MBE Advance Access originally published online on September 21, 2007
Molecular Biology and Evolution 2007 24(11):2525-2534; doi:10.1093/molbev/msm195
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Research Articles |
Human TRIM71 and Its Nematode Homologue Are Targets of let-7 MicroRNA and Its Zebrafish Orthologue Is Essential for Development
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* Genomics Research Center, Academia Sinica, Taipei, Taiwan
Institute of Information Science, Academia Sinica, Taipei, Taiwan
Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan
Department of Pediatrics Hematology-Oncology, University of California, San Diego
|| Department of Ecology and Evolution, University of Chicago
E-mail: whli{at}uchicago.edu.
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Abstract |
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 TOP Abstract IntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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Animal microRNAs (miRNAs) are short RNAs that function as posttranscriptional regulators of gene expression by binding to the target mRNAs. Noting that some miRNAs are highly conserved in evolution, we explored the possibility of evolutionary conservation of their targets. We identified human orthologues of experimentally verified let-7 miRNA target genes in Caenorhabditis elegans and used the luciferase reporter system to examine whether these human genes are still the targets of let-7 miRNA. We found that in some cases, the miRNA–target relationship has indeed been conserved in human. Interestingly, human TRIM71, an orthologue of C. elegans let-7–target lin-41 gene, can be repressed by hsa-let-7a and hsa-let-7c. This repression was abolished when both predicted let-7 target sites of TRIM71 were mutated. Moreover, the zebrafish lin-41 orthologue was also repressed by let-7 to a similar degree as was TRIM71. When the expression of zebrafish lin-41 orthologue was silenced by microinjection of RNA interference or morpholino into zebrafish zygotes, retarded embryonic development was observed, providing direct evidence for an essential role of lin-41 in zebrafish development. Taken together, our results suggest that the regulation of TRIM71 expression by let-7 has been evolutionarily conserved and that TRIM71 likely plays an important role in development.
Key Words: TRIM71 • lin-41 • let-7 • microRNA target
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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MicroRNAs (miRNAs) are endogenous noncoding RNA molecules of
22 nt that negatively regulate the expression of target genes in animals by binding to the 3' untranslated regions (UTRs) of the target mRNAs. The binding generally includes strong base pairing between the 5' end region of an miRNA and the complementary sequence of its target, although extensive base pairing in the 3' end of the miRNA may compensate for insufficient base pairing of the 5' end (reviewed in Carrington and Ambros 2003
; Lai 2003; Ambros 2004; Bartel 2004; Kim and Nam 2006; Niwa and Slack 2007). The importance of miRNAs in animals is underscored by the facts that they are estimated to comprise 1–5% of animal genes (Altschul et al. 1990; Lim et al. 2003), appear to regulate a large proportion of protein-coding genes (Brennecke et al. 2005; Krek et al. 2005; Lewis et al. 2005; Lim et al. 2005), and are involved in the control of a variety of physiological processes including development, cell proliferation, apoptosis, tissue differentiation, and metabolism (Johnston et al. 2001; Lim et al. 2003; Ambros 2004; Plasterk 2006; Valencia-Sanchez et al. 2006).
A number of miRNAs are evolutionarily highly conserved (Pasquinelli et al. 2000; Johnston et al. 2001; Pasquinelli et al. 2003; Ambros 2004; Valencia-Sanchez et al. 2006). For example, the let-7 miRNA gene, which was originally identified as an important regulator involved in the heterochronic pathway controlling developmental timing in Caenorhabditis elegans (Reinhart et al. 2000), is conserved from nematodes to primates. Supplementary figure S1 (Supplementary Material online) shows the sequence alignment of the elements of the let-7 family found in C. elegans, zebra fish, and human. Because each miRNA regulates multiple genes, the binding regions of miRNAs are under tight structural constraints to avoid losing the regulation. This might have contributed to the high conservation of binding regions of these miRNAs across species. In contrast, the binding sites of their targets would not be under such strong constraints and might have more freedom to mutate without affecting many other genes. Moreover, gene duplication and alternative splicing are 2 important sources of gene function diversity and are prevalent in complex organisms (Mironov et al. 1999; Li et al. 2001; Kan et al. 2002; Waterston et al. 2002). Duplicate genes may diverge in their 3' UTRs, and alternatively spliced transcripts of a gene may give rise to different 3' UTRs. These 2 mechanisms may accelerate the evolution of the target sites on 3' UTRs, making the miRNA–target interactions more complex.
In this study, we are interested in the evolution of the target genes of highly conserved miRNAs. We selected all experimentally verified miRNA–target pairs of C. elegans that have orthologues for both miRNA and target genes in human and used the luciferase reporter system to examine whether the human orthologues and their cognate miRNAs remain interacting pairs. In addition, if any of the C. elegans target genes have multiple orthologous copies in human, we also examined these paralogous copies with their cognate miRNAs. For this purpose, we focused on the 4 experimentally verified C. elegans let-7–target pairs that have orthologous pairs in human. For the pair of RAS and let-7, its orthologous pairs have been shown to function in human (Johnson et al. 2005). Our result showed that in the remaining 3 pairs, only the orthologue TRIM71 (human orthologue of lin-41) is still the target of let-7 in human. Thus, all together, 2 of the 4 let-7–target pairs studied have been conserved, suggesting that the interaction relationship of a miRNA–target pair can be conserved during a very long period of evolution. Because the function of TRIM71 has not been documented yet, we silenced the lin-41 orthologue in a zebrafish model and demonstrated developmental defects similar to the effects of let-7 oligo injection in zebrafish embryos reported previously (Kloosterman et al. 2004). Taken together, our results suggest that TRIM71 and let-7 are an evolutionarily conserved miRNA–target pair and that TRIM71 likely plays an important role in embryonic development.
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Materials and Methods |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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Verified miRNA–Target Pairs
Information regarding the miRNA–target pairs verified by experiment was obtained from TarBase version 3 (Sethupathy et al. 2006
). A total of 14 experimentally supported miRNA–target pairs were identified in C. elegans.
Identification of Human Orthologues of C. elegans miRNA–Target Pairs
For each of the verified miRNA–target pairs in C. elegans, we first looked for its miRNA orthologues and target orthologues in human. We obtained the family information on each miRNA of the pairs from miRBase release 9 (Stein et al. 2003) to identify whether the miRNA had orthologues in human or not. Moreover, every C. elegans target protein sequence of the pairs was used as a query to search against the whole set of human protein sequences from Ensembl database release 42.36d (Waterston et al. 2002) using BlastP program (Altschul et al. 1990). If there existed Blast hits with E value < 10–20, we took the hits with the smallest E values as the human orthologues of the target gene.
Definition of Alignable Length and Alignable Length Ratio of a Protein
The "alignable length" between the 2 proteins of a Blast query was defined as the sum of all the lengths of the Blast hits of the query minus the gap lengths. The "alignable length ratio of a protein" was defined as the alignable length divided by the protein length.
Identification of Putative miRNA Target Sites
In each of the human orthologous pairs identified by the above procedure, we used a bioinformatics approach to predict putative miRNA binding sites of the cognate miRNA in the 3' UTR sequence. The 3' UTR sequences were retrieved from the Ensembl database (Homo sapiens core version 42.36d) by the Ensembl Core API (Waterston et al. 2002). We considered 2 factors for judging whether a segment of the 3' UTR sequence of the gene is a potential target site or not. The first factor was the degree of affinity, based on binding energy, between a segment of 3' UTR sequence and miRNA sequence. We used a sliding window of 25 nt to slide over the 3' UTR sequence of the gene and calculated the free energy between the miRNA sequence and the segment sequence within the window. The 2-state hybridization function of the UNAFold package (version 3.0) (Dimitrov and Zuker 2004; Markham and Zuker 2005) was used to perform the core of the calculation. For a segment of interest and the miRNA sequence to have high affinity for each other, we used the criterion that the free energy between them is 
–10 kcal/mol. The second factor was pairing quality between the segment of interest and the miRNA sequence. A number of miRNA–target pairing rules have been deduced in the literature (Lewis et al. 2003; Doench and Sharp 2004; Kiriakidou et al. 2004; Brennecke et al. 2005; Didiano and Hobert 2006). We chose the following rules that are considered to be important for pairing constraints. 1) Extensive base pairing at the 5' end "seed" region of the miRNA is important for target site function (Lewis et al. 2003; Doench and Sharp 2004; Kiriakidou et al. 2004; Brennecke et al. 2005; Didiano and Hobert 2006); the seed region is defined as a stretch of 7 consecutive nucleotides starting from second nucleotide at the 5' end of an miRNA (Lewis et al. 2003). 2) The seed sequence or the seed-match target sequence can have a single nucleotide bulge that is positioned symmetrically in the pairing region (Kiriakidou et al. 2004; Brennecke et al. 2005). 3) The seed pairing region may have G:U base pairs (Kiriakidou et al. 2004; Brennecke et al. 2005; Didiano and Hobert 2006), although its presence in the seed region is usually detrimental (Brennecke et al. 2005). 4) If the seed region is imperfectly base paired or has a shorter stretch of base pairings, extensive base pairings around the 3' end of the miRNA should be present to compensate for the insufficient pairing of the 5' end (Brennecke et al. 2005). Combining the computational results of pairing quality and binding affinity between an miRNA sequence and the segments of the 3' UTR sequence of a gene, we can identify putative target sites for each miRNA–target pair.
Cell Culture and Plasmids
Human hepatocellular carcinoma HepG2 cell line was cultured in modified Eagle medium supplemented with 10% fetal bovine serum and 0.1 mM nonessential amino acids in 5% CO2. As described previously (Kiriakidou et al. 2004; Boutz et al. 2007), we used phRG-TK plasmid (coding for Renilla luciferase; Promega, Madison, WI) to construct the let-7 target site reporters and used the pGL3-control plasmid as a control plasmid (coding for firefly luciferase; Promega) for normalizing the transfection efficiency. Primer sequences are listed in supplementary table S4 (Supplementary Material online). To construct target site reporter plasmids, various target fragments, which were cloned by polymerase chain reaction (PCR) from a cDNA pool of HRK 293 cells, were inserted at the XbaI site, downstream of the luciferase gene in the phRG-TK vector. The target fragment of Vitamin D3 Receptor (VDR) including nt1922 to nt4232 of human VDR cDNA (accession no. NM_001017535) was amplified by PCR with primers VDR1922 and VDR4232R. The target fragment of NR1I2 including nt3535 to nt4366 of human NR1I2 gene (accession no. NM_003889
[GenBank]
) was amplified by PCR with primers NR1I2 F and NR1I2 R from the human genomic DNA of peripheral blood monocytes. The target fragment of FOXA1 including nt1814 to nt2850R of human FOXA1 cDNA (accession no. NM_004496
[GenBank]
) was amplified by PCR with primers FOXA1814 and FOXA2850R. The target fragment of TRIM71 including nt2810 to nt2958 of human TRIM71 cDNA (accession no. NM_001039111) was amplified by PCR with primers TRIM2810 and TRIM2958R. The target fragment of Danio rerio lin-41 (DLIN41) including nt2222 to nt2389 of zebra fish lin-41 cDNA (accession no. XM_685160
[GenBank]
) was amplified by PCR with primers DLIN2222 and DLIN2389R from zebra fish genomic DNA. PCR was performed by using Phusion DNA polymerase (Finnzymes Oy, Espoo, Finland) following manufacturer's instruction. The TRIM71 mutants with deletion of the first target site (nt2828 to nt2849), the second target site (nt2908 to nt2929), or both sites (nt2828 to nt2849 and 2908 to nt2929) were generated by QuickChange II XL Site-directed Mutagensis Kit (Stratagene, La Jolla, CA) with the combination of following primer pairs: TRIM71 mt1 F and TRIM71 mt1 R for the first site; TRIM71 mt2 F and TRIM71 mt2 R for the second site, according to the instruction. The plet7AS reporter plasmid, which was inserted only with the antisense sequence of has-let-7a, was generated by inserting the ds-oligo, which were annealed from primer pairs let7AS F and let7AS R into the XbaI site of phRG-TK vector.
Transfection and Luciferase Reporter Assay
Transient transfection and luciferase activity assays were performed as described previously with modifications (Kiriakidou et al. 2004; Boutz et al. 2007). miRNA precursors for hsa-let-7a (pre-let7a), hsa-let-7c (pre-let7c), and the negative control precursor #1 (pre-ctrl) were purchased from Ambion (Austin, TX). HepG2 cells were seeded in 12-well plates 24 h before transfection. Cells were transfected with indicated amounts of precursor miRNA oligo and reporter plasmids (0.2 µg of target reporter and 0.2 µg of pGL3-control) in the presence of Lipofectamine2000 reagent (Invitrogen, Carlsbad, CA) following manufacturer's manual. Three days posttransfection, the luciferase activity was determined using the Dual-luciferase assay system (Promega) according to the manufacturer's instruction. The Renilla luciferase activity was normalized by dividing the firefly luciferase activity of the same transfection. The normalized luciferase activity of pre-ctrl in vector transfection was set to 1.0, and all others were expressed relative to it.
let-7a and let-7c miRNA Detection and Quantification
Total RNA was extracted by the Trizol reagent (Invitrogen), and quantitative reverse transcriptase–polymerase chain reaction for let-7a (or let-7c) and U6 RNA was performed with the TaqMan Real-time PCR kit (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions and detected with an ABI Prism 7000 sequence detection system (Applied Biosystems). The raw data were analyzed by ABI Prism 7000 SDS software (Applied Biosystems). The cycle threshold, Ct, of each sample was generated with the default setting. The let-7 expression level of each sample was normalized to the expression level of U6 in the same sample by the formula: let-7/U6 = 2–(Ct of let-7a-Ct of U6). The let-7/U6 ratio of the 2 nM pre-ctrl oligo transfection was set to 1.0, and the values of all others relative to it were calculated accordingly. The result represents the average of 3 independent experiments with standard deviations.
lin-41 Silencing in the Zebrafish Model
To silence the lin-41 expression in zebrafish with (small interfering RNA [siRNA]), we used the pcDNA6.2-GW/EmGFP-miR of the Block-iT Pol II miR RNAi Expression Vector Kits (Invitrogen) to construct engineered siRNA-expressing plasmids according to the user manual. Two regions of zebrafish lin-41 orthologue cDNA were chosen for the engineered siRNAs: one from nt716 to nt736 and the other from nt1611 to nt1631. The pcDNA6.2-GW/EmGFP-neg control plasmid, which expresses the engineered siRNA with random sequence, served as a negative control. These 3 resulting plasmids were used as templates for PCR amplifications with 2 primers, siGW F and siGW R, to obtain the inserts for the next transposon plasmid construction. These amplified inserts were digested with ClaI and ligated into the ClaI sites of pretreated transposon pT2KXIG plasmid (Kawakami et al. 2004), which has been digested with ApaI and then self-ligated first, to obtain the engineered siRNA-expressing transposon plasmids, 716RNA interference (RNAi), 1611RNAi, or RNAi control. One hundred picograms of the final transposon plasmid was comicroinjected with 50 pg of pCS-TP plasmid, which expressed the transposase (Kawakami et al. 2004), into 1-cell stage of zebrafish embryos. For each transposon plasmid injection, at least 150 eggs were injected and the abnormal embryos were counted under the dissection microscope.
In addition to the RNAi strategy, morpholino knockdown experiments were also carried out to silence lin-41 expression. Morpholinos are chemically modified oligonucleotides (Nasevicius and Ekker 2000), which have been widely used in zebrafish and frog studies to specifically knockdown gene expression by blocking translation. We injected lin-41 morpholino or control morpholino into the yolk region at the 1- to 4-cell stage of zebrafish embryos. lin-41 morpholino (5'-ATTGAGCATGGAGAAGCTGTTGTGG-3') and nonspecific control morpholino (5'-CCTCTTACCTCAGTTACAATTTATA-3') were purchased from Gene Tools, Philomath, OR.
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Results |
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Caenorhabditis elegans let-7 Targets and Their Human Orthologues Have Similar Functions
There were 14 experimentally verified miRNA–target pairs in C. elegans (supplementary table S1, Supplementary Material online), and we found only 4 of them to have orthologues for both miRNA and target gene in human (see Materials and Methods). These consisted of let-7 miRNA and its 4 target genes in C. elegans.
These 4 verified C. elegans target genes of let-7 are daf-12 (Grosshans et al. 2005), pha-4 (Grosshans et al. 2005), let-60 (Johnson et al. 2005), and lin-41 (Slack et al. 2000). Their human orthologues found by amino acid sequence Blast are listed in table 1, and the Blast results are summarized in supplementary table S2 (Supplementary Material online). It was noted that some of the orthologous genes had paralogues (duplicates). In the case of worm daf-12, both VDR (also termed NR1I1) and NR1I2 (also termed PXR, the pregnane X receptor [Kliewer et al. 1998] and SXR, the steroid and xenobiotic receptor [Blumberg et al. 1998]) are its human orthologues. VDR and NR1I2 are both nuclear receptors (Evans 1988; Kliewer et al. 1998); VDR functions as a ligand-dependent transcription factor to maintain calcium metabolism through the regulation of target genes in response to vitamin D3 (Jones et al. 1998). NR1I2 is also a ligand-dependent transcription factor involved in the regulation of a number of xenobiotic-metabolizing genes (Bertilsson et al. 1998; Blumberg et al. 1998; Kliewer et al. 1998; Lehmann et al. 1998). Similarly, the worm daf-12, which regulates the dauer diapause and developmental age (Antebi et al. 1998, 2000), is also a nuclear receptor (Yeh 1991) and has been seen as being closely related to vertebrate VDR and NR1I2 (Antebi et al. 2000). The worm pha-4, which encodes a protein that most closely resembles a forkhead box A (FOXA) transcription factor (Horner et al. 1998; Kalb et al. 1998), has 3 human orthologues FOXA1, FOXA2, and FOXA3. pha-4 functions in organogenesis of the C. elegans pharynx (Azzaria et al. 1996; Kalb et al. 1998; Gaudet and Mango 2002). In mammals, the FOXA family has been shown to be critical in multiple developmental stages, beginning with early development, continuing during organogenesis, and finally in metabolism and homeostasis in the adult (reviewed in Friedman and Kaestner 2006). let-60, a GTP-binding RAS proto-oncogene (Han and Sternberg 1990), is the C. elegans orthologue of human HRAS, KRAS, and NRAS. Because human RAS genes have recently been verified to be the target genes of let-7 miRNA (Johnson et al. 2005), they were not pursued in this study.
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The C. elegans heterochronic gene lin-41, which encodes a Ring finger–B box–coiled coil (RBCC) protein (Slack et al. 2000
), seems to have only one human orthologue, that is, TRIM71. TRIM71 is a novel gene with unknown function in human; however, its mouse orthologue is also a member of the RBCC protein family (Kanamoto et al. 2006). During mouse embryogenesis, reciprocal expression of the lin-41 orthologue and let-7 has been reported (Schulman et al. 2005). Moreover, in chicken and mouse, the lin-41 orthologues seem to be important in developing limb buds, branchial arches, and tail buds (Lancman et al. 2005; Schulman et al. 2005; Kanamoto et al. 2006). From our survey, the 4 target genes of the let-7 miRNA in C. elegans and their human orthologues have, to some degree, similar functions. This observation lends further support for these human genes as the orthologues of the C. elegans genes.
Putative Target Sites for let-7 on Human Orthologues
The predicted target sites of let-7 on human orthologues are listed in supplementary table S3 (Supplementary Material online). The putative target sites were identified by considering the pairing quality and the degree of binding affinity between an miRNA sequence and the segments of the 3' UTR sequence of an orthologous gene (see Materials and Methods). We did not impose the cross-species conservation requirement, which has been used in many target prediction programs (Enright et al. 2002; Lewis et al. 2003, 2005; John et al. 2004; Kiriakidou et al. 2004; Grun et al. 2005; Krek et al. 2005) because we tried to identify not only the evolutionarily conserved target sites but also the human-specific target sites. In addition, as our pairing constraints were loose (see Materials and Methods), these putative target sites likely include most or all of the target sites. This is useful for identifying a sufficient portion of a 3' UTR sequence that can be tested in the following experiments.
For each of the C. elegans genes we studied, we chose one of its human orthologues that has most putative target sites on its 3' UTR for the first round reporter assay. After surveying the putative target sites of the human genes, we chose VDR, FOXA1, and TRIM71 to examine whether they are targets of let-7. We then chose the paralogues of the human genes used in the first round test for the second round reporter assay. As shown in table 1, FOXA2 and FOXA3 are paralogues of FOXA1 and NR1I2 is a paralogue of VDR, and these 3 genes had not been tested in the first round. By surveying the putative target sites of these 3 genes, we found that NR1I2 had a perfect seed-match site, but FOXA2 and FOXA3 did not have good putative target sites. Therefore, NR1I2 was selected for the second round test. The locations of the putative target sites of these 4 selected human genes are shown in figure 1A. It is noteworthy that 2 of the putative target sites of TRIM71 have perfect base pairing with the seed region of let-7 and have been evolutionarily conserved (see supplementary fig. S2, Supplementary Material online). On the other hand, NR1I2 has a perfect seed-match site, but the site is not evolutionarily conserved (supplementary fig. S2, Supplementary Material online).
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Both hsa-let-7a and hsa-let-7c Repress Human TRIM71
To examine the connection between the target orthologues and their cognate miRNAs, the luciferase reporter assay provides a reliable solution and has been used in many studies. Because the sequences of the let-7 family have been highly conserved from C. elegans to human (supplementary fig. S1, Supplementary Material online), we used hsa-let-7a to perform the reporter assay for 2 reasons. First, the sequences of hsa-let-7a and C. elegans let-7 are identical. Second, hsa-let-7a has been shown to repress RAS effectively (Johnson et al. 2005
). We first constructed the target site reporter plasmids for VDR, NR1I2, FOXA1, TRIM71 (fig. 1A), and plet7AS control reporters; the latter contained the antisense sequence of hsa-let-7a and served as a positive control. Each reporter plasmid was cotransfected with 2 nM hsa-let-7a precursor oligos or control oligos into HepG2 cells, which was reported to have no detectable level of let-7a by microarray analysis (Johnson et al. 2005), and the luciferase reporter assay was performed. As shown in figure 1B, let-7a oligo had a slight inhibitory effect on the reporter vector (pre-let7a/pre-ctrl: 74.6%) compared with the control oligo. In contrast, let-7a markedly repressed the luciferase activity of the TRIM71 reporter to 12.7% of the control. As to the VDR, NR1I2, and FOXA1 reporters, the repressive activities of let-7a (pre-let7a/pre-ctrl: 72.9%, 66.2%, and 86.0%, respectively) were not significantly different from the reporter vector. As expected, the repression was most pronounced with the plet7AS control reporter (pre-let7a/pre-ctrl: 7.1%). Although it is possible that 2 nM pre-let-7a oligo used for transfection may not be sufficient for repressing the VDR and FOXA1 reporters, a dramatically increased amount of mature let-7a was detected in pre-let-7a transfectant in parallel transfection experiments (fig. 1C). To confirm the above results, 100 nM precursor oligos were cotransfected with reporter plasmids into HepG2 cells. As shown in figure 1D, 100 nM let-7a transfections did not repress VDR or FOXA1 reporters (pre-let7a/pre-ctrl: 69.3% and 119.7%, respectively) as compared with the empty reporter vector (pre-let7a/pre-ctrl: 58.4%). However, the repression on TRIM71 reporter was clearly demonstrated to an even greater extent than transfection with 2 nM oligo (pre-let7/pre-ctrl: 8.7 vs. 12.7%) (fig. 1D). These results confirm that the 3' UTR of TRIM71 contains the putative target sites for let-7a, whereas let-7a has little or no effect on the 3' UTRs of VDR, NR1I2, and FOXA1.
To examine whether other members of the hsa-let-7 family are also capable to repress the TRIM71 reporter, we used 2 nM pre-let7c oligo (pre-let7c) to perform the same transient transfection and the reporter assay. Although hsa-let-7a and hsa-let-7c are identical except for the nineteenth nucleotide, which is outside of the seed region, whether there is any difference in their functions has never been addressed. As shown in figure 2C, let-7c effectively repressed TRIM71 reporter as did let-7a (pre-let7/pre-ctrl: 11.5% of let-7c vs. 12.7% of let-7a). Besides, let-7c greatly repressed plet7AS control reporter, too, even though there is a nucleotide mismatch between them. These results suggest that TRIM71 is regulated not only by hsa-let-7a but also by other members of the human let-7 family.
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Mutation in the Predicted Target Sites of TRIM71 Abolishes the Repression by let-7
In our computational prediction, we mapped 2 putative target sites on the 3' UTR of TRIM71 cDNA with perfect match to the seed region of let-7a: the first site was from nt2828 to nt2849 and the second from nt2908 to nt2929. To determine which of the target sites is responsible for interaction with let-7a, we mutated the first (TRIM71m1), the second (TRIM71m2), or both sites (TRIM71m1m2) by deleting 22 bp of the respective sites in the TRIM71 reporter (fig. 2A). These TRIM71 mutants were cotransfected with precursor let-7a or control oligo, and the luciferase reporter assay was performed. As shown in figure 2B, the repression of TRIM71m1 and TRIM71m2 reporters by let-7a diminished to approximately half of the TRIM71 reporter (pre-let7a/pre-ctrl: 24.1% and 26.3% vs. 12.7%). Moreover, the repression of TRIM71 reporter by let-7a was nearly abolished when both sites were mutated (pre-let7/pre-ctrl: m1m2 vs. vector is 70.0% vs. 75.4%). In addition, derepression by let-7c was also observed in the reporter with both sites mutated (pre-let7c/pre-ctrl: m1m2 vs. vector is 69.1% vs. 69.5%) (fig. 2C). These findings suggest that both of the predicted target sites in TRIM71 are targets of let-7 and there are no other target sites in TRIM71.
let-7a Represses Zebrafish lin-41, an Orthologue of TRIM71
The orthologues of C. elegans lin-41 in zebrafish, chicken, and mouse, which were negatively regulated by let-7 by reporter assays, have been described before (Kloosterman et al. 2004; Kanamoto et al. 2006). Using our methods to predict the target sites on the 3' UTR of zebrafish lin-41 gene, we discovered 2 putative target sites (from nt2249 to nt2273 and from nt2330 to nt2354) with perfect match to the seed region, which were identical to the 2 target sites reported by Kloosterman et al. (2004) (figs. 3A and 5B); this finding further confirmed the reliability of our prediction methods. In their study, coinjection of mRNA of green fluorescent protein (GFP) fused to the 3'UTR of zebrafish lin-41 orthologue with ds let-7 oligo into the zebrafish zygotes led to silencing of this GFP reporter. We cloned a 167-bp fragment of the 3'UTR of zebrafish lin-41 to construct the DLIN41 reporter and performed the cotransfection and the luciferase reporter assays in HepG2 cells. The result showed that let-7a repressed the DLIN41 reporter to a similar extent as the TRIM71 reporter (pre-let7/pre-ctrl: 14.2% vs. 12.7%) (fig. 3B), suggesting that both TRIM71 and zebrafish lin-41 are evolutionarily conserved targets of let-7.
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Silencing of Zebrafish lin-41 Orthologue Causes Severe Developmental Defects
The function of human TRIM71 has not been reported, and the effects of silencing lin-41 orthologues in zebrafish, chicken, and mouse models have not been conducted, although lin-41 orthologues have been shown to be downstream of the signal pathways of both the secrete Sonic hedgehog and fibroblast growth factor in chicken and mouse (Lancman et al. 2005
). To examine the function of lin-41, we first constructed the cytomegalovirus (CMV) promoter–based transposon vector, which expressed the engineered siRNA, to perform the RNAi in the zebrafish model (Kawakami et al. 2004). Two different RNAi transposons were constructed to silence the expression of the zebrafish lin-41 orthologue: 716RNAi was specific for nt716 to nt736 in the coding region, and 1611RNAi was specific for nt1611 to nt1631 in the 3'UTR of zebrafish lin-41 orthologue cDNA. A nonspecific RNAi control plasmid, which expressed the engineered siRNA with random sequence, served as a negative control. Twenty-four hours after injecting these RNAi plasmids into the 1-cell stage of zebrafish embryos, we observed a high incidence of severely defective embryos in those injected with 716RNAi or 1611RNAi (34.7% and 49.3%, respectively), as compared with only 1.9% abnormal embryos in those injected with the RNAi control (fig. 4). These severely defective embryos induced by injection of 716RNAi or 1611RNAi displayed the apparent phenotypes with short trunk, deformed yolk, and S-shaped tail, and they began to die in 48–60 h after injection. Furthermore, over half of the remainder of the embryos treated with 716RNAi (65.3%) or 1611RNAi (50.7%) also showed abnormal phenotype with different degrees of defects, especially variations in the curvature of tails. In contrast, all the remaining RNAi control group (98.1%) had normal phenotype.
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To further confirm that the defective phenotype was the consequence of lin-41 silencing, we injected lin-41 morpholino into zebrafish eggs to silence lin-41 expression. After injection of 5-ng lin-41 morpholino, more than 76.1% of embryos (n = 134) exhibited the same developmental retardation, characterized by short trunk, abnormal yolk shape, and S-shaped tail, as those treated with RNAi (fig. 4). In contrast, all embryos developed normally after injection with the same dose of control morpholino (data not shown). We noted that these defective phenotypes of lin-41-silenced zebrafish embryos were similar to those of ds let-7a oligo-injected zebrafish embryos reported previously (Kloosterman et al. 2004
), suggesting that the effects of let-7 injection might have resulted from downregulated lin-41 expression by let-7. Taken together, these results demonstrate that silencing of the zebrafish lin-41 orthologue causes severe developmental defects and thus, for the first time, provided direct evidence that lin-41 plays an essential role in embryo development. |
Discussion |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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In this study, we examined whether the interaction between the C. elegans targets and the evolutionarily highly conserved miRNA let-7 is conserved in human. Among the 4 genes we considered as possibly conserved let-7-target pairs, 3 were investigated and the interaction of the let-7-lin-41 pair was found to be preserved in human. Another predicted target gene, the RAS gene family, had been previously shown to be regulated by let-7 both in C. elegans and in human (Johnson et al. 2005
). The latter study showed that C. elegans let-7/RAS has multiple orthologues in human, and at least 2 orthologous copies, KRAS and NRAS, as well as 2 alternative splicing forms of NRAS preserve the interaction relationship with let-7. In our study, although both C. elegans daf-12 and pha-4 also have multiple orthologues in human, none of them seem to have preserved the relationship. This implies that the interaction of the miRNA–target pairs may not be as well conserved as the miRNA itself during evolution. Based on our survey, each of the C. elegans target genes was closely related to its human orthologues either in structure or in function. In an attempt to explain why the interaction is not always preserved, we compared the conserved systems (the lin-41 and let-60 systems) and the nonconserved systems (the daf-12 and pha-4 systems). It was noted that the alignable length ratios (see Materials and Methods and supplementary table S2 [Supplementary Material online]) of the 2 conserved systems were much higher than that of the 2 nonconserved systems (0.61 1.00 for the conserved systems, whereas 0.20 0.41 for the nonconserved systems). This suggests that the orthologous proteins in the conserved systems may have greater similarities in protein domains and their structure and function, so that the interaction with the highly conserved miRNA may be better preserved. In contrast, the orthologous proteins in the nonconserved systems may have less similar protein domains; some of their ancestral functions may have been lost during evolution and the miRNA–target regulation may have been replaced or compensated by other regulatory pathways. To sum up, it seems that a system that has a higher alignable length ratio may be better conserved. However, a definitive conclusion awaits studies of a larger number of cases.
We noticed that NR1I2 has a perfect let-7 seed-match site in the 3'UTR (supplementary fig. S2, Supplementary Material online), and yet our NR1I2 reporter construct did not provide clear-cut evidence that it could be regulated by let-7. This suggests that the perfect seed-match rule is not the only criterion for the miRNA–target binding. It is known that miRNAs can act in a combinatorial fashion (Krek et al. 2005), and multiple miRNAs may contribute together to an important effect. Thus, we could not exclude the possibility that these genes are regulated by let-7 in concert with other miRNAs acting simultaneously. In contrast, TRIM71, which has 2 evolutionarily conserved perfect seed-match sites in the 3'UTR (supplementary fig. S2, Supplementary Material online), was shown to be the target of let-7. Moreover, it is intriguing that even though the locations of the 2 sites in the 3'UTR in human are different from that in zebra fish, the distance between the 2 sites in human (81 nt) is about the same as that in zebra fish (80 nt) (fig. 5). Further, we notice that the 2 let-7 target sites of lin-41 (the worm orthologoue of TRIM71) (Reinhart et al. 2000; Slack et al. 2000; Vella et al. 2004) are not perfect match to the seed region but have high binding energy (fig. 5 and supplementary fig. S2 [Supplementary material online]). This suggests that even if the interaction relationship of these orthologous miRNA–target pairs is conserved during evolution, the pairing condition for these pairs may be different.
Downregulation of target genes by miRNA may involve one of the 2 posttranscriptional suppression mechanisms: translational repression or mRNA cleavage (reviewed in Bartel 2004). It has been reported that the degradation of lin-41 mRNA is induced by let-7 in C. elegans (Bagga et al. 2005). However, in zebrafish, the plasmid-containing GFP gene fused with the 3'UTR of the lin-41 zebra fish orthologue was translationally repressed by let-7, but its mRNA level was unchanged (Kloosterman et al. 2004). This implies that even though the orthologous miRNA–target pairs are conserved in evolution, their regulatory mechanisms may have changed.
We demonstrated that human TRIM71 is a specific target of let-7a and let-7c by the luciferase reporter assay. Besides the pair of RAS and let-7, the TRIM7 and let-7 pair is another target–miRNA pair that showed evolutionary conservation in both C. elegans and human. In addition to C. elegans and human, the evolutionarily conserved pair of lin-41 and let-7 has also been verified in zebrafish (Kloosterman et al. 2004), chicken, and mouse (Kanamoto et al. 2006), providing additional evidence to support that the regulation of lin-41 mediated by let-7 miRNA is conserved in animals, from C. elegans to human.
Why has this target–miRNA interaction been conserved in distantly related animals? The answer may be linked to the importance of the lin-41 gene in animal development. The lin-41 protein family, from nematodes to mammals, belongs to the RBCC protein family and shares most of the functional domains (Lancman et al. 2005; Schulman et al. 2005; Kanamoto et al. 2006). In C. elegans, late larval activation of let-7 expression downregulates lin-41 to relieve inhibition of the expression of the adult specification transcription factor lin-29 (Slack et al. 2000), and null mutation of let-7 caused precocious expression of adult fates at larval stages. Another study demonstrated a role for the lin-41 gene in C. elegans male tail tip morphogenesis (Del Rio-Albrechtsen et al. 2006). Although the above studies provide no direct in vivo evidence in vertebrate animals, the results of reporter assay and the reciprocal expression pattern of lin-41 and let-7 in chicken and mouse suggest that the negative regulation of lin-41 by let-7 is important in their limb development (Lancman et al. 2005; Schulman et al. 2005; Kanamoto et al. 2006). Moreover, in our zebrafish model, silencing of lin-41 by siRNA or morpholino resulted in developmentally defective embryos, thus providing direct in vivo evidence for the first time that lin-41 plays an important role in the development of vertebrates. To put these results together, we propose that downregulation of lin-41 by let-7 plays a fundamental role in animal development, which may be the reason why the lin-41-let-7 pair has been well preserved during animal evolution.
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Supplementary Material |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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Supplementary figures S1 and S2 and tables S1–S3 are available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/).
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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We thank Arthur Shih for suggestions. This study was supported by grants from National Science Council (NSC95-3114-P-002-005-Y to W.-H.L. and A.L.Y., and NSC 93005P to L.-C.H.) and by Academia Sinica.
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1 Equal contribution to this work.
Takashi Gojobori, Associate Editor
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