Co-transcriptional splicing facilitates transcription of gigantic genes.

Although introns are typically tens to thousands of nucleotides, there are notable exceptions. In flies as well as humans, a small number of genes contain introns that are more than 1000 times larger than typical introns, exceeding hundreds of kilobases (kb) to megabases (Mb). It remains unknown why gigantic introns exist and how cells overcome the challenges associated with their transcription and RNA processing. The Drosophila Y chromosome contains some of the largest genes identified to date: multiple genes exceed 4Mb, with introns accounting for over 99% of the gene span. Here we demonstrate that co-transcriptional splicing of these gigantic Y-linked genes is important to ensure successful transcription: perturbation of splicing led to the attenuation of transcription, leading to a failure to produce mature mRNA. Cytologically, defective splicing of the Y-linked gigantic genes resulted in disorganization of transcripts within the nucleus suggestive of entanglement of transcripts, likely resulting from unspliced long RNAs. We propose that co-transcriptional splicing maintains the length of nascent transcripts of gigantic genes under a critical threshold, preventing their entanglement and ensuring proper gene expression. Our study reveals a novel biological significance of co-transcriptional splicing.

In Multiple Myeloma, High-Risk Secondary Genetic Events Observed at Relapse Are Present From Diagnosis in Tiny, Undetectable Subclonal Populations.

PURPOSE: Multiple myeloma (MM) is characterized by copy number abnormalities (CNAs), some of which influence patient outcomes and are sometimes observed only at relapse(s), suggesting their acquisition during tumor evolution. However, the presence of micro-subclones may be missed in bulk analyses. Here, we use single-cell genomics to determine how often these high-risk events are missed at diagnosis and selected at relapse. MATERIALS AND METHODS: We analyzed 81 patients with plasma cell dyscrasias using single-cell CNA sequencing. Sixty-six patients were selected at diagnosis, nine at first relapse, and six in presymptomatic stages. A total of 956 newly diagnosed patients with MM and patients with first relapse MM have been identified retrospectively with required cytogenetic data to evaluate enrichment of CNA risk events and survival impact. RESULTS: A total of 52,176 MM cells were analyzed. Seventy-four patients (91%) had 2-16 subclones. Among these patients, 28.7% had a subclone with high-risk features (del(17p), del(1p32), and 1q gain) at diagnosis. In a patient with a subclonal 1q gain at diagnosis, we analyzed the diagnosis, postinduction, and first relapse samples, which showed a rise of the high-risk 1q gain subclone (16%, 70%, and 92%, respectively). In our clinical database, we found that the 1q gain frequency increased from 30.2% at diagnosis to 43.6% at relapse (odds ratio, 1.78; 95% CI, 1.58 to 2.00). We subsequently performed survival analyses, which showed that the progression-free and overall survival curves were superimposable between patients who had the 1q gain from diagnosis and those who seemingly acquired it at relapse. This strongly suggests that many patients had 1q gains at diagnosis in microclones that were missed by bulk analyses. CONCLUSION: These data suggest that identifying these scarce aggressive cells may necessitate more aggressive treatment as early as diagnosis to prevent them from becoming the dominant clone.

Primary plasma cell leukemias displaying t(11;14) have specific genomic, transcriptional, and clinical features.

Primary plasma cell leukemia (pPCL) is an aggressive form of multiple myeloma (MM) that has not benefited from recent therapeutic advances in the field. Because it is very rare and heterogeneous, it remains poorly understood at the molecular level. To address this issue, we performed DNA and RNA sequencing of sorted plasma cells from a large cohort of 90 newly diagnosed pPCL and compared with MM. We observed that pPCL presents a specific genomic landscape with a high prevalence of t(11;14) (about half) and high-risk genomic features such as del(17p), gain 1q, and del(1p32). In addition, pPCL displays a specific transcriptome when compared with MM. We then wanted to characterize specifically pPCL with t(11;14). We observed that this subentity displayed significantly fewer adverse cytogenetic abnormalities. This translated into better overall survival when compared with pPCL without t(11;14) (39.2 months vs 17.9 months, P = .002). Finally, pPCL with t(11;14) displayed a specific transcriptome, including differential expression of BCL2 family members. This study is the largest series of patients with pPCL reported so far.

Marine Ultrasmall Prokaryotes Likely Affect the Cycling of Carbon, Methane, Nitrogen, and Sulfur.

Recently, we uncovered the genetic components from six carbon fixation autotrophic pathways in cleaned ultrasmall size fractions from marine samples (<0.22 µm) gathered worldwide by the Tara Oceans Expedition. This first finding suggested that prokaryotic nanoorganisms, phylogenetically distantly related to the known CPR and DPANN groups, could collectively impact carbon cycling and carbon fixation across the world's ocean. To extend our mining of the functional and taxonomic microbial dark matter from the ultrasmall size fraction from the Tara Oceans Expedition, we investigated the distribution of 28 metabolic pathways associated with the cycling of carbon, methane, nitrogen, and sulfur. For all of these pathways, we report the existence not only of novel metabolic homologs in the ultrasmall size fraction of the oceanic microbiome, associated with nanoorganisms belonging to the CPR and DPANN lineages, but also of metabolic homologs exclusively found in marine host taxa belonging to other (still unassigned) microbial lineages. Therefore, we conclude that marine nanoorganisms contribute to a greater diversity of key biogeochemical cycles than currently appreciated. In particular, we suggest that oceanic nanoorganisms may be involved in a metabolic loop around Acetyl-CoA, have an underappreciated genetic potential to degrade methane, contribute to sustaining redox-reactions by producing Coenzyme F420, and affect sulfur cycling, notably as they harbor a complete suite of homologs of enzymes of the SOX system.

del(17p) without TP53 mutation confers a poor prognosis in intensively treated newly diagnosed patients with multiple myeloma.

Despite tremendous improvements in the outcome of patients with multiple myeloma in the past decade, high-risk patients have not benefited from the approval of novel drugs. The most important prognostic factor is the loss of parts of the short arm of chromosome 17, known as deletion 17p (del(17p)). A recent publication (on a small number of patients) suggested that these patients are at very high-risk only if del(17p) is associated with TP53 mutations, the so-called "double-hit" population. To validate this finding, we designed a much larger study on 121 patients presenting del(17p) in > 55% of their plasma cells, and homogeneously treated by an intensive approach. For these 121 patients, we performed deep next generation sequencing targeted on TP53. The outcome was then compared with a large control population (2505 patients lacking del(17p)). Our results confirmed that the "double hit" situation is the worst (median survival = 36 months), but that del(17p) alone also confers a poor outcome compared with the control cohort (median survival = 52.8 months vs 152.2 months, respectively). In conclusion, our study clearly confirms the extremely poor outcome of patients displaying "double hit," but also that del(17p) alone is still a very high-risk feature, confirming its value as a prognostic indicator for poor outcome.

Rich Repertoire of Quorum Sensing Protein Coding Sequences in CPR and DPANN Associated with Interspecies and Interkingdom Communication.

The bacterial candidate phyla radiation (CPR) and the archaeal DPANN superphylum are two novel lineages that have substantially expanded the tree of life due to their large phylogenetic diversity. Because of their ultrasmall size, reduced genome, and lack of core biosynthetic capabilities, most CPR and DPANN members are predicted to be sustained through their interactions with other species. How the few characterized CPR and DPANN symbionts achieve these critical interactions is, however, poorly understood. Here, we conducted an in silico analysis on 2,597 CPR/DPANN genomes to test whether these ultrasmall microorganisms might encode homologs of reference proteins involved in the synthesis and/or the detection of 26 different types of communication molecules (quorum sensing [QS] signals), since QS signals are well-known mediators of intra- and interorganismic relationships. We report the discovery of 5,693 variants of QS proteins distributed across 63 CPR and 6 DPANN phyla and associated with 14 distinct types of communication molecules, most of which were characterized as interspecies QS signals.IMPORTANCE The selection of predicted genes for interspecies communication within the CPR and DPANN genomes sheds some light onto the underlying mechanisms supporting their inferred symbiotic lifestyle. Also, considering the lack of core pathways such as the de novo synthesis of nucleotides or amino acids in the CPR and DPANN lineages, the persistence of these genes highlights how determinant social traits can be for the survival of some microorganisms. Finally, the considerable number of variants of QS proteins identified among the 69 CPR and DPANN phyla substantially expands our knowledge of prokaryotic communication across the tree of life and suggests that the multiplicity of "dialects" in the microbial world is probably larger than previously appreciated.

The Methodology Behind Network Thinking: Graphs to Analyze Microbial Complexity and Evolution.

In the post genomic era, large and complex molecular datasets from genome and metagenome sequencing projects expand the limits of what is possible for bioinformatic analyses. Network-based methods are increasingly used to complement phylogenetic analysis in studies in molecular evolution, including comparative genomics, classification, and ecological studies. Using network methods, the vertical and horizontal relationships between all genes or genomes, whether they are from cellular chromosomes or mobile genetic elements, can be explored in a single expandable graph. In recent years, development of new methods for the construction and analysis of networks has helped to broaden the availability of these approaches from programmers to a diversity of users. This chapter introduces the different kinds of networks based on sequence similarity that are already available to tackle a wide range of biological questions, including sequence similarity networks, gene-sharing networks and bipartite graphs, and a guide for their construction and analyses.

Carbon Fixation by Marine Ultrasmall Prokaryotes.

Autotrophic carbon fixation is a crucial process for sustaining life on Earth. To date, six pathways, the Calvin-Benson-Bassham cycle, the reductive tricarboxylic acid cycle, the 3-hydroxypropionate bi-cycle, the Wood-Ljungdahl pathway, the dicarboxylate/4-hydroxybutyrate cycle, and the 4-hydroxybutyrate cycle, have been described. Nano-organisms such as members of the Candidate Phyla Radiation (CPR) bacterial superphylum and the Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, Nanohalorchaeota (DPANN) archaeal superphylum could deeply impact carbon cycling and carbon fixation in ways that are still to be determined. CPR and DPANN are ubiquitous in the environment but understudied; their gene contents are not exhaustively described; and their metabolisms are not yet fully understood. Here, the completeness of each of the above pathways was quantified and tested for the presence of all key enzymes in nano-organisms from across the World Ocean. The novel marine ultrasmall prokaryotes were demonstrated to collectively harbor the genes required for carbon fixation, in particular the "energetically efficient" dicarboxylate/4-hydroxybutyrate pathway and the 4-hydroxybutyrate pathway. This contrasted with the known carbon metabolic pathways associated with CPR members in aquifers, where they are described as degraders (Castelle CJ, et al. 2015. Genomic expansion of domain archaea highlights roles for organisms from new phyla in anaerobic carbon cycling. Curr Biol. 25(6):690-701; Castelle CJ, et al. 2018. Biosynthetic capacity, metabolic variety and unusual biology in the CPR and DPANN radiations. Nat Rev Microbiol. 16(10):629-645; Anantharaman K, et al. 2016. Thousands of microbial genomes shed light on interconnected biogeochemical processes in an aquifer system. Nat Commun. 7:13219.). Our findings suggest that nano-organisms have a broader contribution to carbon fixation and cycling than currently assumed. Furthermore, CPR and DPANN superphyla are possibly not the only nanosized prokaryotes; therefore, the discovery of new autotrophic marine nano-organisms by future single cell genomics is anticipated.

Does the Presence of Transposable Elements Impact the Epigenetic Environment of Human Duplicated Genes?

Epigenetic modifications have an important role to explain part of the intra- and inter-species variation in gene expression. They also have a role in the control of transposable elements (TEs) whose activity may have a significant impact on genome evolution by promoting various mutations, which are expected to be mostly deleterious. A change in the local epigenetic landscape associated with the presence of TEs is expected to affect the expression of neighboring genes since these modifications occurring at TE sequences can spread to neighboring sequences. In this work, we have studied how the epigenetic modifications of genes are conserved and what the role of TEs is in this conservation. For that, we have compared the conservation of the epigenome associated with human duplicated genes and the differential presence of TEs near these genes. Our results show higher epigenome conservation of duplicated genes from the same family when they share similar TE environment, suggesting a role for the differential presence of TEs in the evolutionary divergence of duplicates through variation in the epigenetic landscape.

Microbial Dark Matter Investigations: How Microbial Studies Transform Biological Knowledge and Empirically Sketch a Logic of Scientific Discovery.

Microbes are the oldest and most widespread, phylogenetically and metabolically diverse life forms on Earth. However, they have been discovered only 334 years ago, and their diversity started to become seriously investigated even later. For these reasons, microbial studies that unveil novel microbial lineages and processes affecting or involving microbes deeply (and repeatedly) transform knowledge in biology. Considering the quantitative prevalence of taxonomically and functionally unassigned sequences in environmental genomics data sets, and that of uncultured microbes on the planet, we propose that unraveling the microbial dark matter should be identified as a central priority for biologists. Based on former empirical findings of microbial studies, we sketch a logic of discovery with the potential to further highlight the microbial unknowns.