Spliceosomal introns in the 5′ untranslated region of plant BTL RING-H2 ubiquitin ligases are evolutionary conserved and required for gene expression
© Aguilar-Hernández and Guzmán; licensee BioMed Central Ltd. 2013
Received: 30 July 2013
Accepted: 11 November 2013
Published: 14 November 2013
Introns located close to the 5′ end of a gene or in the 5′ untranslated region often exert positive effects on gene expression. This effect, known as intron-mediated enhancement (IME), has been observed in diverse eukaryotic organisms, including plants. The sequences involved in IME seem to be spread across the intron and function in an additive manner. The IMEter algorithm was developed to predict plant introns that may enhance gene expression. We have identified several plant members of the BTL class of E3s, which may have orthologs across eukaryotes, that contain a 5′UTR intron. The RING finger E3 ligases are key enzymes of the ubiquitination system that mediate the transfer of ubiquitin to substrates.
In this study, we retrieved BTL sequences from several angiosperm species and found that 5′UTR introns showing a strong IMEter score were predicted, suggesting that they may be conserved by lineage. Promoter-GUS fusion lines were used to confirm the IME effect of these 5′UTR introns on gene expression. IMEter scores of BTLs were compared with the 5′UTR introns of two gene families MHX and polyubiquitin genes.
Analysis performed in two Arabidopsis BTL E3 ligases genes indicated that the 5′UTR introns were essential for gene expression in all the tissues tested. Comparison of the average 5′UTR intron size on three gene families in ten angiosperm species suggests that a prevalent size for a 5′UTR intron is in the range of 600 nucleotides, and that the overall IMEter score within a gene family is preserved across several angiosperms. Our results indicated that gene expression dependent on a 5′UTR intron is an efficient regulatory mechanism in BTL E3 ligases that has been preserved throughout plant evolution.
Spliceosomal introns are evolutionary conserved features ubiquitously found in eukaryotic genomes that interrupt the coding sequences of genes. Introns are transcribed into pre-mRNA, which is then processed to produce mature mRNA. The spliceosome is a conserved and versatile ribonucleoprotein machinery that generates the mature mRNA by splicing the exons via precise excision of the introns at the donor and acceptor sites [1, 2]. Alternative splicing of introns enhances proteome diversity as well as serves as a mechanism for regulating gene expression. Over 40% of plant genes (Arabidopsis thaliana and Oryza sativa) and over 90% of human genes are processed by alternative splicing to generate multiple and assorted mature mRNA molecules [3–6].
Recent studies have identified specific introns that are able to enhance gene expression. In some cases, these introns enhance mRNA accumulation 10-fold higher than normal levels. This effect, known as intron-mediated enhancement (IME), is likely to be a conserved mechanism throughout evolution, as it has been described in diverse plant, animal and fungal species [7–10]. The mechanisms underlying IME have not been clearly established, but a mechanism distinct from that of conventional transcriptional enhancer elements has been envisioned. Introns involved in IME should be located within the transcription unit in the sense orientation and close to the 5′ terminal end of the gene. IME progressively declines as the intron is located farther from the 5′ end, and is inactive when the intron is located at the 3′ untranslated region. Sequences consisting of redundant motifs that are dispersed throughout the intron are shown to be required for IME. These sequences are frequently located toward the 5′ end of the intron and exert additive effects on expression [8, 11, 12].
Spliceosomal introns are also present in the untranslated regions (UTRs) of genes. These 5′UTR introns are generally longer than introns within coding sequences and may influence gene expression, mRNA stability or mRNA export. Conversely, introns located in the 3′UTR are capable of downregulating levels of gene expression [13, 14]. IME has been observed in several plant introns, usually prompted by introns located in the 5′UTR. The extent of IME depends on intron sequence composition and position and on the spatiotemporal expression features of the promoter. The enhancement of expression from 2- to 10-fold or more can be expected, and a stronger effect has been observed in monocots compared to eudicots [12, 15, 16].
The specific sequences in introns involved in IME are not restricted to discrete motifs. On the contrary, the sequences seem to be scattered throughout the intron and function in an additive fashion . Although these sequences have not yet been identified, an algorithm to analyze introns that may stimulate expression in plants has been developed. This algorithm, known as the IMEter is based on word pattern frequency at introns. The IMEter score reveals the similarity between an input intron to introns located proximal to the start of transcription or to distal introns. IMEter gives high scores to sequences that resemble proximal introns and low scores to sequences that resemble distal introns [17, 18].
The BCA2 zinc finger ATL (BTL) family of RING-H2 finger E3 ligases in plants are possible orthologs of the mammalian Rabring7/BCA2 RING-H2 E3 ligases [19, 20]; BTLs share some common structural features with the Arabidopsis Tóxicos en Levadura (ATL) family of RING finger ligases . E3 ligases are components of the ubiquitin proteasome system (UPS) that recognize specific substrates for degradation and mediates the transfer of the ubiquitin . Although functional analysis of Rabring7/BCA2/BTLs is scarce, this class of E3s has been identified across eukaryotes, in plants, animals, fungi, and protozoa genomes, and has shown gene expansion in land plants . The best characterized member of this class in humans is Rab7-interacting RING finger protein (Rabring7), also known as Breast Cancer Associated gene 2 (BCA2), which is involved in intracellular vesicle traffic regulation and was also identified as a differentially expressed gene in cancerous mammary epithelial cell lines [23, 24]. Few putative orthologs have been identified thus far in plants, including CIP8, which supports the function of the E3 ligase COP1 in ubiquitination; AtRDUF1 and AtRZF1 that may function in the response to dehydration and drought, and RHC1 which is involved in root architecture [25–28].
In this report, we evaluated the conservation of BTL 5′UTR introns in fourteen angiosperm species. We confirmed that 5′UTR introns are conserved by lineage, with most showing a strong IMEter score. Analysis of the effect of the 5′UTR intron on the expression of two AthBTL genes showed that these introns are required for gene expression.
Results and discussion
Identification of BTLs in 14 angiosperm species
Retrieved BTL genes in groups A and B from 14 angiosperm genomes containing a 5′UTR intron
Oryza sativa japonica
To confirm spliceosomal intron annotations in BTLs, sequence LOGOs were generated over the nucleotides at the splicing donor and acceptor sites (Figure 1b). Canonical sequences were observed at the generated LOGOs, with GT at the donor site and AG at the acceptor site, indicating that annotated introns from A. thaliana and O. sativa as well as from the selected plant genomes are likely to be valid spliceosomal introns . Moreover, there was no evidence of additional conserved sequences within 40 nucleotides in close proximity to the donor or acceptor sites (Figure 1b).
IMEter scores of 5′UTR introns of group A and B BTL genes
As described for several plant 5′UTR introns, we speculated that the 5′UTR introns in these BTLs might also be involved in gene expression. To evaluate this hypothesis, we used the intron IMEter score. The IMEter score determines whether an intron is more similar to proximal than distal introns, hence more likely to encode elements that enhance gene expression. Although the IMEter was developed with A. thaliana intron sequences, it is also effective in predictions of other plant species. The higher the score of the intron indicates a higher probability that the intron enhances expression. An IMEter score above 20 was established as predicting introns that are likely to have a strong effect on enhancing expression, while an IMEter score between 10 and 20 likely has a moderate effect on gene expression. Approximately 80% of all A. thaliana introns have an IMEter score less than or equal to 9.25 (see http://korflab.ucdavis.edu/cgi-bin/web-imeter2.pl). We found that among the 62 BTLs tested, 39 had a strong IMEter score, 20 showed a moderate score and 3 had a low score of less than 10 (Figure 2). This IMEter scoring predicts that the 5′UTR introns in BTLs may enhance gene expression in a moderate or strong fashion.
Effect of a 5′UTR intron on BTL gene expression in A. thaliana
Comparison of the IMEter scores of 5′UTR introns from BTLs, MHX and polyubiquitin genes in 10 angiosperm species
Several 5′ UTR introns have been previously shown to enhance gene expression. Among them, the MHX transporter that encodes a Mg2+/H+ exchanger and the polyubiquitin genes that encode ubiquitin monomers in tandem disposition, have been the subject of several studies [30–33]. To have an appraisal of the IME effect on the BTL family, we compared IMEter scores from BTL, MHX and polyubiquitin genes. We identified 5′UTR intron sequences from three monocot and seven eudicot species. A single MHX 5′UTR intron sequence was retrieved from each species, whereas one to six intron sequences from polyubiquitin genes (see Additional file 1). The position of the intron in polyubiquitin genes is highly conserved, located just upstream to the translation start codon, as previously inferred .
Many BTL genes have spliceosomal introns located in the 5′UTR. Our findings suggest that this 5′UTR intron arose early in BTL evolution, as it was detected in two lineages that included both monocot and eudicot species. The IMEter score predicted that 5′UTR spliceosomal introns in BTLs encode regulatory elements that enhance gene expression, and our results showed that IMEter evaluation functions across a wide range of plant species. Analysis of promoter-GUS fusions lines indicated that the 5′UTR introns in AthBTL1 and AthBTL4 are important for gene expression in vegetative and reproductive tissues. Indeed, the IMEter scores of the two tested promoters were high, predicting that they may enhance gene expression in a strong manner. Comparison of IMEter scores of BTLs with known gene families, indicated that the IMEter score is conserved in members of a gene family across angiosperms, and that specifically, the 5′UTR intron of polyubiquitin genes resulted in higher values. It would be interesting to compare the extent of gene expression enhancement along members of these gene families to correlate the overall IMEter score with gene expression.
The use of 5′UTR intron for enhancement of gene expression in this class of E3 ligases is likely an effective mechanism, as our analysis suggests it has been preserved in evolution. Although the sizes of the 5′UTR introns in AthBTL1 and AthBTL4 are different, the introns exert a comparable enhancement of gene expression and similar expression domains in transgenic lines. Approximately 15% of BTLs in groups A and B do not have predicted introns in the 5′UTR. It would be interesting to evaluate whether these intronless sequences are expressed or whether the loss of the intron completely abolished gene expression, rendering these genes as pseudogenes.
Identification of 5′UTR introns in BTL, MHX and polyubiquitin genes from angiosperms
The peptide and predicted spliceosomal intron sequences were retrieved from fourteen angiosperm genomes deposited in the Phytozome 9.1 database at http://www.phytozome.net/. The genomes included four monocots and ten eudicot plants. If more than one spliceosomal intron was predicted in the 5′UTR, the largest intron sequence was considered. Only BTLs belonging to the previously identified groups A and B were studied. Spliceosomal intron sequences from the 5′UTR of MHX and polyubiquitin genes were retrieved from three monocot and seven eudicot species. In the polyubiquitin genes, the position of the intron was found immediately upstream to the ATG codon, except in three Eucalyptus grandis sequences, where the first intron within the coding DNA sequence was considered in the analysis.
Multiple alignment of the 81 retrieved BTL sequences was obtained by Clustal X . The phylogenetic tree was based on concatenated RING-H2 and the BZF domains, and was generated using the Neighbor-Joining method with a Bootstrap value of 1000 replicates. The tree phylogeny was displayed on NJplot .
IMEter version 2.0 was used to obtain the IMEter scores: http://korflab.ucdavis.edu/cgi-bin/web-imeter2.pl. The transcribed strand was used in the analysis. A window of 80 nucleotides was used when the complete sequence from A. thaliana genes were scored. IMEter v2.0 scores were considered as: moderate enhancement (mod), between 10 and 20, and strong enhancement, more than 20.
Generation of sequence LOGOs
Sequence LOGOs of donor and acceptor sites were obtained using MEME (Multiple EM for Motif Elicitation) version 4.9.0 at http://meme.nbcr.net/meme/cgi-bin/meme.cgi. Forty nucleotides spanning the donor or the acceptor sites were included in the sequence LOGO. The following parameters were used for analysis: zero or one per sequence, 40 amino acids as minimum and maximum sizes of motifs.
Analysis of AthBTL1 and AthBTL4 expression in transgenic A. thaliana lines
A. thaliana ecotype Columbia (Col-0) was used in the floral dip transformation procedure with Agrobacterium tumefaciens strain GV2260. Transgenic plants were selected on MS agar medium containing 50 μg/ml kanamycin (Km). Plants were grown on MS agar medium or on soil under controlled environmental conditions at 16 h light/8 h dark cycles. A segregation test was performed by growing the seedlings from 25 independent lines of T2 generation under selective conditions. T2 lines that did not show segregation were eliminated from the analysis. Homozygous plants of the third generation (T3) were used for GUS staining experiments.
To obtain pAthBTL1: GUS and pAthBTL4: GUS transcriptional fusions, 2500 and 2200 bp fragments, respectively, were PCR amplified from genomic A. thaliana DNA and cloned into the vector pBI101 . The reverse primer was designed to bind several nucleotides upstream of the assumed translation start codon. Oligonucleotides pairs used for molecular cloning of the promoter regions were as follows: pAthBTL1, 5′-TACTCGAGAGGAGGGGTCGTGTTAGTTG-3′ and 5′-TAGGATCCCAATGCAATGCTTTCCTTGA-3′; pAthBTL4, 5′-TAGTCGACGCAGTCAATAAGCGCAAGGT-3′ and 5′- ATGGATCCTTCCTTACTTCACCCCCACA-3′. The reverse oligonucleotides used for molecular cloning of the promoter regions without the intron sequences were as follows: for pAthBTL1 5′-TAGGATCCTAAGTCAACCCTACGTCTGC-3′, and for pAthBTL4 5′- ATGGATCCAAAATCTGTCCTTGCTTCTT-3′. Restriction site sequences were added at the 5′ and 3′ end of the primers to facilitate directional cloning into the pBI101.1 vector. The constructs were confirmed by sequencing.
GUS histochemical analysis was performed as previously described . Tissues were analyzed under a Leica MZ12 stereomicroscope. Approximately 20 independent T2 segregating lines were initially stained for each of the constructs. For each construct, at least five lines that were followed until the T3 generation were used for the spatial-temporal expression analysis.
We thank Phytozome for making the genome sequences and gene annotations from several sources available. We also thank Laura Aguilar-Henonin for technical assistance. This work was supported by Consejo Nacional de Ciencia y Tecnología México, CONACYT, grants CB-78777 and CB-177528 to P.G.
- Roy SW, Gilbert W: The evolution of spliceosomal introns: patterns, puzzles and progress. Nat Rev Genet. 2006, 7: 211-221.PubMedGoogle Scholar
- Mattick JS: Introns: evolution and function. Curr Opin Genet Dev. 1994, 4: 823-831. 10.1016/0959-437X(94)90066-3.PubMedView ArticleGoogle Scholar
- Filichkin SA, Priest HD, Givan SA, Shen R, Bryant DW, Fox SE, Wong W-K, Mockler TC: Genome-wide mapping of alternative splicing in Arabidopsis thaliana. Gen Res. 2010, 20: 45-58. 10.1101/gr.093302.109.View ArticleGoogle Scholar
- Wang ET, Sandberg R, Luo S, Khrebtukova I, Zhang L, Mayr C, Kingsmore SF, Schroth GP, Burge CB: Alternative isoform regulation in human tissue transcriptomes. Nature. 2008, 456: 470-476. 10.1038/nature07509.PubMedPubMed CentralView ArticleGoogle Scholar
- Nilsen TW, Graveley BR: Expansion of the eukaryotic proteome by alternative splicing. Nature. 2010, 463: 457-463. 10.1038/nature08909.PubMedPubMed CentralView ArticleGoogle Scholar
- Severing EI, van Dijk AD, van Ham RC: Assessing the contribution of alternative splicing to proteome diversity in Arabidopsis thaliana using proteomics data. BMC Plant Biol. 2011, 11: 82-10.1186/1471-2229-11-82.PubMedPubMed CentralView ArticleGoogle Scholar
- Lugones LG, Scholtmeijer K, Klootwijk R, Wessels JG: Introns are necessary for mRNA accumulation in Schizophyllum commune. Mol Microbiol. 1999, 32: 681-689. 10.1046/j.1365-2958.1999.01373.x.PubMedView ArticleGoogle Scholar
- Mascarenhas D, Mettler IJ, Pierce DA, Lowe HW: Intron-mediated enhancement of heterologous gene expression in maize. Plant Mol Biol. 1990, 15: 913-920. 10.1007/BF00039430.PubMedView ArticleGoogle Scholar
- Le Hir H, Nott A, Moore MJ: How introns influence and enhance eukaryotic gene expression. Trends Biochem Sci. 2003, 28: 215-220. 10.1016/S0968-0004(03)00052-5.PubMedView ArticleGoogle Scholar
- Zieler H, Huynh C: Intron-dependent stimulation of marker gene expression in cultured insect cells. Insect Mol Biol. 2002, 11: 87-95. 10.1046/j.0962-1075.2001.00312.x.PubMedView ArticleGoogle Scholar
- Rose AB: The effect of intron location on intron-mediated enhancement of gene expression in Arabidopsis. Plant J. 2004, 40: 744-751. 10.1111/j.1365-313X.2004.02247.x.PubMedView ArticleGoogle Scholar
- Rose AB, Beliakoff JA: Intron-mediated enhancement of gene expression independent of unique intron sequences and splicing. Plant Physiol. 2000, 122: 535-542. 10.1104/pp.122.2.535.PubMedPubMed CentralView ArticleGoogle Scholar
- Barrett LW, Fletcher S, Wilton SD: Regulation of eukaryotic gene expression by the untranslated gene regions and other non-coding elements. Cell Mol Life Sci. 2012, 69: 3613-3634. 10.1007/s00018-012-0990-9.PubMedPubMed CentralView ArticleGoogle Scholar
- Bicknell AA, Cenik C, Chua HN, Roth FP, Moore MJ: Introns in UTRs: Why we should stop ignoring them. Bioessays. 2012, 34: 1025-1034. 10.1002/bies.201200073.PubMedView ArticleGoogle Scholar
- Chung B, Simons C, Firth A, Brown C, Hellens R: Effect of 5′UTR introns on gene expression in Arabidopsis thaliana. BMC genomics. 2006, 7: 120-10.1186/1471-2164-7-120.PubMedPubMed CentralView ArticleGoogle Scholar
- Lu J, Sivamani E, Azhakanandam K, Samadder P, Li X, Qu R: Gene expression enhancement mediated by the 5′-UTR intron of the rice rubi3 gene varied remarkably among tissues in transgenic rice plants. Mol Genet Genomics. 2008, 279: 563-572. 10.1007/s00438-008-0333-6.PubMedView ArticleGoogle Scholar
- Rose AB, Elfersi T, Parra G, Korf I: Promoter-proximal introns in Arabidopsis thaliana are enriched in dispersed signals that elevate gene expression. Plant Cell. 2008, 20: 543-551. 10.1105/tpc.107.057190.PubMedPubMed CentralView ArticleGoogle Scholar
- Parra G, Bradnam K, Rose AB, Korf I: Comparative and functional analysis of intron-mediated enhancement signals reveals conserved features among plants. Nucleic Acids Res. 2011, 39: 5328-5337. 10.1093/nar/gkr043.PubMedPubMed CentralView ArticleGoogle Scholar
- Bacopulos S, Amemiya Y, Yang W, Zubovits J, Burger A, Yaffe M, Seth AK: Effects of partner proteins on BCA2 RING ligase activity. BMC cancer. 2012, 12: 63-10.1186/1471-2407-12-63.PubMedPubMed CentralView ArticleGoogle Scholar
- Aguilar-Hernández V, Medina J, Aguilar-Henonin L, Guzmán P: Expansion and diversification of BTL RING-H2 ubiquitin ligases in angiosperms: putative Rabring7/BCA2 orthologs. PloS ONE. 2013, 8: e72729-10.1371/journal.pone.0072729.PubMedPubMed CentralView ArticleGoogle Scholar
- Guzmán P: The prolific ATL family of RING-H2 ubiquitin ligases. Plant Signal Behav. 2012, 7: 1014-1021. 10.4161/psb.20851.PubMedPubMed CentralView ArticleGoogle Scholar
- Vierstra RD: The ubiquitin-26S proteasome system at the nexus of plant biology. Nat Rev Mol Cell Biol. 2009, 10: 385-397. 10.1038/nrm2688.PubMedView ArticleGoogle Scholar
- Mizuno K, Kitamura A, Sasaki T: Rabring7, a novel Rab7 target protein with a RING finger motif. Mol Biol Cell. 2003, 14: 3741-3752. 10.1091/mbc.E02-08-0495.PubMedPubMed CentralView ArticleGoogle Scholar
- Burger AM, Gao Y, Amemiya Y, Kahn HJ, Kitching R, Yang Y, Sun P, Narod SA, Hanna WM, Seth AK: A novel RING-type ubiquitin ligase breast cancer-associated gene 2 correlates with outcome in invasive breast cancer. Cancer Res. 2005, 65: 10401-10412. 10.1158/0008-5472.CAN-05-2103.PubMedView ArticleGoogle Scholar
- Hardtke CS, Okamoto H, Stoop-Myer C, Deng XW: Biochemical evidence for ubiquitin ligase activity of the Arabidopsis COP1 interacting protein 8 (CIP8). Plant J. 2002, 30: 385-394. 10.1046/j.1365-313X.2002.01298.x.PubMedView ArticleGoogle Scholar
- Kim SJ, Ryu MY, Kim WT: Suppression of Arabidopsis RING-DUF1117 E3 ubiquitin ligases, AtRDUF1 and AtRDUF2, reduces tolerance to ABA-mediated drought stress. Biochem Biophys Res Comm. 2012, 420: 141-147. 10.1016/j.bbrc.2012.02.131.PubMedView ArticleGoogle Scholar
- Jiang K, Zhu T, Diao Z, Huang H, Feldman LJ: The maize root stem cell niche: a partnership between two sister cell populations. Planta. 2010, 231: 411-424. 10.1007/s00425-009-1059-3.PubMedPubMed CentralView ArticleGoogle Scholar
- Ju H-W, Min J-H, Chung M-S, Kim CS: The atrzf1 mutation of the novel RING-type E3 ubiquitin ligase increases proline contents and enhances drought tolerance in Arabidopsis. Plant Sci. 2012, 203-204: 1-7.PubMedView ArticleGoogle Scholar
- Munch K, Krogh A: Automatic generation of gene finders for eukaryotic species. BMC Bioinform. 2006, 7: 263-10.1186/1471-2105-7-263.View ArticleGoogle Scholar
- Akua T, Berezin I, Shaul O: The leader intron of AtMHX can elicit, in the absence of splicing, low-level intron-mediated enhancement that depends on the internal intron sequence. BMC Plant Biol. 2010, 10: 93-10.1186/1471-2229-10-93.PubMedPubMed CentralView ArticleGoogle Scholar
- Akua T, Shaul O: The Arabidopsis thaliana MHX gene includes an intronic element that boosts translation when localized in a 5‚-UTR intron. J Exp Botany. 2013, 64: 4255-4270. 10.1093/jxb/ert235.View ArticleGoogle Scholar
- Emami S, Arumainayagam D, Korf I, Rose AB: The effects of a stimulating intron on the expression of heterologous genes in Arabidopsis thaliana. Plant Biotechnol J. 2013, 11: 555-563. 10.1111/pbi.12043.PubMedView ArticleGoogle Scholar
- Norris SR, Meyer SE, Callis J: The intron of Arabidopsis thaliana polyubiquitin genes is conserved in location and is a quantitative determinant of chimeric gene expression. Plant Mol Biol. 1993, 21: 895-906. 10.1007/BF00027120.PubMedView ArticleGoogle Scholar
- Larkin M, Blackshields G, Brown N, Chenna R, McGettigan P, McWilliam H, Valentin F, Wallace I, Wilm A, Lopez R: Clustal W and Clustal X version 2.0. Bioinform. 2007, 23: 2947-2948. 10.1093/bioinformatics/btm404.View ArticleGoogle Scholar
- Perriere G, Gouy M: WWW-query: an on-line retrieval system for biological sequence banks. Biochimie. 1996, 78: 364-369. 10.1016/0300-9084(96)84768-7.PubMedView ArticleGoogle Scholar
- Jefferson RA, Kavanagh TA, Bevan MW: GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 1987, 6: 3901-3907.PubMedPubMed CentralGoogle Scholar
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