Transcription of the rat testis-specific Rtdpoz-T1 and -T2 retrogenes during embryo development: co-transcription and frequent exonisation of transposable element sequences
© Huang et al; licensee BioMed Central Ltd. 2009
Received: 20 March 2009
Accepted: 25 July 2009
Published: 25 July 2009
Retrotransposition is an important evolutionary force for the creation of new and potentially functional intronless genes which are collectively called retrogenes. Many retrogenes are expressed in the testis and the gene products have been shown to actively participate in spermatogenesis and other unique functions of the male germline. We have previously reported a cluster of retrogenes in the rat genome that encode putative TRAF- and POZ-domain proteins. Two of the genes, Rtdpoz-T1 and -T2 (abbreviated as T1 and T2), have further been shown to be expressed specifically in the rat testis.
We show here that the T1 and T2 genes are also expressed in the rat embryo up to days 16–17 of development when the genes are silenced until being re-activated in the adult testis. On database interrogation, we find that some T1/T2 exons are chromosomally duplicated as cassettes of 2 or 3 exons consistent with retro-duplication. The embryonic T1/T2 transcripts, characterised by RT-PCR-cloning and rapid amplification of cDNA ends, are further found to have acquired one or more noncoding exons in the 5'-untranslated region (5'-UTR). Most importantly, the T1/T2 locus is embedded within a dense field of relics of transposable element (TE) derived mainly from LINE1 and ERV sequences, and the TE sequences are frequently exonised through alternative splicing to form the 5'-UTR sequences of the T1/T2 transcripts. In a case of T1 transcript, the 3'-end is extended into and terminated within an L1 sequence. Since the two genes share a common exon 1 and are, therefore, regulated by a single promoter, a T2-to-T1 co-transcription model is proposed. We further demonstrate that the exonised 5'-UTR TE sequences could lead to the creation of upstream open reading frames resulting in translational repression.
Exonisation of TE sequences is a frequent event in the transcription of retrogenes during embryonic development and in the testis and may contribute to post-transcriptional regulation of expression of retrogenes.
Retrotransposition is an important evolutionary driving force for the creation of new genes with novel lineage- and species-specific phenotypic traits. New genes created through retrotransposition are retrogenes that are devoid of introns. Furthermore, paralogues are subsequently created through segmental duplications and sequence modifications. Retrogenes could be re-activated by putative promoters and other transcription regulatory elements suitably located upstream of the retrogene insertion sites [1–3]. In the process of transcriptional re-activation, the newly arisen transcript may acquire one or more noncoding exons in the 5'-untranslated region (5'-UTR). In the context of our current knowledge of the generation of multiple transcripts from a single gene through alternative splicing [4, 5], the term "retrogene" is used throughout this paper to mean the "genomic copy" of a gene that is consituted of a complete coding sequence without intron interruption taking no consideration on whether the resulting transcripts carry exonised sequences through alternative splicing. Whether or not "retrogene" should be redefined as such is debatable.
Many retrogenes are functional . It has further been estimated that there are in excess of 1,000 transcribed retrocopies in the human genome over a tenth of which is biologically active . Interestingly, the bulk of retrogenes is preferably expressed in the testis where the retrogene products actively participate in the spermatogenesis process and serve to further enhance biological functions unique to the male germline [8, 9]. Transcription in the testis is not as tightly regulated as in other somatic tissues due to hyper transcription rates which could result in non-discriminatory activation of otherwise imperfect or weak promoters . Such a mode of promiscuous transcription, and possibly erratic alternative splicing processes, could lead to the generation of fortuitous testicular transcripts. Promiscuous transcription and transient transcriptional gene activation have also been shown to occur at the crucial stage of zygotic genome activation . One outstanding feature of promiscuos transcription is excessive transcription of highly repetitive genomic sequences [11, 12]. The bulk of genomic repetitive sequences are transposable elements (TEs). A significant number of mammalian genes has been shown to be regulated by transcriptional elements of the endogenous retroviruses (ERVs) or long-terminal repeats (LTRs) of TEs . Evidence is emerging to suggest that ERVs and other TEs may constitute a critical driving force in speciation .
We have previously proposed the existence of a novel bipartite TDPOZ protein family members of which carry the TD (TRAF domain), also called MATH (meprin and TRAF homology, and POZ (poxvirus and zinc finger)/BTB (Broad complex, Tramtrack, Bric à brac) . Almost all known eukaryotic TD proteins are known to be involved in the regulation of protein processing and ubiquitination . The representative TD proteins, the tumour necrosis factor receptor-associated factors (TRAFs), bind to the tumour necrosis factor receptors or other adaptor molecules to participate in cellular proliferation and survival, and in cell-death signalling [17, 18]. On the other hand, POZ proteins have been implicated in biological processes including DNA damage responses, cell cycle progression and in embryonic developmental events and hematopoietic stem cell fate determination . The TD and POZ domains are found in separate proteins in association with other DNA-binding or protein-protein interacting domains except for the TDPOZ bipartite proteins that we have first reported [15, 20]. Tdpoz genes are found in both higher and lower animals and in plants suggesting important biological functions. To date, the only functionally characterised mammalian TDPOZ protein is the nuclear speckle-associated protein SPOP. SPOP acts as an adaptor of Daxx in the ubiquitination process involving CUL3-based ubiquitin ligase contributing to regulation of Hedgehog/Gli signalling [21–24].
Developmental regulation of T1 and T2 expression
To determine the expression status of the T1 and T2 genes during development, RNA was prepared from developmental stages between day 12 (E12) to day 20 (E20) just before birth. RT-PCR was performed using T1- or T2-discriminating primers located in different exon sequences of the genes (Figure 1A). The RT-PCR results show that T1 was expressed only at stages E14 to E16 of development (Figure 1B, lanes 3–5) with a distinctive expression profile for each stage indicating differential T1 transcription at these developmental stages. On the other hand, T2 was expressed up to E17 and the expression profile was rather consistent and was largely similar to that of the testis  except that at E16, extra bands were detected, and at E17 only the lower band was present (Figure 1B, lanes 9–14). The transcription profiling experiments, hence, establish that expression of the testicular T1 and T2 genes is developmentally regulated, and that there exists a notable disparity in the expression patterns of the genes at specific developmental stages. This suggests that transcription of the two genes, despite the sharing of the leader exon 1a, is differentially regulated. The T1 and T2 genes, are silenced at day 17 (E17) and day 18 (E18) of development, respectively, when organogenesis is now completed and the foetus enters the active phase of growth and expansion.
Assorted 5'-UTR structures derived from alternative splicing involving transposable element sequences
The uncovering in the 3'-RACE experiments of the extended 3'-UTR sequence of T1 warranted further RT-PCR analysis to confirm the authenticity of the 3'-extension and to further investigate if other T1 transcripts carried similar 3'-extension. To achieve this goal, two-round nested RT-PCR was performed across the entire gene sequences stretching from the 5'-terminal exon 1a to the extended L1 sequence using primers Ex1a-B and 3096R followed by the use of primers Ex1a-A and 2965R (Figure 1A, T1 primer map). In the three T1-expressing developmental stages, one or two RT-PCR bands were discerned indicating, indeed, the existence of multiple T1 transcripts with the 3'-extension (Figure 1C). The PCR products were subsequently cloned and sequenced and the sequences are designated with the prefix "XT" (Figure 2A). Full-length XT transcripts are constructed assuming they share the same testicular 5'- and the 3'-end of T1E16-B (Figure 2A). A notable exception in the XT transcripts was T1E14-XT2 which included a new 45-bp exon the sequence of which was located 887-bp downstream of the leader exon 1a; this transcript also lacks the ubiquitous exons 2 and 3 (T1–2 and -3) of T1 (Figure 2A).
On the other hand, the developmental T2 transcripts are uncomplicated in exon organisation (Figure 2B). All developmental T2 transcripts also carry the leader exon 1a and the uninterrupted coding exon 3 (exon T2-3). The testicular exon 2 (T2-2), which itself is an ERV remnant, may or may not be associated with the developmental T2 transcripts. In T2E16-RT1, a superfluous exon derived from L1 is found located between the constitutive exon 1a and the coding exon 3 replacing exon T2-2 (Figure 2B). The T2 transcripts may be simpler in exon organisation but they are still tinted by TE-derived exons.
In summary, sequence analysis of the T1 and T2 transcripts indicates extensive alternative splicing events involving sequences of various highly repetitive TE sequences contributing to the 5'-UTR of the transcripts, particularly the T1 transcripts. In the 3'-UTR, two major termination sites were elucidated for the T1 transcripts with the 3'-distal termination site contributed by L1. It is also observed that the overall exon organisation of the T2 transcripts is uncomplicated whereas the T1 transcripts vary extensively in the 5'-UTR structure when expressed during development.
Detection of T1-T2 chimeric transcripts
The PCR bands were excised and cloned; two or more clones derived from each of these bands were sequenced; the sequences are given the prefixes T21 or T12 in the order of appearance of the T1 and T2 sequences. Exon mapping of the sequences derived clearly shows that the mix-gene PCR products are T2–T1 or T1–T2 chimeric transcripts defined by accurate splice-site demarcation (Figure 4B). The 1.7-kb T21Te-RT1 sequence is composed of the T2 exon 2 and partial exon 3 truncated within the coding sequence, and the T2 exons are accurately spliced to the T1 exons 2, 3 and the full coding sequence of exon 4. The 1.4-kb T21Te-RT2 sequence carries T2 exon 2 (T2-2) which is spliced to exons 2, 3 and 4 of T1 (T1–2, -3, and -4). Conversely, the 1.2-kb T12Te-RT1 sequence is composed of the T1 exon 2 (T1–2) spliced to exon 2 and the coding exon 3 of T2 (T2–3 and -3). The T12Te-RT2 sequence was simplest in structure in being composed of T1-exon 2 coupling with the coding T2-exon 3. Since these transcript sequences were RT-PCR products obtained rather forcefully through two rounds of PCR, they most likely represent minor populations of authentic T1–T2 chimeric transcripts. The naturally occurring 5'- and 3'-ends had not been determined for these chimeric transcripts. We have, thus, produced further experimental evidence that T1–T2 or T2-T1 chimeric transcripts do exist and their existence raises the question on how these transcripts are generated
Embedment of T1 and T2 exons in a minefield of TE sequences
Rtdpoz-T1 and -T2 exon assembly
Rn_2148 position (nt)
15,707,883 – 15,707,814
Major exon 1 for T1 and T2
15,668,417 – 15,666,808
nonLTR-L1 & ERV TE sequences
15,635,659 – 15,635,503
ERV-MT2A TE sequence
15,634,940 – 15,633,391
T2 coding sequence
15,553,789 – 15,550,897
Unique sequences amidst SINE/hAT
15,540,915 – 15,539,386
T1 coding sequence
(3' L1 extension)
15,474,800 – 15,474,727
15,474,193 – 15,473,900
ERV-MT2B TE sequence
15,115,847 – 15,115,693
ERV-MT2A TE sequence
15,115,223 – 15,113,674
T2 coding sequence
15,101,224 – 15,101,151
15,100,615 – 15,100,323
ERV-MT2B TE sequence
15,095,393 – 15,093,863
T1 coding sequence
(3' L1 extension)
As presented in the preceding sections, numerous T1 and T2 exons are derivatives of the highly repetitive sequences of the L1 and ERV transposable elements (Table 1 and Figures 2 and 3). When the ~700-kb genomic sequence that harbours all the T1 and T2 exons were subjected to a RepBase query, the sequence was found to be heavily mined with relics of TE sequences. An average TE occupancy of ~60% is computed; some segments contain as high as ~70% TE sequences (Figure 5B). In this genomic region, the unique T1 and T2 exons are precariously embedded within the TE minefield. When a transcription read-through primary transcript carries such a heavy loading of redundant TE sequences, TE sequences that have developed favourable splice junctions could readily be harvested as exons and be inducted into mature transcripts as typically exemplified by the T1 transcripts T1E16-A and T1E16-B (Figures 2 and 3). In the more simplistic T2 splicing, the ERV-MT2A has become an almost permanent landmark of the T2 transcripts harvested as exon 3a (T2-2) (Figure 5C, panel I). In the same token, the ERV-MT2B-derived exon 8a or 8b (T1-3) has also become a permanent fixture of the T1 transcripts (Figure 5C, panel II). At the 3'-end, transcription read-though of the regular transcription termination site into a downstream L1 sequence has also resulted in an extended 3'-UTR in a significant population of T1 transcripts (Figure 2A).
A model of T2 and T1 transcription and post-transcriptional processing
Only one copy of exon 1 (previously called exon 1a) could be identified in the ~700-kb genomic sequence and also in the entire rat genome. When the completed mouse genome was interrogated for possible presence of the rat exon 1-like sequence, only one exon 1 copy was found on chromosome 3 [GenBank:NT_039240.7] with a 75.1% sequence identity but there were short ~50-bp segments showing >90% identities between the two sequences (see Additional File 2). When longer (1.5 kb) genomic sequences encompassing exon 1 and about 1-kb upstream sequences of the two rodents were aligned, sequence identity remained high at 69.4% (data not shown) indicating evolutionary relatedness. The described mouse-rat genomic sequence identity further supports the uniqueness of the exon 1 sequence in the rat genome. Interestingly, this exon 1 sequence is found only in the rodent genomes and not in the genomes of other animals and plants examined (data not shown) suggesting that the exon had evolved independently since the branching out of the rodent lineage.
Taken together, the proposed exon organisation offers a model of co-transcription and post-transcriptional processing to explain the structure of the T1 and T2 transcripts. Firstly, the close proximity of the leader exon 1 with the T2 exons (exons 1, 3a and 4a) explains why the T2 transcripts are uncomplicated with fewer splice variants whereas the T1 transcripts are highly erratic and are frequently infused with TE sequences due to the extended size of the proposed primary transcripts. Secondly, the model also dissembles that chimeric transcripts in both the T1–T2 and T2-T1 orientations are generated as rogue transcripts that have acquired illegitimate exons of the cousin gene through erratic alternative splicing. Although there is no evidence of alternative trans splicing, this possibility could not, however, be completely ruled out.
Translational repression by TE-derived uAUGs and uORFs of T1 transcripts
To investigate if TE-containing 5'-UTR sequences of different T1 transcripts contribute to regulation of gene expression, the 5'-UTR sequences of the T1, T1E16-RT4 and T1E16-A transcripts were inserted before the luciferase gene under the regulation of the SV40 promoter and an SV40 enhancer; the constructs were used in transient transfection of the Chinese hamster ovary cell line, CHO-K1, followed by luciferase activity assays (Figure 7B). The results show that the luciferase activities under the regulation of the uAUG- and uORF-free T1E16-RT4 were maximal in the presence or absent of the SV40 enhancer. On the other hand, the uORF-abundant 5'-UTR of T1E16-A resulted in the lowest levels of luciferase activity whereas the T1 construct with two uORFs showed intermediate level of luciferase activity. Similar albeit lower relative luciferase activities were obtained using the testicular cancer cell line LC-540 in similar transfection and luciferase assays (data not shown).
To determine if the varied luciferase activities observed are associated with differential RNA stabilities, total RNA was prepared from the transfected cells at different post-transfection time points for RT-PCR analysis. At each of the time points examined, the luciferase mRNA level was found to be comparable for the three 5'-UTR constructs indicating similar luciferase mRNA stability despite the presence of different 5'-UTR sequences (Figure 7C). To discern possible regulation at the translation step, western blot analysis of lysate of the same sets of transfected cells was performed using an anti-luciferase antibody. The results show that the luciferase protein level was maximal for T1E16-RT4, minimal for T1E16-A and intermediate for T1, in direct agreement with the relative luciferase activities determined above (Figure 7D). The effects of uAUGs and uORFs on translation were further supported by data derived from mutagenesis analysis of the two uORFs of T1 in the luciferase constructs (Figure 7E). On removal of either or both the uORFs of T1, luciferase activities were partially restored. Our data collectively indicate that different 5'-UTR sequences in the T1 transcript variants carrying different numbers of TE-derived uAUGs and uORFs could result in repressed translation of the T1 gene.
In a previous work, we first described testis-specific transcription of the Rtdpoz-T1 and -T2 genes . In this work, we show that T1 and T2 are also transcribed in the developing embryo (Figure 1). More significantly, we show that each of the uninterrupted T1 and T2 coding exons are duplicated and the exons are embedded in a dense field of TE sequences. Consequently, the embryonic T1/T2 transcription displays two novel features: co-transcription of the two genes and frequent exonisation of TE sequences into the 5'-UTRs of the transcripts.
Developmentally-regulated T2 and T1 co-transcription
Co-transcription of T2 and T1, in this gene order, is proposed based on the observation that all testicular and developmental T2 and T1 transcripts discerned share a unique exon 1 that resides upstream of the T2/T1 exon assemblage (Figures 2 and 5A). The exon 1 sequence is also found to be conserved as a unique sequence in the mouse genome (see Additional file 2) but not in the genomes of other animals and plants examined, consistent with evolutionary relatedness between the mouse and rat genomes. T2-T1 co-transcription implies that the genes are co-ordinately controlled by a common promoter and associated regulatory sequences.
T2 transcription is found to occur throughout the E12 to E17 developmental stages analysed and is silenced beyond E17. On the other hand, T1 transcription is restricted only to E14–E16. In rodents, the organogenesis phase of embryonic development comes to an end at about E14–E15 from which point on active foetal growth occurs, a process that involves active cellular proliferation as opposed to active differentiation during the organogenesis phase . Our data collectively suggest that T2 expression is a normal monogenic transcriptional event that uses the AAUAAA polyadenylation signal located 10 nucleotides upstream of the polyA tract of the mature T2 transcripts [GenBank:AY902367 and ref. ] and. On the other hand, T1 expression is only realised when T2-to-T1 transcription read-through occurs to transcribe the T1 coding exons (Figure 6). The occurrence of T2-to-T1 co-transcription may be attributed to high rates of transcription associated with active foetal growth similar to hyper transcription rates that have been shown in the testis . The silencing of T1/T2 transcription at E17–E18 does not seem to involve hypermethylation of the exon 1 promoter which is unmethylated, despite the presence of a CpG island, in the testis and in all the developmental stages examined irrespective of T1/T2 expression (unpublished data). It remains to be investigated if developmental and testicular transcription of the T2/T1 locus involves chromatin remodelling or the availability of positive or negative trans-acting factors.
Exonisation of TE sequences into 5'-UTRs of the T1 and T2 transcripts
The most notable finding of this work is the high rate of exonisation of TE sequences into the 5'-UTRs of the T1 and T2 transcripts through alternative splicing. In some T1 transcripts, an alternative transcription termination site is found in an L1 sequence located downstream of the constitutive site (Figure 2A). Frequent TE exonisation is clearly associated with the embedment of the constitutive exon 1 and the uninterrupted T1 and T2 coding exons in a ~700-kb chromosomal segment that is heavily populated with TE sequences (Figure 5). In this segment, the computed average TE content is 60.7%, much higher than the mean TE content of 40% in the rat genome [29, 30]. Notably, the second exon of the T2 gene (T2-2 or exon 3a/3b) and that of T1 (T1–2 or exon 8a/8b) are relics of the ERV-MT2A and -MT2B sequences, respectively; these TE relics have developed strong and stable splice sites to be frequently recruited into 5'-UTRs of the transcripts of the respective gene (Table 1 and Figure 5). We also detected apparent T2-T1 "chimeric" transcripts that involve only 5'-UTRs (Figure 4). On closer examination, all the discerned T2 and T1 5'-UTR exons, with the exception of the constitutive exon 1 and the exon 2 of T1 (T1–2, or exon 7a/7b), are TE remnants (Figures 2A and 2B, see bottom TE annotations). However, we cannot rule out the possibility that the T1–2 exon was also originally derived from a TE sequence but had lost its TE features through evolution for recognition. In other words, all 5'-UTR exons, except exon 1, may, in fact, be products of exonised TE sequences recruited through alternative splicing.
Several salient features of TE insertions in the human and mouse genomes have been described based on bioinformatics analysis: (i) the TE exons are mostly intronic; (ii) all TE families can be exonised; (iii) TE exons are found mostly in the UTR, and (iv) potential tissue-specific association [31, 32]. In this report, the depicted exon organization of the T1 and T2 transcripts has provided direct experimental evidence to support all of the above features of TE-derived exons. An important mechanism that contributes to the exonisation of Alu, a highly repetitive and primate-specific TE, is the RNA-editing-mediated adenosine-to-inosine (A-to-I) modification [33–35]. A-to-I RNA editing is catalysed by adenosine deaminase acting on double-stranded RNA stretches of primary transcripts formed by annealing of inverted-repeat sequences in the pre-mRNA . The dense field of predominantly LINE1 and ERV sequences in the T2/T1 locus provides ample opportunities for the T2/T1 pre-mRNA species to form double-stranded structures for adenosine deaminase-mediated RNA editing. Furthermore, the TE exonization may be driven by the use of cryptic exonic splicing enhancers (ESEs) as proposed by Lin et al. . The exact mechanism that is responsible for exonisation of TE sequences into the T1 and T2 transcripts is a subject for further investigation.
Biologically, TE insertions into 5'-UTRs of transcripts have been shown to influence gene expression at the level of transcription through the creation of new transcription factor binding sites or by other transcriptional mechanisms [36–38]. Alternatively, the presence of TE sequences could introduce deleterious uAUGs and uORFs to repress translational initiation [39–41] as we have demonstrated for selected T1 transcripts (Figure 7). The complexity of the T1 transcript population in the developing embryo impedes detailed determination of the relative abundances of the discerned transcripts.
This work provides evidence to indicate that exonisation of TE sequences is a frequent event in the transcription of retrogenes during embryonic development and in the testis and TE exonisation may contribute to post-transcriptional regulation of expression of retrogenes through translational repression. The T2/T1 locus, thus, provide a spatio-temporal model for further dissection of developmentally-regulated and testis-specific transcription and possible biological significance of TE exonisation of retrogenes.
Cell lines and rats
The rat insulinoma cell line RIN-m5F was acquired from the Bioresource Collection and Research Centre, Taiwan. Sprague Dawley rats were used throughout this work and were obtained from the Laboratory Animal Centre, National Yang-Ming University, Taiwan. This study was approved by the Institutional Animal Care and Use Committee (IACUC) of the Taipei Veterans General Hospital. The animals were sacrificed according to the IACUC guidelines.
RNA preparation and expression profiling by RT-PCR
Oligonucleotide primers used in this study
(A) Primers for RT-PCR expression profiling
(B) 5'- and 3'-RACE gene-specific primers
(C) Primers for cloning of 5'-UTR for uORF assays
T1 uORF-R NcoI
(D) Primers for site-directed mutagenesis
(E) Primers for luciferase RT-PCR
5'- and 3'-RACE and bioinformatics analysis
The procedure of rapid amplification of cDNA ends (RACE) was used to derive sequences of the 5'- and 3'-halves of T1 and T2 mRNAs for construction of full-length transcript sequences. For the RACE experiments, a SMART® RACE cDNA Amplification Kit (BD Biosciences) was used according to the manufacturer's instructions and as described [1, 20]. To increase specificity and sensitivity, nested PCR was routinely performed using the Nested Universal Primer A included in the SMART kit and T1- or T2-gene specific nested primers (5'RACE-GSP-R2 or 3'RACE-GSP-F1 for 5'- or 3'-RACE, respectively, see Table 2). All RACE-generated sequences were cloned into the pGEM®-TEasy vector for sequence analysis. Nucleotide sequences were subjected to BLAST searches of the GenBank rat resources database [http://www.ncbi.nlm.nih.gov/genome/guide/rat/; assembly version RGSC v3.4, as on December 1, 2008] using default parameters and filters. The Lasergene® software programs package obtained from DNAstar® was used for in-house sequence alignment and nucleotide sequence analysis.
Plasmid construction, site-specific mutagenesis, transient transfection and luciferase activity assay
The 5'-UTR sequences of selected T1 transcripts were derived from the RNA by PCR amplification using oligo(dT)-primed RT products and reverse primers flanked with NcoI recognition sequence for cloning into the SV40 promoter-driven pGL3-Promoter and the SV40 promoter-plus-enhancer pGL3-Control luciferase reporter plasmids (Promega). For site-directed mutagenesis, oligonucleotides encompassing the mutations and containing restriction cloning sites were used as primers in PCR amplification reactions as described . For transient transfection experiments, CHO-K1 cells were seeded onto 24-well Petri dishes 24 h prior to transfection. Cells were co-transfected in duplicates with the luciferase constructs and the thymidine kinase promoter-driven Renilla Luciferase plasmid using the Lipofectamine Plus reagent (Life Technologies) as described [20, 43]. Forty-eight hours post-transfection, cells were lysed and duplicates of 20 μl aliquots of the cell lysate were removed for measurement of the luciferase activities in a luminometer using the Dual-Luciferase Reporter Assay Kit according to the user's manual (Promega).
Western blot analysis
CHO-K1 cells transfected with Luciferase reporter constructs were harvested 24, 36 and 48 h after transfection. Subsequent processing of the lysed cells for western blot analysis using an anti-luciferase antibody (Novus) was performed as previously described . Signals were visualised by chemiluminescence after treating the membrane blot with a Western Lightning Plus-ECL reagent (Perkin-Elmer) according to the manufacturer's recommendations.
The authors thank Yao-Hui Tsai for technical assistance and Tsung-Sheng Su for discussion. This work was supported by grants V97C1-055 and V98C1-020 to KBC and CJH from the Taipei Veterans General Hospital, Taipei, Taiwan.
- Chen HH, Liu TY, Huang CJ, Choo KB: Generation of two homologous and intronless zinc-finger protein genes, Zfp352 and Zfp353, with different expression patterns by retrotransposition. Genomics. 2002, 79 (1): 18-23.View ArticlePubMedGoogle Scholar
- Chen HH, Liu TY, Li H, Choo KB: Use of a common promoter by two juxtaposed and intronless mouse early embryonic genes, Rnf33 and Rnf35: implications in zygotic gene expression. Genomics. 2002, 80 (2): 140-143.View ArticlePubMedGoogle Scholar
- Choo KB, Chen HH, Liu TY, Chang CP: Different modes of regulation of transcription and pre-mRNA processing of the structurally juxtaposed homologs, Rnf33 and Rnf35, in eggs and in pre-implantation embryos. Nucleic Acids Res. 2002, 30 (22): 4836-4844.PubMed CentralView ArticlePubMedGoogle Scholar
- Maniatis T, Tasic B: Alternative pre-mRNA splicing and proteome expansion in metazoans. Nature. 2002, 418 (6894): 236-243.View ArticlePubMedGoogle Scholar
- Kim E, Goren A, Ast G: Alternative splicing: current perspectives. Bioessays. 2008, 30 (1): 38-47.View ArticlePubMedGoogle Scholar
- Marques AC, Dupanloup I, Vinckenbosch N, Reymond A, Kaessmann H: Emergence of young human genes after a burst of retroposition in primates. PLoS Biol. 2005, 3 (11): e357-PubMed CentralView ArticlePubMedGoogle Scholar
- Vinckenbosch N, Dupanloup I, Kaessmann H: Evolutionary fate of retroposed gene copies in the human genome. Proc Natl Acad Sci USA. 2006, 103 (9): 3220-3225.PubMed CentralView ArticlePubMedGoogle Scholar
- Kleene KC, Mulligan E, Steiger D, Donohue K, Mastrangelo MA: The mouse gene encoding the testis-specific isoform of Poly(A) binding protein (Pabp2) is an expressed retroposon: intimations that gene expression in spermatogenic cells facilitates the creation of new genes. J Mol Evol. 1998, 47 (3): 275-281.View ArticlePubMedGoogle Scholar
- Swanson WJ, Vacquier VD: The rapid evolution of reproductive proteins. Nat Rev Genet. 2002, 3 (2): 137-144.View ArticlePubMedGoogle Scholar
- Schmidt EE: Transcriptional promiscuity in testes. Curr Biol. 1996, 6 (7): 768-769.View ArticlePubMedGoogle Scholar
- Ma J, Svoboda P, Schultz RM, Stein P: Regulation of zygotic gene activation in the preimplantation mouse embryo: global activation and repression of gene expression. Biol Reprod. 2001, 64 (6): 1713-1721.View ArticlePubMedGoogle Scholar
- Ko MS, Kitchen JR, Wang X, Threat TA, Hasegawa A, Sun T, Grahovac MJ, Kargul GJ, Lim MK, Cui Y, et al: Large-scale cDNA analysis reveals phased gene expression patterns during preimplantation mouse development. Development. 2000, 127 (8): 1737-1749.PubMedGoogle Scholar
- Conley AB, Piriyapongsa J, Jordan IK: Retroviral promoters in the human genome. Bioinformatics. 2008, 24 (14): 1563-1567.View ArticlePubMedGoogle Scholar
- Yohn CT, Jiang Z, McGrath SD, Hayden KE, Khaitovich P, Johnson ME, Eichler MY, McPherson JD, Zhao S, Paabo S, et al: Lineage-specific expansions of retroviral insertions within the genomes of African great apes but not humans and orangutans. PLoS Biol. 2005, 3 (4): e110-PubMed CentralView ArticlePubMedGoogle Scholar
- Huang CJ, Chen CY, Chen HH, Tsai SF, Choo KB: TDPOZ, a family of bipartite animal and plant proteins that contain the TRAF (TD) and POZ/BTB domains. Gene. 2004, 324: 117-127.View ArticlePubMedGoogle Scholar
- Zapata JM, Martinez-Garcia V, Lefebvre S: Phylogeny of the TRAF/MATH domain. Adv Exp Med Biol. 2007, 597: 1-24.View ArticlePubMedGoogle Scholar
- Wajant H, Henkler F, Scheurich P: The TNF-receptor-associated factor family: scaffold molecules for cytokine receptors, kinases and their regulators. Cell Signal. 2001, 13 (6): 389-400.View ArticlePubMedGoogle Scholar
- Bradley JR, Pober JS: Tumor necrosis factor receptor-associated factors (TRAFs). Oncogene. 2001, 20 (44): 6482-6491.View ArticlePubMedGoogle Scholar
- Kelly KF, Daniel JM: POZ for effect – POZ-ZF transcription factors in cancer and development. Trends Cell Biol. 2006, 16 (11): 578-587.View ArticlePubMedGoogle Scholar
- Choo KB, Hsu MC, Chong KY, Huang CJ: Testis-specific expression and genomic multiplicity of the rat Rtdpoz genes that encode bipartite TRAF- and POZ/BTB-domain proteins. Gene. 2007, 387 (1–2): 141-149.View ArticlePubMedGoogle Scholar
- Hernandez-Munoz I, Lund AH, Stoop van der P, Boutsma E, Muijrers I, Verhoeven E, Nusinow DA, Panning B, Marahrens Y, van Lohuizen M: Stable X chromosome inactivation involves the PRC1 Polycomb complex and requires histone MACROH2A1 and the CULLIN3/SPOP ubiquitin E3 ligase. Proc Natl Acad Sci USA. 2005, 102 (21): 7635-7640.PubMed CentralView ArticlePubMedGoogle Scholar
- Kwon JE, La M, Oh KH, Oh YM, Kim GR, Seol JH, Baek SH, Chiba T, Tanaka K, Bang OS, et al: BTB domain-containing speckle-type POZ protein (SPOP) serves as an adaptor of Daxx for ubiquitination by Cul3-based ubiquitin ligase. J Biol Chem. 2006, 281 (18): 12664-12672.View ArticlePubMedGoogle Scholar
- Di Marcotullio L, Ferretti E, Greco A, De Smaele E, Screpanti I, Gulino A: Multiple ubiquitin-dependent processing pathways regulate hedgehog/gli signaling: implications for cell development and tumorigenesis. Cell Cycle. 2007, 6 (4): 390-393.View ArticlePubMedGoogle Scholar
- Zhang Q, Zhang L, Wang B, Ou CY, Chien CT, Jiang J: A hedgehog-induced BTB protein modulates hedgehog signaling by degrading Ci/Gli transcription factor. Dev Cell. 2006, 10 (6): 719-729.View ArticlePubMedGoogle Scholar
- Choo KB, Chen HH, Cheng WT, Chang HS, Wang M: In silico mining of EST databases for novel pre-implantation embryo-specific zinc finger protein genes. Mol Reprod Dev. 2001, 59 (3): 249-255.View ArticlePubMedGoogle Scholar
- Kempken F, Windhofer F: The hAT family: a versatile transposon group common to plants, fungi, animals, and man. Chromosoma. 2001, 110 (1): 1-9.View ArticlePubMedGoogle Scholar
- Rubin E, Lithwick G, Levy AA: Structure and evolution of the hAT transposon superfamily. Genetics. 2001, 158 (3): 949-957.PubMed CentralPubMedGoogle Scholar
- Ko MS: Embryogenomics: developmental biology meets genomics. Trends Biotechnol. 2001, 19 (12): 511-518.View ArticlePubMedGoogle Scholar
- Gibbs RA, Weinstock GM, Metzker ML, Muzny DM, Sodergren EJ, Scherer S, Scott G, Steffen D, Worley KC, Burch PE, et al: Genome sequence of the Brown Norway rat yields insights into mammalian evolution. Nature. 2004, 428 (6982): 493-521.View ArticlePubMedGoogle Scholar
- Bourque G, Leong B, Vega VB, Chen X, Lee YL, Srinivasan KG, Chew JL, Ruan Y, Wei CL, Ng HH, et al: Evolution of the mammalian transcription factor binding repertoire via transposable elements. Genome Res. 2008, 18 (11): 1752-1762.PubMed CentralView ArticlePubMedGoogle Scholar
- Sela N, Mersch B, Gal-Mark N, Lev-Maor G, Hotz-Wagenblatt A, Ast G: Comparative analysis of transposed element insertion within human and mouse genomes reveals Alu's unique role in shaping the human transcriptome. Genome Biol. 2007, 8 (6): R127-PubMed CentralView ArticlePubMedGoogle Scholar
- Lin L, Jiang P, Shen S, Sato S, Davidson BL, Xing Y: Large-scale analysis of exonized mammalian-wide interspersed repeats in primate genomes. Hum Mol Genet. 2009, 18 (12): 2204-2214.PubMed CentralView ArticlePubMedGoogle Scholar
- Rueter SM, Dawson TR, Emeson RB: Regulation of alternative splicing by RNA editing. Nature. 1999, 399 (6731): 75-80.View ArticlePubMedGoogle Scholar
- Lev-Maor G, Sorek R, Shomron N, Ast G: The birth of an alternatively spliced exon: 3' splice-site selection in Alu exons. Science. 2003, 300 (5623): 1288-1291.View ArticlePubMedGoogle Scholar
- Moller-Krull M, Zemann A, Roos C, Brosius J, Schmitz J: Beyond DNA: RNA editing and steps toward Alu exonization in primates. J Mol Biol. 2008, 382 (3): 601-609.View ArticlePubMedGoogle Scholar
- Landry JR, Medstrand P, Mager DL: Repetitive elements in the 5' untranslated region of a human zinc-finger gene modulate transcription and translation efficiency. Genomics. 2001, 76 (1–3): 110-116.View ArticlePubMedGoogle Scholar
- Belancio VP, Hedges DJ, Deininger P: LINE-1 RNA splicing and influences on mammalian gene expression. Nucleic Acids Res. 2006, 34 (5): 1512-1521.PubMed CentralView ArticlePubMedGoogle Scholar
- Deininger PL, Batzer MA: Mammalian retroelements. Genome Res. 2002, 12 (10): 1455-1465.View ArticlePubMedGoogle Scholar
- Morris DR, Geballe AP: Upstream open reading frames as regulators of mRNA translation. Mol Cell Biol. 2000, 20 (23): 8635-8642.PubMed CentralView ArticlePubMedGoogle Scholar
- Churbanov A, Rogozin IB, Babenko VN, Ali H, Koonin EV: Evolutionary conservation suggests a regulatory function of AUG triplets in 5'-UTRs of eukaryotic genes. Nucleic Acids Res. 2005, 33 (17): 5512-5520.PubMed CentralView ArticlePubMedGoogle Scholar
- Iacono M, Mignone F, Pesole G: uAUG and uORFs in human and rodent 5'untranslated mRNAs. Gene. 2005, 349: 97-105.View ArticlePubMedGoogle Scholar
- Huang CJ, Chang JG, Wu SC, Choo KB: Negative transcriptional modulation and silencing of the bi-exonic Rnf35 gene in the preimplantation embryo. Binding of the CCAAT-displacement protein/Cux to the untranslated exon 1 sequence. J Biol Chem. 2005, 280 (35): 30681-30688.View ArticlePubMedGoogle Scholar
- Huang CJ, Wu SC, Choo KB: Transcriptional modulation of the pre-implantation embryo-specific Rnf35 gene by the Y-box protein NF-Y/CBF. Biochem J. 2005, 387 (Pt 2): 367-375.PubMed CentralView ArticlePubMedGoogle Scholar
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