Characterization of spliced leader trans-splicing in a photosynthetic rhizarian amoeba, Paulinella micropora, and its possible role in functional gene transfer

Paulinella micropora is a rhizarian thecate amoeba, belonging to a photosynthetic Paulinella species group that has a unique organelle termed chromatophore, whose cyanobacterial origin is distinct from that of plant and algal chloroplasts. Because acquisition of the chromatophore was quite a recent event compared with that of the chloroplast ancestor, the Paulinella species are thought to be model organisms for studying the early process of primary endosymbiosis. To obtain insight into how endosymbiotically transferred genes acquire expression competence in the host nucleus, here we analyzed the 5′ end sequences of the mRNAs of P. micropora MYN1 strain with the aid of a cap-trapper cDNA library. As a result, we found that mRNAs of 27 genes, including endosymbiotically transferred genes, possessed the common 5′ end sequence of 28–33 bases that were posttranscriptionally added by spliced leader (SL) trans-splicing. We also found two subtypes of SL RNA genes encoded by the P. micropora MYN1 genome. Differing from the other SL trans-splicing organisms that usually possess poly(A)-less SL RNAs, this amoeba has polyadenylated SL RNAs. In this study, we characterize the SL trans-splicing of this unique organism and discuss the putative merits of SL trans-splicing in functional gene transfer and genome evolution

Characterization of spliced leader trans-splicing in a photosynthetic rhizarian amoeba, Paulinella micropora, and its possible role in functional gene transfer.

Paulinella micropora is a rhizarian thecate amoeba, belonging to a photosynthetic Paulinella species group that has a unique organelle termed chromatophore, whose cyanobacterial origin is distinct from that of plant and algal chloroplasts. Because acquisition of the chromatophore was quite a recent event compared with that of the chloroplast ancestor, the Paulinella species are thought to be model organisms for studying the early process of primary endosymbiosis. To obtain insight into how endosymbiotically transferred genes acquire expression competence in the host nucleus, here we analyzed the 5' end sequences of the mRNAs of P. micropora MYN1 strain with the aid of a cap-trapper cDNA library. As a result, we found that mRNAs of 27 genes, including endosymbiotically transferred genes, possessed the common 5' end sequence of 28-33 bases that were posttranscriptionally added by spliced leader (SL) trans-splicing. We also found two subtypes of SL RNA genes encoded by the P. micropora MYN1 genome. Differing from the other SL trans-splicing organisms that usually possess poly(A)-less SL RNAs, this amoeba has polyadenylated SL RNAs. In this study, we characterize the SL trans-splicing of this unique organism and discuss the putative merits of SL trans-splicing in functional gene transfer and genome evolution

Functional and taxonomical classification of SL <i>trans</i>-spliced genes.

<p><b>A</b>. Functional categorization of 27 <i>trans</i>-spliced genes of <i>P</i>. <i>micropora</i> according to COG and KOG classification [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200961#pone.0200961.ref044" target="_blank">44</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200961#pone.0200961.ref045" target="_blank">45</a>] with manual editing; a class of photosynthesis related function is added. <b>B.</b> Taxonomical classification of 27 SL <i>trans</i>-spliced genes based on the BLASTX annotation of the NCBI-nr database.</p

Characteristics of SL RNA genes in <i>P</i>. <i>micropora</i>.

<p><b>A.</b> ClustalW alignment of SL-I RNA gene candidates. pmSL1 represents the Sanger sequence of a PCR-amplified clone, and the others are <i>de novo</i> assembled contig sequences from <i>P</i>. <i>micropora</i> genomic shotgun sequencing. Transcription start sites (arrowheads) of pmSL1 were determined by 5′ end mapping of cap-trapper cDNA reads, and the cleavage sites (asterisks) were determined by 3′ RACE analysis. Canonical poly(A) signals (AATAAA) are indicated by red boxes. The regions forming the stem-loop structure in pmSL1 are represented by dashed lines. <b>B.</b> Relative levels of SL-I RNA, SL-II RNA, and calmodulin (CALM) mRNA to 18S rRNA. Bulk expression levels of SL-I and SL-II genes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200961#pone.0200961.s005" target="_blank">S2 Fig</a>) were analyzed by real-time RT-PCR using conserved sequences of the respective SL RNA gene group. <b>C.</b> Secondary structure of the conserved region of pmSL1 RNA predicted by Mfold [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200961#pone.0200961.ref046" target="_blank">46</a>]. The shaded area indicates the candidate of Sm binding site, when the consensus rule is loosened to pyrimidine rich sequence sandwiched by adenosine and guanosine.</p

Alignment of the spliced leader <i>trans</i>-spliced mRNAs of <i>P</i>. <i>micropora</i>.

<p>cDNAs contain 28–33 base common sequences at their 5′ ends. These contigs were annotated using COG and NCBI-nr database (PsbN; photosystem II PsbN, Histone H3, Rac1; Ras-related protein Rac1, Rab1; GTP-binding protein, PHF5; PHF5-domain-containing protein, Rab11; GTPase Rab11/YPT3, CALM; Calmodulin, RRP43; Exosome complex exonuclease RRP43, CypA; Cyclophilin, LASP1; LIM and SH3 domain protein, GDI1; Rab GDP dissociation inhibitor alpha, SARS; seryl-tRNA synthetase, ATOX1; copper transport protein ATOX1, TRAPPC3; trafficking protein particle complex subunit 3, Rsf1; RNA-binding protein Rsf1). The common sequence is shaded, with the UUU triplet and 3′ terminal G indicated by the gray and black arrows, respectively.</p

Structure of the calmodulin gene of <i>P</i>. <i>micropora</i>.

<p><b>A.</b> Schematic model of the calmodulin gene and its mRNA. Primers used for PCR- and RT-PCR analyses are shown by small arrows a, b, c, and d. The outron and spliced leader are shown as hashed and black boxes, respectively. The thick arrow indicates the transcription start site (TSS). <b>B.</b> Genomic sequence of the calmodulin gene, with the ATG initiation codon boxed. The junction of the outron and exon is indicated by the vertical bar. TSS and the pyrimidine rich region are indicated by the thick arrow and dots, respectively. The underlines show the position of primer b (consisting of two nested primers). <b>C.</b> PCR profiles of the calmodulin gene using genomic and cDNA templates.</p