Laboratory study of Fritillaria lifecycle reveals key morphogenetic events leading to genus-specific anatomy.

A fascinating variety of adult body plans can be found in the Tunicates, the closest existing relatives of vertebrates. A distinctive feature of the larvacean class of pelagic tunicates is the presence of a highly specialized surface epithelium that produces a cellulose test, the "larvacean house". While substantial differences exist between the anatomy of larvacean families, most of the ontogeny is derived from the observations of a single genus, Oikopleura. We present the first study of Fritillaria development based on the observation of individuals reproduced in the laboratory. Like the other small epipelagic species Oikopleura dioica, the larvae of Fritillaria borealis grow rapidly in the laboratory, and they acquire the adult form within a day. We could show that major morphological differences exhibited by Fritillaria and Oikopleura adults originate from a key developmental stage during larval organogenesis. Here, the surface epithelium progressively retracts from the posterior digestive organs of Fritillaria larvae, and it establishes house-producing territories around the pharynx. Our results show that the divergence between larvacean genera was associated with a profound rearrangement of the mechanisms controlling the differentiation of the larval ectoderm.

Conservative route to genome compaction in a miniature annelid.

The causes and consequences of genome reduction in animals are unclear because our understanding of this process mostly relies on lineages with often exceptionally high rates of evolution. Here, we decode the compact 73.8-megabase genome of Dimorphilus gyrociliatus, a meiobenthic segmented worm. The D. gyrociliatus genome retains traits classically associated with larger and slower-evolving genomes, such as an ordered, intact Hox cluster, a generally conserved developmental toolkit and traces of ancestral bilaterian linkage. Unlike some other animals with small genomes, the analysis of the D. gyrociliatus epigenome revealed canonical features of genome regulation, excluding the presence of operons and trans-splicing. Instead, the gene-dense D. gyrociliatus genome presents a divergent Myc pathway, a key physiological regulator of growth, proliferation and genome stability in animals. Altogether, our results uncover a conservative route to genome compaction in annelids, reminiscent of that observed in the vertebrate Takifugu rubripes.

Evolution of the U2 Spliceosome for Processing Numerous and Highly Diverse Non-canonical Introns in the Chordate Fritillaria borealis.

An overwhelming majority of eukaryotic introns have GT/AG ends, whose identities play a critical role for their recognition and removal by the U2 spliceosome, a well-conserved complex of protein and RNAs. Introns with other splice sites exist at very low frequencies in various genomes, and some of them are processed by the U12 spliceosome. Here, we show that, in the chordate Fritillaria borealis, the majority of old introns have been lost and replaced by introns with highly divergent splice sites. The new introns of F. borealis are exceptionally diverse, though more frequently AG/AC or AG/AT, and features of thousands of them support an origin from transposons. They cannot be processed in human cells, but their splicing is rescued by mutating terminal dinucleotides to GT/AG. With lariat sequencing and splicing inhibitor assays, we show that F. borealis introns are spliced by the U2 spliceosome, which thus evolved to a different selectivity, with neither novel U1 small nuclear RNA (snRNA) types nor major remodeling of its protein and snRNA complements. This genome-wide recolonization by non-canonical introns emphasizes the importance of transposons as a resource of novel introns in a context of massive intron loss. An evolution of the spliceosome may also permit to neutralize harmful transposons through their conversion into introns.

Massive Changes of Genome Size Driven by Expansions of Non-autonomous Transposable Elements.

In eukaryotes, genome size correlates little with the number of coding genes or the level of organismal complexity (C-value paradox). The underlying causes of variations in genome size, whether adaptive or neutral, remain unclear, although several biological traits often covary with it [1-5]. Rapid increases in genome size occur mainly through whole-genome duplications or bursts in the activity of transposable elements (TEs) [6]. The very small and compact genome of Oikopleura dioica, a tunicate of the larvacean class, lacks elements of most ancient families of animal retrotransposons [7, 8]. Here, we sequenced the genomes of six other larvaceans, all of which are larger than that of Oikopleura (up to 12 times) and which increase in size with greater body length. Although no evidence was found for whole-genome duplications within the group of species, the global amount of TEs strongly correlated with genome size. Compared to other metazoans, however, the TE diversity was reduced in all species, as observed previously in O. dioica, suggesting a common ancestor with a compacted genome. Strikingly, non-autonomous elements, particularly short interspersed nuclear elements (SINEs), massively contributed to genome size variation through species-specific independent amplifications, ranging from 3% in the smallest genome up to 49% in the largest. Variations in SINE abundance explain as much as 83% of interspecific genome size variation. These data support an indirect influence of autonomous TEs on genome size via their ability to mobilize non-autonomous elements.

Prevalence of Mutation-Prone Microhomology-Mediated End Joining in a Chordate Lacking the c-NHEJ DNA Repair Pathway.

Classical non-homologous end joining (c-NHEJ), a fundamental pathway that repairs double-strand breaks in DNA, is almost universal in eukaryotes and involves multiple proteins highly conserved from yeast to human [1]. The genes encoding these proteins were not detected in the genome of Oikopleura dioica, a new model system of tunicate larvaceans known for its very compact and highly rearranged genome [2-4]. After showing their absence in the genomes of six other larvacean species, the present study examined how O. dioica oocytes and embryos repair double-strand DNA breaks (DSBs), using two approaches: the injection of linearized plasmids, which resulted in their rapid end joining, and a newly established CRISPR Cas9 technique. In both cases, end joining merged short microhomologous sequences surrounding the break (mainly 4 bp long), thus inducing deletions larger than for the tunicate ascidian Ciona intestinalis and human cells. A relatively high frequency of nucleotide insertions was also observed. Finally, a survey of genomic indels supports the involvement of microhomology-mediated repair in natural conditions. Overall, O. dioica repairs DSBs as other organisms do when their c-NHEJ pathway is experimentally rendered deficient, using another mode of end joining with the same effect as alternative NHEJ (a-NHEJ) or microhomology-mediated end joining (MMEJ) [5-7]. We discuss how the exceptional loss of c-NHEJ and its replacement by a more mutation-prone mechanism may have contributed to reshaping this genome and even been advantageous under pressure for genome compaction.

From Cells to Virus Particles: Quantitative Methods to Monitor RNA Packaging.

In cells, positive strand RNA viruses, such as Retroviridae, must selectively recognize their full-length RNA genome among abundant cellular RNAs to assemble and release particles. How viruses coordinate the intracellular trafficking of both RNA and protein components to the assembly sites of infectious particles at the cell surface remains a long-standing question. The mechanisms ensuring packaging of genomic RNA are essential for viral infectivity. Since RNA packaging impacts on several essential functions of retroviral replication such as RNA dimerization, translation and recombination events, there are many studies that require the determination of RNA packaging efficiency and/or RNA packaging ability. Studies of RNA encapsidation rely upon techniques for the identification and quantification of RNA species packaged by the virus. This review focuses on the different approaches available to monitor RNA packaging: Northern blot analysis, ribonuclease protection assay and quantitative reverse transcriptase-coupled polymerase chain reaction as well as the most recent RNA imaging and sequencing technologies. Advantages, disadvantages and limitations of these approaches will be discussed in order to help the investigator to choose the most appropriate technique. Although the review was written with the prototypic simple murine leukemia virus (MLV) and complex human immunodeficiency virus type 1 (HIV-1) in mind, the techniques were described in order to benefit to a larger community.

Embryonic expression of endogenous retroviral RNAs in somatic tissues adjacent to the Oikopleura germline.

Selective pressure to maintain small genome size implies control of transposable elements, and most old classes of retrotransposons are indeed absent from the very compact genome of the tunicate Oikopleura dioica. Nonetheless, two families of retrotransposons are present, including the Tor elements. The gene organization within Tor elements is similar to that of LTR retrotransposons and retroviruses. In addition to gag and pol, many Tor elements carry a third gene encoding viral envelope-like proteins (Env) that may mediate infection. We show that the Tor family contains distinct classes of elements. In some classes, env mRNA is transcribed from the 5'LTR as in retroviruses. In others, env is transcribed from an additional promoter located downstream of the 5'LTR. Tor Env proteins are membrane-associated glycoproteins which exhibit some features of viral membrane fusion proteins. Whereas some elements are expressed in the adult testis, many others are specifically expressed in embryonic somatic cells adjacent to primordial germ cells. Such embryonic expression depends on determinants present in the Tor elements and not on their surrounding genomic environment. Our study shows that unusual modes of transcription and expression close to the germline may contribute to the proliferation of Tor elements.

Plasticity of animal genome architecture unmasked by rapid evolution of a pelagic tunicate.

Genomes of animals as different as sponges and humans show conservation of global architecture. Here we show that multiple genomic features including transposon diversity, developmental gene repertoire, physical gene order, and intron-exon organization are shattered in the tunicate Oikopleura, belonging to the sister group of vertebrates and retaining chordate morphology. Ancestral architecture of animal genomes can be deeply modified and may therefore be largely nonadaptive. This rapidly evolving animal lineage thus offers unique perspectives on the level of genome plasticity. It also illuminates issues as fundamental as the mechanisms of intron gain.

HIV-1 Vif binds to APOBEC3G mRNA and inhibits its translation.

The HIV-1 viral infectivity factor (Vif) allows productive infection of non-permissive cells (including most natural HIV-1 targets) by counteracting the cellular cytosine deaminases APOBEC-3G (hA3G) and hA3F. The Vif-induced degradation of these restriction factors by the proteasome has been extensively studied, but little is known about the translational repression of hA3G and hA3F by Vif, which has also been proposed to participate in Vif function. Here, we studied Vif binding to hA3G mRNA and its role in translational repression. Filter binding assays and fluorescence titration curves revealed that Vif tightly binds to hA3G mRNA. Vif overall binding affinity was higher for the 3'UTR than for the 5'UTR, even though this region contained at least one high affinity Vif binding site (apparent K(d) = 27 +/- 6 nM). Several Vif binding sites were identified in 5' and 3'UTRs using RNase footprinting. In vitro translation evidenced that Vif inhibited hA3G translation by two mechanisms: a main time-independent process requiring the 5'UTR and an additional time-dependent, UTR-independent process. Results using a Vif protein mutated in the multimerization domain suggested that the molecular mechanism of translational control is more complicated than a simple physical blockage of scanning ribosomes.

Tumultuous relationship between the human immunodeficiency virus type 1 viral infectivity factor (Vif) and the human APOBEC-3G and APOBEC-3F restriction factors.

The viral infectivity factor (Vif) is dispensable for human immunodeficiency virus type 1 (HIV-1) replication in so-called permissive cells but is required for replication in nonpermissive cell lines and for pathogenesis. Virions produced in the absence of Vif have an aberrant morphology and an unstable core and are unable to complete reverse transcription. Recent studies demonstrated that human APOBEC-3G (hA3G) and APOBEC-3F (hA3F), which are selectively expressed in nonpermissive cells, possess strong anti-HIV-1 activity and are sufficient to confer a nonpermissive phenotype. Vif induces the degradation of hA3G and hA3F, suggesting that its main function is to counteract these cellular factors. Most studies focused on the hypermutation induced by the cytidine deaminase activity of hA3G and hA3F and on their Vif-induced degradation by the proteasome. However, recent studies suggested that several mechanisms are involved both in the antiviral activity of hA3G and hA3F and in the way Vif counteracts these antiviral factors. Attempts to reconcile the studies involving Vif in virus assembly and stability with these recent findings suggest that hA3G and hA3F partially exert their antiviral activity independently of their catalytic activity by destabilizing the viral core and the reverse transcription complex, possibly by interfering with the assembly and/or maturation of the viral particles. Vif could then counteract hA3G and hA3F by excluding them from the viral assembly intermediates through competition for the viral genomic RNA, by regulating the proteolytic processing of Pr55(Gag), by enhancing the efficiency of the reverse transcription process, and by inhibiting the enzymatic activities of hA3G and hA3F.

RNA and DNA binding properties of HIV-1 Vif protein: a fluorescence study.

The HIV-1 viral infectivity factor (Vif) is a small basic protein essential for viral fitness and pathogenicity. Some "non-permissive" cell lines cannot sustain replication of Vif(-) HIV-1 virions. In these cells, Vif counteracts the natural antiretroviral activity of the DNA-editing enzymes APOBEC3G/3F. Moreover, Vif is packaged into viral particles through a strong interaction with genomic RNA in viral nucleoprotein complexes. To gain insights into determinants of this binding process, we performed the first characterization of Vif/nucleic acid interactions using Vif intrinsic fluorescence. We determined the affinity of Vif for RNA fragments corresponding to various regions of the HIV-1 genome. Our results demonstrated preferential and moderately cooperative binding for RNAs corresponding to the 5'-untranslated region of HIV-1 (5'-untranslated region) and gag (cooperativity parameter omega approximately 65-80, and K(d) = 45-55 nM). In addition, fluorescence spectroscopy allowed us to point out the TAR apical loop and a short region in gag as primary strong affinity binding sites (K(d) = 9.5-14 nM). Interestingly, beside its RNA binding properties, the Vif protein can also bind the corresponding DNA oligonucleotides and their complementary counterparts with an affinity similar to the one observed for the RNA sequences, while other DNA sequences displayed reduced affinity. Taken together, our results suggest that Vif binding to RNA and DNA offers several non-exclusive ways to counteract APOBEC3G/3F factors, in addition to the well documented Vif-induced degradation by the proteasome and to the Vif-mediated repression of translation of these antiviral factors.

Cooperative and specific binding of Vif to the 5' region of HIV-1 genomic RNA.

The viral infectivity factor (Vif) protein of human immunodeficiency virus type 1 (HIV-1) is essential for viral replication in vivo. Packaging of Vif into viral particles is mediated by an interaction with viral genomic RNA and association with viral nucleoprotein complexes. Despite recent findings on the RNA-binding properties of Vif suggesting that Vif could be involved in retroviral assembly, no RNA sequence or structure specificity has been determined so far. To gain further insight into the mechanisms by which Vif might regulate viral replication, we studied the interactions of Vif with HIV-1 genomic RNA in vitro. Using extensive biochemical analysis, we have measured the affinity of recombinant Vif proteins for synthetic RNAs corresponding to various regions of the HIV-1 genome. We found that recombinant Vif proteins bind specifically to HIV-1 viral RNA fragments corresponding to the 5'-untranslated region (5'-UTR), gag and the 5' part of pol (K(d) between 45 nM and 65 nM). RNA encompassing nucleotides 1-497 or 499-996 of the HIV-1 genomic RNA bind 9+/-2 and 21+/-3 Vif molecules, respectively, and at least some of these proteins bind in a cooperative manner (Hill constant alpha(H) = 2.3). In contrast, RNAs corresponding to other parts of the HIV-1 genome or heterologous RNAs showed poor binding capacity and weak cooperativity (K(d) > 200 nM). Moreover, RNase T1 footprinting revealed a hierarchical binding of Vif, pointing to TAR and the poly(A) stem-loop structures as primary strong affinity targets, and downstream structures as secondary sites with moderate affinity. Taken together, our findings suggest that Vif may assist other proteins to maintain a correct folding of the genomic RNA in order to facilitate its packaging and further steps such as reverse transcription. Interestingly, our results suggest also that Vif could bind the viral RNA in order to protect it from the action of the antiviral factor APOBEC-3G/3F.