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Supplemental information Dissection of the genomic structure of the miR-183/96/182 gene. Previously, we showed that the miR-183/96/182 cluster is an intergenic miRNA cluster, located in a ~60-kb interval between the genes encoding nuclear respiratory factor-1 (Nrf1) and ubiquitin-conjugating enzyme E2H (Ube2h) on mouse chr6qA3.3 (1). To start to uncover the genomic structure of the miR- 183/96/182 gene, we first studied genomic features around miR-183/96/182 in the UCSC genome browser (http://genome.UCSC.edu/), and identified two CpG islands 3.4-6.5 kb 5’ of pre-miR-183, the most 5’ miRNA of the cluster (Fig. 1A; Fig. S1 and Seq. S1). A cDNA clone, AK044220, located at 3.2-4.6 kb 5’ to pre-miR-183, encompasses the second CpG island (Fig. 1A; Fig. S1). We hypothesized that this cDNA clone was derived from 5’ exon(s) of the primary transcript of the miR-183/96/182 gene, as CpG islands are often associated with promoters (2). Supporting this hypothesis, multiple expressed sequences detected by gene-trap clones, including clone D016D06 (3, 4), were co-localized with the cDNA clone AK044220 (Fig. 1A; Fig. S1). Clone D016D06, deposited by the German GeneTrap Consortium (GGTC) (http://tikus.gsf.de) (3, 4), was derived from insertion of a retroviral construct, rFlpROSAβgeo in 129S2 ES cells (Fig. 1A and C). The rFlpROSAβgeo construct carries a promoterless reporter gene, the β−geo cassette - an in-frame fusion of the β-galactosidase and neomycin resistance (Neor) gene (5), with a splicing acceptor (SA) immediately upstream, and a polyA signal downstream of the β−geo cassette (Fig. 1C). When integrated into an intron, it would result in a fusion transcript with the upstream exon(s) of the “trapped” endogenous gene spliced to the β−geo cassette. Therefore, the expressed sequence detected in the D016D06 clone may represent a 5’ exon of the miR-183/96/182 gene. To identify the 5’ end of the miR-183/96/182 gene, we obtained the D016D06 ES clone from the GGTC and performed 5’ Rapid Amplification of cDNA ends (RACE) using primers on the gene-trap vector (353 and 354 in Fig. 1C). 5’RACE produced a single, ~600-bp product (Fig. S2). Sequencing of this 5’RACE product confirmed the “trapped” sequence deposited in the GGTC database (Seq. S1). Alignment with the genomic sequence upstream of miR-183/96/182 revealed that the β−geo cassette of the gene-trap construct was spliced to a G nucleotide (nt), 3943 nt 5’ to pre-miR-183 (-3943G) (Fig. 1C; Seq. S1). The 5’ end of the RACE product extended to 4389 nt upstream of pre-miR-183 (-4389G), without any intronic interruption, suggesting a 446-bp exon – putatively, the first exon of the miR-183/96/182 gene (Fig. 1B and C); and -4389G was the transcription start site (TSS) of the gene. To test whether the same TSS is used in the retina, we designed primers in Exon 1 and performed 5’RACE with Primer 2R as the gene-specific primer (Fig. 1B; Seq. S1) using mouse total retinal RNA. Sequencing of the RACE product confirmed that it extended to the same nucleotide -4389G (Seq. S1). To define the genomic structure of the 3’ end of the miR-183/96/182 gene, we first performed 3’RACE (with Primer 2F in Exon 1. Fig. 1B) using total retinal RNA and showed that Exon 1 was spliced to a second exon, starting from nt 731 5’ to pre- miR-183 (-731G), revealing a 3213-bp intron (nt -3942 to -729) with classical splicing donor and acceptor sites (Fig. 1B. Seq. S1). However, this 3’RACE product stopped at 198 nt 5’ to pre-miR-183 (-198C) and did not extend beyond the miR-183/96/182 cluster (Fig. 1B; Seq. S1). To further explore the 3’ end of the miR-183/96/182 gene, we revisited the mouse genome database and identified an EST clone, BB709579, approximately 6 kb 3’ to miR-182, the most 3’ miRNA of the miR-183/96/182 cluster (Fig. 1A). We hypothesized that this EST clone may be derived from a downstream exon of the miR-183/96/182 gene. Therefore, we designed reverse primers, 5R, and nested primer 5Rnest within the sequence of BB709579 (Fig. 1A; Seq. S1), and performed RT-PCR using primers 2F and 5R with mouse total retinal RNA followed by nested PCR using either 2F/5Rnested or 2.1F/5Rnest (Fig. 1B), which produced 850-bp and 550-bp products, respectively (Fig. S3). Subsequent sequencing of the RT-PCR produced revealed that the putative Exon 1 jumped 14 kb, and spliced to nucleotide G, 6293 bp downstream of pre-miR-182 (Fig. 1B; Seq. S1), suggesting an alternative Exon 2, which we designated as exon 2’ (Fig. 1B; Seq. S1). A poly A signal (AATAAA) was identified ~495 bp downstream of Primer 5R, suggesting the 3’ end of this transcript. To test this, we designed a primer, 6R, further downstream of 5R (Fig. 1B; Seq. S1), and proved that Exon 2 extended further downstream of 5R to 6R by RT-PCR using primers 2F/6R and nested PCR with 2.1F/5Rnested (Fig. 1B; Fig. S3). This result confirms that the putative Exon 1 is indeed the 5’ end of the miR-183/96/182 gene; and the miR-183/96/182 cluster resides in the intron of the miR-183/96/182 gene. These alternative splicing events result in at least two transcripts (Fig. 1B; Seq. S2). Transcript I is at least 979 nt, and is composed by exons 1 and 2. However, the 3’ end of this transcript is yet to be fully defined. Transcript II is about 1432 nt, and is consisted of Exon 1 and alternatively spliced Exon 2’. Sequence analysis showed that Transcript I has no extended ORF (>30 codons), while transcript II has at least 4 ORFs, all of which reside in exon 2’ (Fig. 1B; Seq. S2 and S3). In phase I of the sequence, two ORFs consist of 98 (P98-1) and 70 codons (P70-1), respectively. Phases 2 and 3 each have an ORF with 63 codons (P63-2 and P63-3) (Fig. 1B; Seq. S2 and S3). None of the putative peptides contain known conserved functional domain; however, putative peptide (P98-1) showed sequence conservation with multiple proteins from different species, including proteins of Clostridiales bacterium, Plasmodium berghei, and several proteins in mouse and Chinese hamster (Fig. S4). Although whether any of these putative peptides were translated in vivo needs to be experimentally tested, these data suggested that the miR-183/96/182 cluster gene may be a protein-coding gene. Characterization of the ES cell clone, D016D06 ES clone. 1) Mycoplasma testing using the LookOut Mycoplasma PCR detection kit, following manufacturer’s protocol (Cat: MP0035. Sigma) confirmed that the D016D06 ES cells were free of mycoplasma contamination (Fig. S5). 2) Metaphase chromosomal cytogenetic analysis on 33 metaphase spreads confirmed normal karyotype distribution of the genetrap ES cell clone, D016D06. The assay was performed by the Transgenic Production Core Facility of the Research Resources Center, University of Illinois at Chicago (Fig. S6). 3) FirstChoice RLM-RACE kit (Ambion) was used for 5’RACE following manufacturer’s protocol. 5’RACE was performed using Primer 353 in the GeneTrap construct (Fig. 1C) and 5’RACE Adapter Outer Primer, followed by nested PCR using Primer 354 in the GeneTrap construct (Fig. 1C) and 5’RACE Adaptor Inner Primer produced a single ~580 bp product (Fig. S2), which was subcloned in a TA cloning vector (Invitrogen) and sequenced using TA cloning vector primers, M13 Forward and Reverse primers, as well as Primer 361 in the Genetrap construct (Fig. 1C). The sequence showed that exon 1 of the miR-183/96/182 gene is spliced to the Genetrap vector expressed sequence through the Splicing Adaptor (SA) on the GeneTrap construct (Fig. 1C; Seq. S1). Chimeric mouse production: ES cells were cultured to 80-85% confluent in a 12-well plate, trypsinized, resuspended in 2 ml ES media without LIF plus 20 mM HEPES, and were sent to the Transgenic Core Facility of University of Illinois at Chicago (UIC) for chimeric production. Standard blastocyst injection protocol was followed, with 10-15 cells injected in each blastocyst. Chimeric mouse production was carried out using C57BL6 (Jackson Lab, Stock number: 000664) as surrogate females, with 7-9 embryos injected in each surrogate, following standard protocol (6). 19 chimeric animals (16 males and 3 females) were produced (Table S1), and are mated with C57BL6 mice (Jackson Lab, Stock number: 000664). Seven out of the 16 male chimeric mice produced total of 46 germline-transmitted F1 mice. Using a PCR-based genotyping strategy (Fig. S7), we identified four heterozygous males (miR-183GT/+) and six heterozygous females (miR-183GT/+) among the 46 F1 agouti mice. Subsequently, we used these heterozygous mice for F2 breeding. The genotypes of F2 followed classical Mendelian inheritance pattern (Fig. S8). Genotyping PCR. Mouse tail genomic DNA was prepared using the DNeasy kit (Qiagen). Duplex PCR is performed using primers, 5Fintron1, 3Rintron 1 and 353 (Fig. 1C). Sequences of the primers are listed below. The miR-183GT allele is detected by 5Fintron1/ 353 with a 1.2-kb amplicon; the wild-type allele is detected by 5Fintron1/ 3Rintron1 with a 523-bp amplicon (SI Appendix (Fig. S7)). Amplification conditions are: 95 °C, 30 sec; 59.5 °C, 1 min; 72 °C, 2 min for 30 cycles. Primers of the miR-183/96/182 gene and gene-trap related primers.
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  • Downloaded from URL: Δ Δ Nated ALG-2 GF122) and GST-ALG-2 GF122 Was Cium.Uhnres.Utoronto.Ca/Vgm

    Downloaded from URL: Δ Δ Nated ALG-2 GF122) and GST-ALG-2 GF122 Was Cium.Uhnres.Utoronto.Ca/Vgm

    Inuzuka et al. BMC Structural Biology 2010, 10:25 http://www.biomedcentral.com/1472-6807/10/25 RESEARCH ARTICLE Open Access Molecular basis for defect in Alix-binding by alternatively spliced isoform of ALG-2 (ALG-2ΔGF122) and structural roles of F122 in target recognition Tatsutoshi Inuzuka1, Hironori Suzuki1,2, Masato Kawasaki2, Hideki Shibata1, Soichi Wakatsuki2, Masatoshi Maki1* Abstract Background: ALG-2 (a gene product of PDCD6) belongs to the penta-EF-hand (PEF) protein family and Ca2 +-dependently interacts with various intracellular proteins including mammalian Alix, an adaptor protein in the ESCRT system. Our previous X-ray crystal structural analyses revealed that binding of Ca2+ to EF3 enables the side chain of R125 to move enough to make a primary hydrophobic pocket (Pocket 1) accessible to a short fragment of Alix. The side chain of F122, facing a secondary hydrophobic pocket (Pocket 2), interacts with the Alix peptide. An alternatively spliced shorter isoform, designated ALG-2ΔGF122, lacks Gly121Phe122 and does not bind Alix, but the structural basis of the incompetence has remained to be elucidated. Results: We solved the X-ray crystal structure of the PEF domain of ALG-2ΔGF122 in the Ca2+-bound form and compared it with that of ALG-2. Deletion of the two residues shortened a-helix 5 (a5) and changed the configuration of the R125 side chain so that it partially blocked Pocket 1. A wall created by the main chain of 121- GFG-123 and facing the two pockets was destroyed. Surprisingly, however, substitution of F122 with Ala or Gly, but not with Trp, increased the Alix-binding capacity in binding assays.