Homogenates of transfected cells were assayed in duplicate for either D2 or D1 activity as described (21). In brief, D2 assays contained [125I]T4, 2 nM T4, and 20 mM dithiothreitol (DTT) in phosphate-EDTA (PE) buffer while for D1 measurements [125I]rT3, 1 µM rT3 and 10 mM DTT was used in a 300-µl volume. Activity was expressed as pmol/min·mg protein. A telencephalon sample from an adult chicken was homogenized in PE buffer containing 0.25 sucrose and 10 mM DTT. The sample was taken from the same animal used earlier for liver D2 measurements (21). For the maximum velocity and Michaelis-Menten constant (T4) determination, 200 µg protein were assayed in duplicate for 5'-deiodinase activity in a final volume of 300 µl PE buffer containing 20 mM DTT, [125I]T4, 1, 1.5, 3, and 10 nM T4 and 1 mM 6-n-propylthiouracil. Incubation was for 120 min at 37 C. Activity was expressed as fmol T4 deiodinated/h·mg protein.
Northern Blots
D10 based constructs were transfected into HEK-293 cells as described. On the second day after transfection, 1 µg/ml tetracycline was added to the culture medium to block transcription (47). Cells were harvested after 13 h after tetracycline addition and RNA was isolated using Trizol LS (Life Technologies, Inc.). RNA was treated using ribonuclease-free deoxyribonuclease I (Life Technologies, Inc.), following the instructions of the manufacturer. Ten micrograms of total RNA were processed for Northern blot. The experiment was performed on two separate samples for each construct. The blot was probed with a 450-bp cD2 coding region probe (21) then stripped and probed with a 330-bp probe generated by PCR (Bp135-Bp136) containing the SECIS region of cD2. The blot was exposed for 20 h at -80 C on Biomax film (Eastman Kodak, Rochester, NY). Northern blots were performed as described (21). As denominator for densitometry, both the density of the 28S subunit and the amount of the hGH in the media were used to monitor loading and transfection efficiency, respectively.
Na2(75Se)O3 Labeling and Immunoprecipitation of Transiently Expressed FLAG-D2
The FLAG D2+108-SelP or D2+108-SelP constructs were transfected into HEK-293 cells. The transfected cells were cultured in the presence of 46 µCi of Na2(75Se)O3/dish on d 2 after transfection in the presence of DMEM supplemented with 10% fetal bovine serum. Na2(75Se)O3 was kindly provided by the University of Missouri Research Reactor, courtesy of Drs. Marla Berry and Dolph L. Hatfield. On d 3, the cells were washed with PBS and sonicated in a lysis buffer [1% Triton X-100, 1% bovine hemoglobin, 0.2 U aprotinin/ml, 0.2 U leupeptin/ml 1 mM phenylmethylsulfonyl fluoride in Tris-saline-azide (TSA) buffer (0.01 M Tris-HCl, pH 8.0; 0.14 M NaCl)]. Sonication was followed by lysis under slow agitation at 4 C for 30 min. After centrifugation of the lysate at 4000 rpm for 15 min, each supernatant was incubated under slow agitation at 4 C for 1 h with preimmune mouse sera to a final dilution of 1:100 and 20 µl Protein G Plus Protein A Agarose (Oncogene Research Products, San Diego, CA) by centrifugation at 1500 rpm for 15 min. The supernatant was incubated with 2 µl of anti-FLAG M2 antibody (Sigma) and 20 µl Protein G Plus Protein A Agarose (Oncogene Research Products) for 1218 h under slow agitation at 4 C. After centrifugation at 2500 rpm for 15 min, the pellet was washed three times in 1:10 diluted lysis buffer, twice in 0.3 M TSA, and twice in 0.14 M TSA. The pellets were then heated at 95 C for 4 min in sample loading buffer, spun and resolved by SDS-PAGE. The gels were subjected to autoradiography for 12 d.
Sequences
The chicken and human 5'UTR are formed in the GenBank accession nos. AF125575 and AF188709 (11, 21). The missing 5' portion of the mouse 5'UTR was recently published by Song et al. (44) (GenBank accession no. AF195885). The rat D2 5'UTR is contained in the GenBank entry nos. U53505 and AF249274 (1, 12). The TSS of the chicken and mouse D2 mRNA was determined by 5'RACE and of the human by 5'RACE, S1 digestion and primer extension (11, 21, 44). The putative TSS of rat D2 was assigned by sequence alignment. The sequence of the
77cD2 coding region was deposited into the GenBank under accession no. AF401753.
 |
FOOTNOTES
|
---|
This work was supported by NIH Grant DK-36256. B.G. is a Magyary Zoltán postdoctoral fellow of the Hungarian Education Ministry and supported by an FKFP grant. This work was presented in part at the 12th International Thyroid Congress, 2000 (Kyoto, Japan).
Abbreviations: c, Chicken; D2, type 2 deiodinase; dio2, type 2 iodothyronine deiodinase gene; DTT, dithiothreitol; h, human; hD2, human D2; HEK, human embryonic kidney; hGH, human GH; mut, mutant; nt, nucleotide; ORF, open reading frame; rT3, reverse T3; SECIS, selenocysteine insertion sequence; sORF, short ORF; TSS, transcription start site; UTR, untranslated region; wt, wild-type.
Received for publication January 23, 2002.
Accepted for publication March 14, 2002.
 |
REFERENCES
|
---|
- Croteau W, Davey JC, Galton VA, St Germain DL 1996 Cloning of the mammalian type II iodothyronine deiodinase. A selenoprotein differentially expressed and regulated in human and rat brain and other tissues. J Clin Invest 98:405417[Abstract/Free Full Text]
- Bianco AC, Salvatore D, Gereben B, Berry MJ, Larsen PR 2002 Biochemistry, cellular and molecular biology and physiological roles of the iodothyronine selenodeiodinases. Endocr Rev 23:3889[Abstract/Free Full Text]
- Berry MJ, Banu L, Chen YY, Mandel SJ, Kieffer JD, Harney JW, Larsen PR 1991 Recognition of UGA as a selenocysteine codon in type I deiodinase requires sequences in the 3' untranslated region. Nature 353:273276[CrossRef][Medline]
- Buettner C, Harney JW, Larsen PR 2000 The role of selenocysteine 133 in catalysis by the human type 2 iodothyronine deiodinase. Endocrinology 141:46064612[Abstract/Free Full Text]
- Pallud S, Lennon AM, Ramauge M, Gavaret JM, Croteau W, Pierre M, Courtin F, St Germain DL 1997 Expression of the type II iodothyronine deiodinase in cultured rat astrocytes is selenium-dependent. J Biol Chem 272:1810418110[Abstract/Free Full Text]
- Curcio C, Baqui MMA, Salvatore D, Rihn BH, Mohr S, Harney JW, Larsen PR, Bianco AC 2001 The human type 2 iodothyronine deiodinase is a selenoprotein highly expressed in a mesothelioma cell line. J Biol Chem 276:3018330187[Abstract/Free Full Text]
- Tujebajeva RM, Copeland PR, Xu XM, Carlson BA, Harney JW, Driscoll DM, Hatfield DL, Berry MJ 2000 Decoding apparatus for eukaryotic selenocysteine incorporation. EMBO Rep 2:158163[CrossRef]
- Salvatore D, Bartha T, Harney JW, Larsen PR 1996 Molecular biological and biochemical characterization of the human type 2 selenodeiodinase. Endocrinology 137:33083315[Abstract]
- Salvatore D, Tu H, Harney JW, Larsen PR 1996 Type 2 iodothyronine deiodinase is highly expressed in human thyroid. J Clin Invest 98:962968[Abstract/Free Full Text]
- Murakami M, Araki O, Hosoi Y, Kamiya Y, Morimura T, Ogiwara T, Mizuma H, Mori M 2001 Expression and regulation of type II iodothyronine deiodinase in human thyroid gland. Endocrinology 142:29612967[Abstract/Free Full Text]
- Bartha T, Kim SW, Salvatore D, Gereben B, Tu HM, Harney JW, Rudas P, Larsen PR 2000 Characterization of the 5'-flanking and 5'-untranslated regions of the cyclic adenosine 3',5'-monophosphate-responsive human type 2 iodothyronine deiodinase gene. Endocrinology 141:229237[Abstract/Free Full Text]
- Gereben B, Salvatore D, Harney JW, Tu HM, Larsen PR 2001 The human, but not rat, dio2 gene is stimulated by thyroid transcription factor-1 (TTF-1). Mol Endocrinol 15:112124[Abstract/Free Full Text]
- Burmeister LA, Pachucki J, St Germain DL 1997 Thyroid hormones inhibit type 2 iodothyronine deiodinase in the rat cerebral cortex by both pre- and posttranslational mechanisms. Endocrinology 138:52315237[Abstract/Free Full Text]
- Kim SW, Harney JW, Larsen PR 1998 Studies of the hormonal regulation of type 2 5'-iodothyronine deiodinase messenger ribonucleic acid in pituitary tumor cells using semiquantitative reverse transcription-polymerase chain reaction. Endocrinology 139:48954905[Abstract/Free Full Text]
- Smith M, Burke Z, Humphries A, Wells T, Klein D, Carter D, Baler R 2001 Tissue-specific transgenic knockdown of fos-related antigen 2 (fra-2) expression mediated by dominant negative fra-2. Mol Cell Biol 21:37043713[Abstract/Free Full Text]
- Steinsapir J, Harney J, Larsen PR 1998 Type 2 iodothyronine deiodinase in rat pituitary tumor cells is inactivated in proteasomes. J Clin Invest 102:18951899[Abstract/Free Full Text]
- Steinsapir J, Bianco AC, Buettner C, Harney J, Larsen PR 2000 Substrate-induced down-regulation of human type 2 deiodinase (hD2) is mediated through proteasomal degradation and requires interaction with the enzymes active center. Endocrinology 141:11271135[Abstract/Free Full Text]
- Gereben B, Goncalves C, Harney JW, Larsen PR, Bianco AC 2000 Selective proteolysis of human type 2 deiodinase: a novel ubiquitin-proteasomal mediated mechanism for regulation of hormone activation. Mol Endocrinol 14:16971708[Abstract/Free Full Text]
- Buettner C, Harney JW, Larsen PR 1998 The 3'-untranslated region of human type 2 iodothyronine deiodinase mRNA contains a functional selenocysteine insertion sequence element. J Biol Chem 273:3337433378[Abstract/Free Full Text]
- Davey JC, Schneider MJ, Becker KB, Galton VA 1999 Cloning of a 5.8 kb cDNA for a mouse type 2 deiodinase. Endocrinology 140:10221025[Abstract/Free Full Text]
- Gereben B, Bartha T, Tu HM, Harney JW, Rudas P, Larsen PR 1999 Cloning and expression of the chicken type 2 iodothyronine 5'-deiodinase. J Biol Chem 274:1376813776[Abstract/Free Full Text]
- McCaughan KK, Brown CM, Dalphin ME, Berry MJ, Tate WP 1995 Translational termination efficiency in mammals is influenced by the base following the stop codon. Proc Natl Acad Sci USA 92:54315435[Abstract]
- Chen CY, Shyu AB 1995 AU-rich elements: characterization and importance in mRNA degradation. Trends Biochem Sci 20:465470[CrossRef][Medline]
- Hinnebusch AG 1994 Translational control of GCN4: an in vivo barometer of initiation-factor activity. Trends Biochem Sci 19:409414[CrossRef][Medline]
- Werner M, Feller A, Messenguy F, Piérard A 1987 The leader peptide of yeast gene CPA1 is essential for the translational repression of its expression. Cell 49:805813[Medline]
- Jackson RJ, Kaminski A 1995 Internal initiation of translation in eukaryotes: the picornavirus paradigm and beyond. RNA 1:9851000[Medline]
- Geballe AP, Morris DR 1994 Initiation codons within 5'-leaders of mRNAs as regulators of translation. Trends Biochem Sci 19:159164[CrossRef][Medline]
- Kozak M 1987 An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res 15:81258148[Abstract]
- Kozak M 1991 An analysis of vertebrate mRNA sequences: intimations of translational control. J Cell Biol 115:887903[Abstract]
- Mori Y, Matsubara H, Murasawa S, Kijima K, Maruyama K, Tsukaguchi H, Okubo N, Hamakubo T, Inagami T, Iwasaka T, Inada M 1996 Translational regulation of angiotensin II type 1A receptor. Role of upstream AUG triplets. Hypertension 28:810817[Abstract/Free Full Text]
- Lincoln AJ, Monczak Y, Williams SC, Johnson PF 1998 Inhibition of CCAAT/enhancer-binding protein
and ß translation by upstream open reading frames. J Biol Chem 273:95529560[Abstract/Free Full Text]
- Manzella JM, Blackshear PJ 1990 Regulation of rat ornithine decarboxylase mRNA translation by its 5'- untranslated region. J Biol Chem 265:1181711822[Abstract/Free Full Text]
- Bates B, Hardin J, Zhan X, Drickamer K, Goldfarb M 1991 Biosynthesis of human fibroblast growth factor-5. Mol Cell Biol 11:18401845[Medline]
- Kwon HS, Lee DK, Lee JJ, Edenberg HJ, Ahn YH, Hur MW 2001 Posttranscriptional regulation of human ADH5/FDH and Myf6 gene expression by upstream AUG codons. Arch Biochem Biophys 386:163171[CrossRef][Medline]
- Byrne PC, Sanders PG, Snell K 1995 Translational control of mammalian serine hydroxymethyltransferase expression. Biochem Biophys Res Commun 214:496502[CrossRef][Medline]
- Child SJ, Miller MK, Geballe AP 1999 Translational control by an upstream open reading frame in the HER-2/neu transcript. J Biol Chem 274:2433524341[Abstract/Free Full Text]
- Parola AL, Kobilka BK 1994 The peptide product of a 5' leader cistron in the ß2 adrenergic receptor mRNA inhibits receptor synthesis. J Biol Chem 269:44974505[Abstract/Free Full Text]
- McGraw DW, Forbes SL, Kramer LA, Liggett SB 1998 Polymorphisms of the 5' leader cistron of the human beta2-adrenergic receptor regulate receptor expression. J Clin Invest 102:19271932[Abstract/Free Full Text]
- Kozak M 1986 Influences of mRNA secondary structure on initiation by eukaryotic ribosomes. Proc Natl Acad Sci USA 83:28502854[Abstract]
- Tanaka K, Murakami M, Greer MA 1986 Type-II thyroxine 5'-deiodinase is present in the rat pineal gland. Biochem Biophys Res Commun 137:863868[Medline]
- Kamiya Y, Murakami M, Araki O, Hosoi Y, Ogiwara T, Mizuma H, Mori M 1999 Pretranslational regulation of rhythmic type II iodothyronine deiodinase expression by ß-adrenergic mechanism in the rat pineal gland. Endocrinology 140:12721278[Abstract/Free Full Text]
- Campos-Barros A, Amma LL, Faris JS, Shailam R, Kelley MW, Forrest D 2000 Type 2 iodothyronine deiodinase expression in the cochlea before the onset of hearing. Proc Natl Acad Sci USA 97:12871292[Abstract/Free Full Text]
- Berry MJ, Banu L, Harney JW, Larsen PR 1993 Functional characterization of the eukaryotic SECIS elements which direct selenocysteine insertion at UGA codons. EMBO J 12:33153322[Abstract]
- Song S, Adachi K, Katsuyama M, Sorimachi K, Oka T 2000 Isolation and characterization of the 5'-upstream and untranslated regions of the mouse type II iodothyronine deiodinase gene. Mol Cell Endocrinol 165:189198[CrossRef][Medline]
- Ohba K, Yoshioka T, Muraki T 2001 Identification of two novel splicing variants of human type II iodothyronine deiodinase mRNA. Mol Cell Endocrinol 172:169175[CrossRef][Medline]
- Brent GA, Larsen PR, Harney JW, Koenig RJ, Moore DD 1989 Functional characterization of the rat growth hormone promoter elements required for induction by thyroid hormone with and without a co-transfected ß type thyroid hormone receptor. J Biol Chem 264:178182[Abstract/Free Full Text]
- Gossen M, Bujard H 1992 Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA 89:55475551[Abstract]
- Martin GW, Harney JW, Berry MJ 1996 Selenocysteine incorporation in eukaryotes: Insights into mechanism and efficiency from sequence, structure, and spacing proximity studies of the type 1 deiodinase SECIS element. RNA 2:171182[Abstract]
- Martin GW, Harney JW, Berry MJ 1998 Functionality of mutations at conserved nucleotides in eukaryotic SECIS elements is determined by the identify of a single non-conserved nucleotide. RNA 4:6573[Abstract/Free Full Text]
- Prost E, Koenig RJ, Moore DD, Larsen PR, Whalen RG 1988 Multiple sequences encoding potential thyroid hormone receptors isolated from mouse skeletal muscle cDNA libraries. Nucleic Acids Res 16:6248[Medline]