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.
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 genetics growth & development MeSH
- Transcription, Genetic * MeSH
- RNA, Messenger chemistry metabolism MeSH
- Nucleotide Motifs MeSH
- Oocytes metabolism MeSH
- Protein Biosynthesis * MeSH
- Gene Expression Regulation * MeSH
- Mammals genetics MeSH
- TOR Serine-Threonine Kinases antagonists & inhibitors metabolism MeSH
- Trans-Splicing * MeSH
- Urochordata genetics MeSH
- Animals MeSH
- Check Tag
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article 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.
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
- Chromosomes chemistry MeSH
- Euglenozoa genetics MeSH
- Transcription, Genetic MeSH
- Genome, Mitochondrial MeSH
- DNA, Mitochondrial chemistry MeSH
- Genes, Mitochondrial MeSH
- Mitochondrial Proteins genetics metabolism MeSH
- Mitochondria genetics metabolism MeSH
- Molecular Sequence Data MeSH
- Sequence Analysis, DNA MeSH
- Trans-Splicing MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't 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.
- MeSH
- Molecular Sequence Annotation MeSH
- Chlorophyta enzymology genetics MeSH
- DNA, Chloroplast genetics MeSH
- DNA, Plant MeSH
- Euglenida genetics MeSH
- Exons genetics MeSH
- Phylogeny * MeSH
- Genetic Heterogeneity MeSH
- Genome, Mitochondrial genetics MeSH
- Introns genetics MeSH
- RNA, Messenger genetics MeSH
- DNA, Mitochondrial genetics MeSH
- Mitochondrial Proteins classification genetics MeSH
- Electron Transport Complex IV genetics MeSH
- RNA, Ribosomal, 18S genetics MeSH
- Plant Proteins classification genetics MeSH
- Plants genetics MeSH
- Base Sequence MeSH
- Trans-Splicing MeSH
- Publication type
- Journal Article MeSH
Mutations in the C1 inhibitor (C1INH) encoding gene, SERPING1, are associated with hereditary angioedema (HAE) which manifests as recurrent submucosal and subcutaneous edema episodes. The major C1INH function is the complement system inhibition, preventing its spontaneous activation. The presented study is focused on SERPING1 exon 3, an alternative and extraordinarily long exon (499 bp). Endogenous expression analysis performed in the HepG2, human liver, and human peripheral blood cells revealed several exon 3 splicing variants alongside exon inclusion: a highly prevalent exon skipping variant and less frequent +38 and -15 variants with alternative 3' splice sites (ss) located 38 and 15 nucleotides downstream and upstream from the authentic 3' ss, respectively. An exon skipping variant introducing a premature stop codon, represented nearly one third of all splicing variants and surprisingly appeared not to be degraded by NMD. The alternative -15 3' ss was used to a small extent, although predicted to be extremely weak. Its use was shown to be independent of its strength and highly sensitive to any changes in the surrounding sequence. -15 3' ss seems to be co-regulated with the authentic 3' ss, whose use is dependent mainly on its strength and less on the presence of intronic regulatory motifs. Subtle SERPING1 exon 3 splicing regulation can contribute to overall C1INH plasma levels and HAE pathogenesis.
- MeSH
- Alternative Splicing genetics MeSH
- Cell Nucleus genetics MeSH
- Hep G2 Cells MeSH
- Exons genetics MeSH
- Complement C1 Inhibitor Protein genetics MeSH
- Humans MeSH
- RNA, Small Interfering metabolism MeSH
- RNA Splice Sites genetics MeSH
- Mutation genetics MeSH
- Nonsense Mediated mRNA Decay genetics MeSH
- Base Sequence MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The nonsense-mediated mRNA decay (NMD) pathway rapidly detects and degrades mRNA containing premature termination codons (PTCs). UP-frameshift 1 (UPF1), the master regulator of the NMD process, has two alternatively-spliced isoforms; one carries 353-GNEDLVIIWLR-363 insertion in the 'regulatory loop (involved in mRNA binding)'. Such insertion can induce catalytic and/or ATPase activity, as determined experimentally; however, the kinetics and molecular level information are not fully understood. Herein, applying all-atom molecular dynamics, we probe the binding specificity of UPF1 with different GC- and AU-rich mRNA motifs and the influence of insertion to the viable control over UPF1 catalytic activity. Our results indicate two distinct conformations between 1B and RecA2 domains of UPF1: 'open (isoform_2; without insertion)' and 'closed (isoform_1; with insertion)'. These structural movements correspond to an important stacking pattern in mRNA motifs, i.e., absence of stack formation in mRNA, with UPF1 isoform_2 results in the 'open conformation'. Particularly, for UPF1 isoform_1, the increased distance between 1B and RecA2 domains has resulted in reducing the mRNA-UPF1 interactions. Lower fluctuating GC-rich mRNA motifs have better binding with UPF1, compared with AU-rich sequences. Except CCUGGGG, all other GC-rich motifs formed a 4-stack pattern with UPF1. High occupancy R363, D364, T627, and G862 residues were common binding GC-rich motifs, as were R363, N535, and T627 for the AU-rich motifs. The GC-rich motifs behave distinctly when bound to either of the isoforms; lower stability was observed with UPF1 isoform_2. The cancer-associated UPF1 variants (P533L/T and A839T) resulted in decreased protein-mRNA binding efficiency. Lack of mRNA stacking poses in the UPF1P533T system significantly decreased UPF1-mRNA binding efficiency and increased distance between 1B-RecA2. These novel findings can serve to further inform NMD-associated mechanistic and kinetic studies.
- MeSH
- Alternative Splicing * MeSH
- Phosphorylation MeSH
- Humans MeSH
- RNA, Messenger genetics metabolism MeSH
- Nonsense Mediated mRNA Decay * MeSH
- Protein Isoforms MeSH
- Gene Expression Regulation * MeSH
- RNA Helicases genetics metabolism MeSH
- Trans-Activators genetics metabolism MeSH
- Protein Binding MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
p73, a member of the p53 family, exhibits activities similar to those of p53, including the ability to induce growth arrest and apoptosis. p73 influences chemotherapeutic responses in human cancer patients, in association with p53. Alternative splicing of the TP73 gene produces many p73 C- and N-terminal isoforms, which vary in their transcriptional activity towards p53-responsive promoters. In this paper, we show that the C-terminal spliced isoforms of the p73 protein differ in their DNA-binding capacity, but this is not an accurate predictor of transcriptional activity. In different p53-null cell lines, p73beta induces either mitochondrial-associated or death receptor-mediated apoptosis, and these differences are reflected in different gene expression profiles. In addition, p73 induces cell cycle arrest and p21(WAF1) expression in H1299 cells, but not in Saos-2. This data shows that TAp73 isoforms act differently depending on the tumour cell background, and have important implications for p73-mediated therapeutic responses in individual human cancer patients.
- MeSH
- Transcriptional Activation MeSH
- Alternative Splicing MeSH
- Apoptosis physiology MeSH
- Cell Line MeSH
- Cell Cycle physiology MeSH
- DNA-Binding Proteins genetics metabolism MeSH
- Financing, Organized MeSH
- Transcription, Genetic MeSH
- Nuclear Proteins genetics metabolism MeSH
- Humans MeSH
- Tumor Suppressor Proteins metabolism MeSH
- Tumor Suppressor Protein p53 genetics metabolism MeSH
- Protein Isoforms genetics metabolism MeSH
- Oligonucleotide Array Sequence Analysis MeSH
- Signal Transduction physiology MeSH
- Gene Expression Profiling MeSH
- Trans-Activators genetics metabolism MeSH
- Protein Binding MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
Bidirectional interactions between astrocytes and neurons have physiological roles in the central nervous system and an altered state or dysfunction of such interactions may be associated with neurodegenerative diseases, such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS). Astrocytes exert structural, metabolic and functional effects on neurons, which can be either neurotoxic or neuroprotective. Their neurotoxic effect is mediated via the senescence-associated secretory phenotype (SASP) involving pro-inflammatory cytokines (e.g., IL-6), while their neuroprotective effect is attributed to neurotrophic growth factors (e.g., NGF). We here demonstrate that the p53 isoforms Δ133p53 and p53β are expressed in astrocytes and regulate their toxic and protective effects on neurons. Primary human astrocytes undergoing cellular senescence upon serial passaging in vitro showed diminished expression of Δ133p53 and increased p53β, which were attributed to the autophagic degradation and the SRSF3-mediated alternative RNA splicing, respectively. Early-passage astrocytes with Δ133p53 knockdown or p53β overexpression were induced to show SASP and to exert neurotoxicity in co-culture with neurons. Restored expression of Δ133p53 in near-senescent, otherwise neurotoxic astrocytes conferred them with neuroprotective activity through repression of SASP and induction of neurotrophic growth factors. Brain tissues from AD and ALS patients possessed increased numbers of senescent astrocytes and, like senescent astrocytes in vitro, showed decreased Δ133p53 and increased p53β expression, supporting that our in vitro findings recapitulate in vivo pathology of these neurodegenerative diseases. Our finding that Δ133p53 enhances the neuroprotective function of aged and senescent astrocytes suggests that the p53 isoforms and their regulatory mechanisms are potential targets for therapeutic intervention in neurodegenerative diseases.
- MeSH
- Alternative Splicing MeSH
- Alzheimer Disease metabolism pathology MeSH
- Amyotrophic Lateral Sclerosis metabolism pathology MeSH
- Astrocytes cytology drug effects metabolism MeSH
- Autophagy drug effects MeSH
- Genetic Vectors genetics metabolism MeSH
- Interleukin-6 genetics metabolism MeSH
- Coculture Techniques MeSH
- Cells, Cultured MeSH
- Leupeptins pharmacology MeSH
- Humans MeSH
- RNA, Small Interfering metabolism MeSH
- Brain metabolism pathology MeSH
- Tumor Suppressor Protein p53 antagonists & inhibitors genetics metabolism MeSH
- Neurons cytology metabolism MeSH
- Neuroprotection physiology MeSH
- Protein Isoforms antagonists & inhibitors genetics metabolism MeSH
- RNA Interference MeSH
- Sequestosome-1 Protein antagonists & inhibitors genetics metabolism MeSH
- Serine-Arginine Splicing Factors antagonists & inhibitors genetics metabolism MeSH
- Cellular Senescence MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural MeSH
The HIV-1 protein Rev is essential for virus replication and ensures the expression of partially spliced and unspliced transcripts. We identified a ULM (UHM ligand motif) motif in the Arginine-Rich Motif (ARM) of the Rev protein. ULMs (UHM ligand motif) mediate protein interactions during spliceosome assembly by binding to UHM (U2AF homology motifs) domains. Using NMR, biophysical methods and crystallography we show that the Rev ULM binds to the UHMs of U2AF65 and SPF45. The highly conserved Trp45 in the Rev ULM is crucial for UHM binding in vitro, for Rev co-precipitation with U2AF65 in human cells and for proper processing of HIV transcripts. Thus, Rev-ULM interactions with UHM splicing factors contribute to the regulation of HIV-1 transcript processing, also at the splicing level. The Rev ULM is an example of viral mimicry of host short linear motifs that enables the virus to interfere with the host molecular machinery.
- MeSH
- Alternative Splicing genetics MeSH
- Amino Acid Motifs genetics MeSH
- Arginine genetics MeSH
- rev Gene Products, Human Immunodeficiency Virus genetics MeSH
- HIV Infections genetics virology MeSH
- HIV-1 genetics pathogenicity MeSH
- Host-Pathogen Interactions genetics MeSH
- Humans MeSH
- Gene Expression Regulation, Viral genetics MeSH
- Virus Replication genetics MeSH
- RNA Splicing Factors genetics MeSH
- Splicing Factor U2AF genetics MeSH
- Spliceosomes genetics MeSH
- Protein Binding genetics MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
