The Candidate Histocompatibility Locus of a Basal Chordate Encodes Two Highly Polymorphic Proteins

<div><p>The basal chordate <i>Botryllus schlosseri</i> undergoes a natural transplantation reaction governed by a single, highly polymorphic locus called the <i>fuhc</i>. Our initial characterization of this locus suggested it encoded a single gene alternatively spliced into two transcripts: a 555 amino acid–secreted form containing the first half of the gene, and a full-length, 1008 amino acid transmembrane form, with polymorphisms throughout the ectodomain determining outcome. We have now found that the locus encodes two highly polymorphic genes which are separated by a 227 bp intergenic region: first, the secreted form as previously described, and a second gene encoding a 531 amino acid membrane-bound gene containing three extracellular immunoglobulin domains. While northern blotting revealed only these two mRNAs, both PCR and mRNA-seq detect a single capped and polyadenylated transcript that encodes processed forms of both genes linked by the intergenic region, as well as other transcripts in which exons of the two genes are spliced together. These results might suggest that the two genes are expressed as an operon, during which both genes are co-transcribed and then trans-spliced into two separate messages. This type of transcriptional regulation has been described in tunicates previously; however, the membrane-bound gene does not encode a typical Splice Leader (SL) sequence at the 5′ terminus that usually accompanies trans-splicing. Thus, the presence of stable transcripts encoding both genes may suggest a novel mechanism of regulation, or conversely may be rare but stable transcripts in which the two mRNAs are linked due to a small amount of read-through by RNA polymerase. Both genes are highly polymorphic and co-expressed on tissues involved in histocompatibility. In addition, polymorphisms on both genes correlate with outcome, although we have found a case in which it appears that the secreted form may be major allorecognition determinant.</p></div

A stable transcript encoding both <i>fuhc</i> sec and <i>fuhc</i> tm can be detected by mRNA-seq and RT-PCR.

<p><i>Top</i> Alignment of Illumina mRNA-seq reads from a single genotype to the <i>fuhc</i> genomic region spanning from <i>fuhc</i>^sec exon 17 to the intron following <i>fuhc</i>^tm exon 2. Multiple sequences span the 227bp region between the genes (arrow in center), however, there are no aligned sequences in introns between <i>fuhc</i>^tm exons 1 and 2, or 2 and 3 (right two arrows). <i>Bottom</i> RT-PCR on cDNA isolated from 4 wild-type individuals. The right 4 lanes are from the exon 2 of <i>fuhc</i>^sec to the intergenic region, the left 4 lanes are from the intergenic region to the exon 13 of <i>fuhc</i>^tm. Only single products are amplified using these primers.</p

the candidate <i>fuhc</i> encodes two tightly linked genes.

<p><b>A.</b> Schematic of the region between <i>fuhc</i>^sec and <i>fuhc</i>^tm. The new 86 bp region is shown in hatched bars, and encodes a new start codon, and a portion of exon 2 of the tm form (exon number in parentheses are the original exon designation). Black arrows show correct intron/exon boundaries, the red arrow outlines the start of the first exon of <i>fuhc</i>^tm, which does not have the correct intron/exon boundary sequence. <b>B.</b> Illustration of the structure of <i>fuhc</i>^sec (left) and <i>fuhc</i>^tm (center) and original full-length protein (right) as predicted by ELM (17). The blue line on <i>fuhc</i>^tm (center) represents the new coding sequence <b>C.</b> Northern blot of total RNA hybridized with probes specific for <i>fuhc</i>^sec (left) and <i>fuhc</i>^tm (rt). Left: lanes 1 and 2 are RNA isolated from two wild-type genotypes, – indicates no RNA. Right. Lane 1 is a positive control using RNA isolated from 293T cells expressing a construct encoding exons 1–27 of the original full-length <i>fuhc</i>/EGFP fusion protein (expressed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065980#pone-0065980-g001" target="_blank">Figure 1D</a>). Lane 2 is a separate genotype.</p

Segregation of the A1 and A2 intron alleles.

<p>Shown are individuals from our mariculture pedigree which were phenotyped as a <i>fuhcA</i> homozygote by fusibility assays, and their corresponding genotype by the STS1 PCR-RFLP marker, which detects polymorphisms in an intron between exons 4 and 5 of the transmembrane gene. Yw1023 was a parental line in our main mapping cross. Pedigree placements of these genotypes in these <i>fuhc</i> inbred lines can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065980#pone.0065980-DeTomaso4" target="_blank">[23]</a>.</p

Original characterization of the candidate <i>fuhc</i>.

<p><b>A.</b> Top shows an illustration of the genomic structure of the <i>fuhc</i> locus using our original exon numbering. Middle illustration represents the original full-length cDNA which encoded exons 1–14 spliced to 18–31. The secreted cDNA (exons 1–17) is shown on the bottom. Primers used in panels B and C are represented by arrows. Gray arrows represent primers which spanned the exon 14/18 boundary (see text). The red arrow was used for 5′ RACE shown in panel B. <b>B.</b> Initial RACE results using original exon numbering. Sense primers for 3′ RACE: lane 1, sense primer in exon 20; lane 2, sense primer in exon 9. For 5′ RACE, Lane 3 is an amplicon using an antisense primer in exon 10, In lane 4 the antisense primer was in exon 21 (red arrow in panel A; middle). Sequence from the RACE product in Lane 4 (white arrow) was the original evidence for alternative splicing. It should have amplified a 373 bp fragment (to the front of the <i>fuhc</i>^tm mRNA), but instead amplified a product that spanned the two genes. <b>C.</b> Amplification of the full-length gene from primers in exon 2 and exon 30 from cDNA isolated from 4 wild-type genotypes. The expected 3.2 Kb full-length product is amplified (asterisks), as well as several smaller products which encoded splice variants missing various exons but still linking the two genes (arrows). <b>D.</b> Retroviral expression of exons 1–27 of the original transmembrane gene fused to EGFP in mammalian 293T cells. The protein (green) is targeted to the plasma membrane, suggesting it is folding correctly and transiting the secretory pathway normally. Nuclei are counterstained with DAPI.</p

<i>fuhc</i>^sec and <i>fuhc</i>^tm alleles correlate with fusion/rejection outcomes in wild-type individuals.

<p>Wild-type individuals were reared in the lab, grown and subcloned into multiple naïve pieces. One group of subclones from each genotype was paired and fusion/rejection outcomes visually assessed. Naïve subclones of the same individuals were isolated for one week to ensure they were not pregnant, then sacrificed for RNA isolation and subsequent cloning and sequencing of the both alleles. cDNA and protein alleles are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065980#pone.0065980.s001" target="_blank">Figure S1</a>.</p

qPCR expression levels of the <i>fuhc</i> in ampullae and blood.

<p>Expression was measured using a ddCt methodology with the housekeeping gene ef1α as the standard. Values and SEM are shown for 5 biological replicates from a single genotype.</p

Localization of <i>fuhc</i>^sec and <i>fuhc</i>^tm using whole mount double-labeled in situ hybridization.

<p>Probes specific for <i>fuhc</i>^sec and <i>fuhc</i>^tm were hybridized to whole mount preparations as described in the methods, and results are shown for <i>fuhc</i>^tm (green) and <i>fuhc</i>^sec (red) as well as the merged image. Both genes are expressed in the epithelium of the ampullae as well as a subset of blood cells. Note that while detection of <i>fuhc</i>^sec is repeatable, detection of <i>fuhc</i>^tm was variable, in that we saw either this staining pattern, or no detection of <i>fuhc</i>^tm mRNA at all. This is most likely due to low levels of expression (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065980#pone-0065980-t001" target="_blank">Table 1</a>).</p

Example of the Type of Data Available from the Online Database (http://www.bruinfly.ucla.edu)

<p>Example of the Type of Data Available from the Online Database (<a href="http://www.bruinfly.ucla.edu" target="_blank">http://www.bruinfly.ucla.edu</a>)</p

Discovery-Based Science Education: Functional Genomic Dissection in Drosophila by Undergraduate Researchers

Discovery-Based Science Education: Functional Genomic Dissection in Drosophila by Undergraduate Researcher