Evolution of variable lymphocyte receptor B antibody loci in jawless vertebrates

Three types of variable lymphocyte receptor (VLR) genes, VLRA, VLRB, and VLRC, encode antigen recognition receptors in the extant jawless vertebrates, lampreys and hagfish. The somatically diversified repertoires of these VLRs are generated by serial stepwise copying of leucine-rich repeat (LRR) sequences into an incomplete germline VLR gene. Lymphocytes that express VLRA or VLRC are T cell–like, while VLRB-expressing cells are B cell–like. Here, we analyze the composition of the VLRB locus in different jawless vertebrates to elucidate its configuration and evolutionary modification. The incomplete germline VLRB genes of two hagfish species contain short noncoding intervening sequences, whereas germline VLRB genes in six representative lamprey species have much longer intervening sequences that exhibit notable genomic variation. Genomic clusters of potential LRR cassette donors, fragments of which are copied to complete VLRB gene assembly, are identified in Japanese lamprey and sea lamprey. In the sea lamprey, 428 LRR cassettes are located in five clusters spread over a total of 1.7 Mbp of chromosomal DNA. Preferential usage of the different donor cassettes for VLRB assemblage is characterized in our analysis, which reveals evolutionary modifications of the lamprey VLRB genes, elucidates the organization of the complex VLRB locus, and provides a comprehensive catalog of donor VLRB cassettes in sea lamprey and Japanese lamprey.

Internally Symmetrical Stwintrons and Related Canonical Introns in Hypoxylaceae Species

Spliceosomal introns are pervasive in eukaryotes. Intron gains and losses have occurred throughout evolution, but the origin of new introns is unclear. Stwintrons are complex intervening sequences where one of the sequence elements (5′-donor, lariat branch point element or 3′-acceptor) necessary for excision of a U2 intron (external intron) is itself interrupted by a second (internal) U2 intron. In Hypoxylaceae, a family of endophytic fungi, we uncovered scores of donor-disrupted stwintrons with striking sequence similarity among themselves and also with canonical introns. Intron–exon structure comparisons suggest that these stwintrons have proliferated within diverging taxa but also give rise to proliferating canonical introns in some genomes. The proliferated (stw)introns have integrated seamlessly at novel gene positions. The recently proliferated (stw)introns appear to originate from a conserved ancestral stwintron characterised by terminal inverted repeats (45–55 nucleotides), a highly symmetrical structure that may allow the formation of a double-stranded intron RNA molecule. No short tandem duplications flank the putatively inserted intervening sequences, which excludes a DNA transposition-based mechanism of proliferation. It is tempting to suggest that this highly symmetrical structure may have a role in intron proliferation by (an)other mechanism(s).

Mutations in Drosophila tRNA processing factors cause phenotypes similar to Pontocerebellar Hypoplasia

Mature tRNAs are generated by multiple RNA processing events, which can include the excision of intervening sequences. The tRNA splicing endonuclease (TSEN) complex is responsible for cleaving these intron-containing pre-tRNA transcripts. In humans, TSEN copurifies with CLP1, an RNA kinase. Despite extensive work on CLP1, its in vivo connection to tRNA splicing remains unclear. Interestingly, mutations in CLP1 or TSEN genes cause neurological diseases in humans that are collectively termed Pontocerebellar Hypoplasia (PCH). In mice, loss of Clp1 kinase activity results in premature death, microcephaly and progressive loss of motor function. To determine if similar phenotypes are observed in Drosophila, we characterized mutations in crowded-by-cid (cbc), the CLP1 ortholog, as well as in the fly ortholog of human TSEN54. Analyses of organismal viability, larval locomotion and brain size revealed that mutations in both cbc and Tsen54 phenocopy those in mammals in several details. In addition to an overall reduction in brain lobe size, we also found increased cell death in mutant larval brains. Ubiquitous or tissue-specific knockdown of cbc in neurons and muscles reduced viability and locomotor function. These findings indicate that we can successfully model PCH in a genetically-tractable invertebrate.

Massive colonization of protein-coding exons by selfish genetic elements in Paramecium germline genomes

Ciliates are unicellular eukaryotes with both a germline genome and a somatic genome in the same cytoplasm. The macronucleus (MAC), responsible for gene expression, is not sexually transmitted but develops from a copy of the micronucleus (MIC) at each sexual generation. In the MIC genome of Paramecium tetraurelia, genes are interrupted by tens of thousands of unique intervening sequences, called Internal Eliminated Sequences (IESs), that have to be precisely excised during the development of the new MAC to restore functional genes. To understand the evolutionary origin of this peculiar genomic architecture, we sequenced the MIC genomes of nine Paramecium species (from ~100 Mb in P. aurelia species to > 1.5 Gb in P. caudatum). We detected several waves of IES gains, both in ancestral and in more recent lineages. Remarkably, we identified 24 families of mobile IESs that generated tens to thousands of new copies. The most active families show the signature of horizontal transfer. These examples illustrate how mobile elements can account for the massive proliferation of IESs in the germline genomes of Paramecium, both in non-coding regions and within exons. We also provide evidence that IESs represent a substantial burden for their host, presumably because of excision errors. Interestingly, we observe that IES excision pathways vary according to the age of IESs, and that older IESs tend to be more efficiently excised. This suggests that once fixed in the genome, the presence of IESs imposes a selective pressure on their host, both in cis (on the excision signals of each IES) and in trans (on the cellular excision machinery), to ensure efficient and precise removal. Finally, we identified 69 IESs that are under strong purifying selection across the P. aurelia clade, which indicates that a small fraction of IESs provide a function beneficial for their host. All these features highlight the major role played by selfish elements in shaping the complexity of gene expression processes and in driving genome architecture.

Complex intron generation in the yeast genus Lipomyces

In primary transcripts of eukaryotic nuclear genes, coding sequences are often interrupted by U2-type introns. Such intervening sequences can constitute complex introns excised by consecutive splicing reactions. The origin of spliceosomal introns is a vexing problem. Sequence variation existent across fungal taxa provides means to study their structure and evolution. In one class of complex introns called [D] stwintrons, an (internal) U2 intron is nested within the 5'-donor element of another (external) U2 intron. In the gene for a reticulon-like protein in species of the ascomycete yeast genus Lipomyces, the most 5' terminal intron position is occupied by one of three complex intervening sequences consistent of differently nested U2 intron units, as demonstrated in L. lipofer, L. suomiensis, and L. starkeyi. In L. starkeyi, the donor elements of the constituent introns are abutting and the complex intervening sequence can be excised alternatively either with one standard splicing reaction or, as a [D] stwintron, by two consecutive reactions. Our work suggests how [D] stwintrons could emerge by the appearance of new functional splice sites within an extant intron. The stepwise stwintronisation mechanism may involve duplication of the functional intron donor element of the ancestor intron.

Downregulation of the essential Rrp44 ribonuclease causes extensive ultra-structure cell modifications in Trypanosoma brucei

Rrp44 is a conserved eukaryotic protein showing endonuclease and exoribonuclease activity that plays essential functions in RNA maturation and degradation. In Trypanosoma brucei, depletion of Rrp44 (TbRrp44) blocks processing of large subunit ribosomal RNA precursors, leading to disruption of ribosome synthesis and inhibition of cell proliferation. We used a combination of molecular and chemical markers to investigate the fate of T. brucei cells upon knockdown of TbRrp44. In addition, synchrotron deep UV microscopy and cryo soft X-ray tomography were used to investigate cell morphology and ultra-structure modifications. Downregulation of TbRrp44 results in induction of autophagy, inactivation of mitochondria and expansion of acidic and lysosome-derived vacuoles in parallel with enlargement of cell size. Nuclei also increase in size without changes in DNA content. 3D tomographic reconstructions revealed extreme vacuolation of the cytoplasm of TbRrp44 knockdown cells and general alteration of organelles. Calcium-containing vesicles (acidocalcisomes) were identified by X-ray absorption near-edge structure spectra (XANES) of calcium L2,3 edges on fully hydrated cells. The volumes of acidocalcisomes and lipid droplet were quantified from 3D reconstructions. Both were found in higher number and with larger volumes in TbRrp44 knockdown cells. These multiple defects indicate that a combination of signals, starting from nucleolar stress and activation of autophagy, converge to induce lysosome expansion. With time, the cytoplasm is taken up by lysosome-derived vacuoles, which may be one of the final stages leading to cell death triggered by TbRrp44 depletion. These studies provide the first evidence on the ultra-structure cell modifications caused by Rrp44 ribonuclease deficiency.Author summaryTrypanosoma brucei is a parasitic protozoan belonging to the Kinetoplastidae Class, which displays distinct cellular, genomic and molecular features. These features include extra intervening sequences in the large subunit ribosomal RNA (rRNA) precursor that are not found in the homologous rRNA precursor of host cells. Genetic downregulation of the T. brucei Rrp44 (TbRrp44) ribonuclease shows that it is required for accurate excision of these intervening sequences to produce mature rRNA molecules. Here, we have used a multidisciplinary approach based on molecular and chemical markers, synchrotron deep UV microscopy and cryo soft X-ray tomography to show that downregulation of TbRrp44 leads to a series of cellular alterations that eventually result in cell death. Mitochondrial activity is particularly affected in parallel with induction of autophagy response, increase in size of the cell and of organelles such as acidocalcisomes and lipid droplets, and with a massive expansion of lysosome-derived vacuoles. The ultrastructural modifications that take place in T. brucei cells are nicely highlighted in the 3D reconstructions generated using cryo soft X-ray tomography images. This study provides new insights into the multiple cellular consequences of Rrp44 ribonuclease depletion in T. brucei parasites.

Import of a major mitochondrial enzyme depends on synergy between two distinct helices of its presequence

Mammalian glutamate dehydrogenase (GDH), a nuclear-encoded enzyme central to cellular metabolism, is among the most abundant mitochondrial proteins (constituting up to 10% of matrix proteins). To attain such high levels, GDH depends on very efficient mitochondrial targeting that, for human isoenzymes hGDH1 and hGDH2, is mediated by an unusually long cleavable presequence (N53). Here, we studied the mitochondrial transport of these proteins using isolated yeast mitochondria and human cell lines. We found that both hGDHs were very rapidly imported and processed in isolated mitochondria, with their presequences (N53) alone being capable of directing non-mitochondrial proteins into mitochondria. These presequences were predicted to form two α helices (α1: N 1–10; α2: N 16–32) separated by loops. Selective deletion of the α1 helix abolished the mitochondrial import of hGDHs. While the α1 helix alone had a very weak hGDH mitochondrial import capacity, it could direct efficiently non-mitochondrial proteins into mitochondria. In contrast, the α2 helix had no autonomous mitochondrial-targeting capacity. A peptide consisting of α1 and α2 helices without intervening sequences had GDH transport efficiency comparable with that of N53. Mutagenesis of the cleavage site blocked the intra-mitochondrial processing of hGDHs, but did not affect their mitochondrial import. Replacement of all three positively charged N-terminal residues (Arg3, Lys7 and Arg13) by Ala abolished import. We conclude that the synergistic interaction of helices α1 and α2 is crucial for the highly efficient import of hGDHs into mitochondria.

Spliceosomal intronogenesis

The presence of intervening sequences, termed introns, is a defining characteristic of eukaryotic nuclear genomes. Once transcribed into pre-mRNA, these introns must be removed within the spliceosome before export of the processed mRNA to the cytoplasm, where it is translated into protein. Although intron loss has been demonstrated experimentally, several mysteries remain regarding the origin and propagation of introns. Indeed, documented evidence of gain of an intron has only been suggested by phylogenetic analyses. We report the use of a strategy that detects selected intron gain and loss events. We have experimentally verified, to our knowledge, the first demonstrations of intron transposition in any organism. From our screen, we detected two separate intron gain events characterized by the perfect transposition of a reporter intron into the yeast genes RPL8B and ADH2, respectively. We show that the newly acquired introns are able to be removed from their respective pre-mRNAs by the spliceosome. Additionally, the novel allele, RPL8Bint, is functional when overexpressed within the genome in a strain lacking the Rpl8 paralogue RPL8A, demonstrating that the gene targeted for intronogenesis is functional.