Kinetoplastids are a clade of eukaryotic protozoans that include human parasitic pathogens like trypanosomes and Leishmania species. In these organisms, protein-coding genes are transcribed as polycistronic pre-mRNAs, which need to be processed by the coupled action of trans-splicing and polyadenylation to yield monogenic mature mRNAs. During trans-splicing, a universal RNA sequence, the spliced leader RNA (SL RNA) mini-exon, is added to the 5'-end of each mRNA. The 5'-end of this mini-exon carries a hypermethylated cap structure and is bound by a trypanosomatid-specific cap-binding complex (CBC). The function of three of the kinetoplastid CBC subunits is unknown, but an essential role in cap-binding and trans-splicing has been suggested. Here, we report cryo-EM structures that reveal the molecular architecture of the Trypanosoma brucei CBC (TbCBC) complex. We find that TbCBC interacts with two distinct features of the SL RNA. The TbCBP20 subunit interacts with the m7G cap while TbCBP66 recognizes double-stranded portions of the SL RNA. Our findings pave the way for future research on mRNA maturation in kinetoplastids. Moreover, the observed structural similarities and differences between TbCBC and the mammalian cap-binding complex will be crucial for considering the potential of TbCBC as a target for anti-trypanosomatid drug development.
- MeSH
- elektronová kryomikroskopie MeSH
- molekulární modely MeSH
- protozoální proteiny * metabolismus chemie genetika MeSH
- RNA čepičky * metabolismus MeSH
- RNA protozoální * metabolismus genetika chemie MeSH
- RNA se sestřihovou vedoucí sekvencí * metabolismus chemie genetika MeSH
- trans-splicing MeSH
- Trypanosoma brucei brucei * metabolismus genetika MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- protozoální proteiny * MeSH
- RNA čepičky * MeSH
- RNA protozoální * MeSH
- RNA se sestřihovou vedoucí sekvencí * MeSH
BACKGROUND: In trypanosomatids, a group of unicellular eukaryotes that includes numerous important human parasites, cis-splicing has been previously reported for only two genes: a poly(A) polymerase and an RNA helicase. Conversely, trans-splicing, which involves the attachment of a spliced leader sequence, is observed for nearly every protein-coding transcript. So far, our understanding of splicing in this protistan group has stemmed from the analysis of only a few medically relevant species. In this study, we used an extensive dataset encompassing all described trypanosomatid genera to investigate the distribution of intron-containing genes and the evolution of splice sites. RESULTS: We identified a new conserved intron-containing gene encoding an RNA-binding protein that is universally present in Kinetoplastea. We show that Perkinsela sp., a kinetoplastid endosymbiont of Amoebozoa, represents the first eukaryote completely devoid of cis-splicing, yet still preserving trans-splicing. We also provided evidence for reverse transcriptase-mediated intron loss in Kinetoplastea, extensive conservation of 5' splice sites, and the presence of non-coding RNAs within a subset of retained trypanosomatid introns. CONCLUSIONS: All three intron-containing genes identified in Kinetoplastea encode RNA-interacting proteins, with a potential to fine-tune the expression of multiple genes, thus challenging the perception of cis-splicing in these protists as a mere evolutionary relic. We suggest that there is a selective pressure to retain cis-splicing in trypanosomatids and that this is likely associated with overall control of mRNA processing. Our study provides new insights into the evolution of introns and, consequently, the regulation of gene expression in eukaryotes.
- Klíčová slova
- Introns, Kinetoplastea, Poly(A) polymerase, RNA helicase, RNA-binding protein, Splicing, Trypanosomatidae,
- MeSH
- fylogeneze MeSH
- introny * genetika MeSH
- Kinetoplastida genetika MeSH
- molekulární evoluce MeSH
- protozoální geny genetika MeSH
- protozoální proteiny genetika MeSH
- trans-splicing * genetika MeSH
- Trypanosomatina genetika MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- protozoální proteiny MeSH
Giardia lambliacauses giardiasis, one of the most common human infectious diseases globally. Previous studies from our lab have shown that hsp90 gene ofGiardia is split into two halves, namely hspN and hspC. The independent pre-mRNAs of these split genes join by trans-splicing, producing a full-length Hsp90 (FlHsp90) mRNA. Genetic manipulation of the participating genes is necessary to understand the mechanism and significance of such trans-splicing based expression of Hsp90. In this study, we have performed transfection based exogenous expression of hspN and/or hspC in G. lamblia. We electroporated a plasmid containing the Avi-tagged hspN component of Hsp90 and examined its fate in G. lamblia. We show that the exogenously expressed hspN RNA gets trans-spliced to endogenously expressed hspC RNA, giving rise to a hybrid-FlHsp90. We highlight the importance of cis-elements in this trans-splicing reaction through mutational analysis. The episomal plasmid carrying deletions in the intronic region of hspN, showed inhibition of the trans-splicing reaction.Additionally, exogenous hspC RNA also followed the same fate as of exogenous hspN, while upon co-transfection with episomal hspN, they underwent trans-splicing with each other. Using eGFP as a test protein, we have shown that intronic sequences of hsp90 gene can guide trans-splicing mediated repair of any associated exonic sequences. Our study provides in vivo validation of Hsp90 trans-splicing, showing crucial role of cis-elements and importantly highlights the potential of hsp90 intronic sequences to function as a minimal splicing tool.
- Klíčová slova
- Gene expression, Giardia lamblia, Hsp90, RNA splicing, Transfection,
- MeSH
- Giardia lamblia * genetika MeSH
- introny genetika MeSH
- prekurzory RNA genetika MeSH
- proteiny tepelného šoku HSP90 * genetika MeSH
- protozoální proteiny * genetika MeSH
- trans-splicing * genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- prekurzory RNA MeSH
- proteiny tepelného šoku HSP90 * MeSH
- protozoální proteiny * MeSH
BACKGROUND: In phylogenetically diverse organisms, the 5' ends of a subset of mRNAs are trans-spliced with a spliced leader (SL) RNA. The functions of SL trans-splicing, however, remain largely enigmatic. RESULTS: We quantified translation genome-wide in the marine chordate, Oikopleura dioica, under inhibition of mTOR, a central growth regulator. Translation of trans-spliced TOP mRNAs was suppressed, consistent with a role of the SL sequence in nutrient-dependent translational control of growth-related mRNAs. Under crowded, nutrient-limiting conditions, O. dioica continued to filter-feed, but arrested growth until favorable conditions returned. Upon release from unfavorable conditions, initial recovery was independent of nutrient-responsive, trans-spliced genes, suggesting animal density sensing as a first trigger for resumption of development. CONCLUSION: Our results are consistent with a proposed role of trans-splicing in the coordinated translational down-regulation of nutrient-responsive genes under growth-limiting conditions.
- MeSH
- Caenorhabditis elegans genetika růst a vývoj MeSH
- genetická transkripce * MeSH
- messenger RNA chemie metabolismus MeSH
- nukleotidové motivy MeSH
- oocyty metabolismus MeSH
- proteosyntéza * MeSH
- regulace genové exprese * MeSH
- savci genetika MeSH
- TOR serin-threoninkinasy antagonisté a inhibitory metabolismus MeSH
- trans-splicing * MeSH
- Urochordata genetika MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- messenger RNA MeSH
- TOR serin-threoninkinasy MeSH
The mitochondrial DNA of diplonemid and kinetoplastid protists is known for its suite of bizarre features, including the presence of concatenated circular molecules, extensive trans-splicing and various forms of RNA editing. Here we report on the existence of another remarkable characteristic: hyper-inflated DNA content. We estimated the total amount of mitochondrial DNA in four kinetoplastid species (Trypanosoma brucei, Trypanoplasma borreli, Cryptobia helicis, and Perkinsela sp.) and the diplonemid Diplonema papillatum. Staining with 4',6-diamidino-2-phenylindole and RedDot1 followed by color deconvolution and quantification revealed massive inflation in the total amount of DNA in their organelles. This was further confirmed by electron microscopy. The most extreme case is the ∼260 Mbp of DNA in the mitochondrion of Diplonema, which greatly exceeds that in its nucleus; this is, to our knowledge, the largest amount of DNA described in any organelle. Perkinsela sp. has a total mitochondrial DNA content ~6.6× greater than its nuclear genome. This mass of DNA occupies most of the volume of the Perkinsela cell, despite the fact that it contains only six protein-coding genes. Why so much DNA? We propose that these bloated mitochondrial DNAs accumulated by a ratchet-like process. Despite their excessive nature, the synthesis and maintenance of these mtDNAs must incur a relatively low cost, considering that diplonemids are one of the most ubiquitous and speciose protist groups in the ocean. © 2018 IUBMB Life, 70(12):1267-1274, 2018.
- Klíčová slova
- DNA content, kinetoplast DNA, mitochondrial DNA, protist,
- MeSH
- Euglenozoa genetika MeSH
- fylogeneze MeSH
- Kinetoplastida genetika MeSH
- mitochondriální DNA genetika izolace a purifikace ultrastruktura MeSH
- mitochondrie genetika MeSH
- trans-splicing genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- mitochondriální DNA MeSH
Trans-splicing is a process by which 5'- and 3'-ends of two pre-RNA molecules transcribed from different sites of the genome can be joined together to form a single RNA molecule. The spliced leader (SL) trans-splicing is mediated by the spliceosome and it allows the replacement of 5'-end of pre-mRNA by 5'(SL)-end of SL-RNA. This form of splicing has been observed in many phylogenetically unrelated eukaryotes. Either the SL trans-splicing (SLTS) originated in the last eukaryotic common ancestor (LECA) (or even earlier) and it was lost in most eukaryotic lineages, or this mechanism of RNA processing evolved several times independently in various unrelated eukaryotic taxa. The bioinformatic comparisons of SL-RNAs from various eukaryotic taxonomic groups have revealed the similarities of secondary structures of most SL-RNAs and a relative conservation of their splice sites (SSs) and Sm-binding sites (SmBSs). We propose that such structural and functional similarities of SL-RNAs are unlikely to have evolved repeatedly many times. Hence, we favor the scenario of an early evolutionary origin for the SLTS and multiple losses of SL-RNAs in various eukaryotic lineages.
- Klíčová slova
- Intron, RNA secondary structure, SL-RNA, Sm-binding site, Spliceosome,
- MeSH
- Eukaryota genetika metabolismus MeSH
- fylogeneze MeSH
- molekulární evoluce * MeSH
- prekurzory RNA metabolismus MeSH
- RNA se sestřihovou vedoucí sekvencí genetika metabolismus MeSH
- trans-splicing * MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
- Názvy látek
- prekurzory RNA MeSH
- RNA se sestřihovou vedoucí sekvencí MeSH
Prasinophytes are a paraphyletic assemblage of nine heterogeneous lineages in the Chlorophyta clade of Archaeplastida. Until now, seven complete mitochondrial genomes have been sequenced from four prasinophyte lineages. Here, we report the mitochondrial genome of Pyramimonas parkeae, the first representative of the prasinophyte clade I. The circular-mapping molecule is 43,294 bp long, AT rich (68.8%), very compact and it comprises two 6,671 bp long inverted repeat regions. The gene content is slightly smaller than the gene-richest prasinophyte mitochondrial genomes. The single identified intron is located in the cytochrome c oxidase subunit 1 gene (cox1). Interestingly, two exons of cox1 are encoded on the same strand of DNA in the reverse order and the mature mRNA is formed by trans-splicing. The phylogenetic analysis using the data set of 6,037 positions assembled from 34 mtDNA-encoded proteins of 48 green algae and plants is not in compliance with the branching order of prasinophyte clades revealed on the basis of 18S rRNA genes and cpDNA-encoded proteins. However, the phylogenetic analyses based on all three genomic elements support the sister position of prasinophyte clades Pyramimonadales and Mamiellales.
- Klíčová slova
- cox1, intron, inverted repeats, phylogeny, trans-splicing,
- MeSH
- anotace sekvence MeSH
- Chlorophyta enzymologie genetika MeSH
- DNA chloroplastová genetika MeSH
- DNA rostlinná MeSH
- Euglenida genetika MeSH
- exony genetika MeSH
- fylogeneze * MeSH
- genetická heterogenita MeSH
- genom mitochondriální genetika MeSH
- introny genetika MeSH
- messenger RNA genetika MeSH
- mitochondriální DNA genetika MeSH
- mitochondriální proteiny klasifikace genetika MeSH
- respirační komplex IV genetika MeSH
- RNA ribozomální 18S genetika MeSH
- rostlinné proteiny klasifikace genetika MeSH
- rostliny genetika MeSH
- sekvence nukleotidů MeSH
- trans-splicing MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA chloroplastová MeSH
- DNA rostlinná MeSH
- messenger RNA MeSH
- mitochondriální DNA MeSH
- mitochondriální proteiny MeSH
- respirační komplex IV MeSH
- RNA ribozomální 18S MeSH
- rostlinné proteiny MeSH
Arguably, the most bizarre mitochondrial DNA (mtDNA) is that of the euglenozoan eukaryote Diplonema papillatum. The genome consists of numerous small circular chromosomes none of which appears to encode a complete gene. For instance, the cox1 coding sequence is spread out over nine different chromosomes in non-overlapping pieces (modules), which are transcribed separately and joined to a contiguous mRNA by trans-splicing. Here, we examine how many genes are encoded by Diplonema mtDNA and whether all are fragmented and their transcripts trans-spliced. Module identification is challenging due to the sequence divergence of Diplonema mitochondrial genes. By employing most sensitive protein profile search algorithms and comparing genomic with cDNA sequence, we recognize a total of 11 typical mitochondrial genes. The 10 protein-coding genes are systematically chopped up into three to 12 modules of 60-350 bp length. The corresponding mRNAs are all trans-spliced. Identification of ribosomal RNAs is most difficult. So far, we only detect the 3'-module of the large subunit ribosomal RNA (rRNA); it does not trans-splice with other pieces. The small subunit rRNA gene remains elusive. Our results open new intriguing questions about the biochemistry and evolution of mitochondrial trans-splicing in Diplonema.
- MeSH
- chromozomy chemie MeSH
- Euglenozoa genetika MeSH
- genetická transkripce MeSH
- genom mitochondriální * MeSH
- mitochondriální DNA chemie MeSH
- mitochondriální geny * MeSH
- mitochondriální proteiny genetika metabolismus MeSH
- mitochondrie genetika metabolismus MeSH
- molekulární sekvence - údaje MeSH
- sekvenční analýza DNA MeSH
- trans-splicing * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- mitochondriální DNA MeSH
- mitochondriální proteiny MeSH
