Extracellular vesicles are involved in the paracrine communication between epithelial cells in different regions of the domestic cat epididymis.

Sperm maturation depends on exposure to specific microenvironments within the different segments of the epididymis, but mechanisms underlying how these microenvironments are produced or maintained are not well understood. We hypothesized that epididymal extracellular vesicles (EVs) could play a role in the process of maintaining microenvironments in different regions of the epididymis. Specifically, we tested whether the EVs from different regions of the epididymis can serve as a form of paracrine communication between cells in different segments. Domestic cat tissues were used to develop a reproducible in vitro culture system for corpus epididymis explants that were then exposed to EVs collected from upstream (i.e. caput) segments. The impacts of different culture or exposure conditions were compared by analyzing the morphology, apoptosis, transcriptional activity, and gene expression in the explants. Here, we report the development of the first in vitro culture system for epididymal tissue explants in the domestic cat model. Using this system, we found that EVs from the caput segment have a significant effect on the transcriptional profile of tissue from the corpus segment (1233 differentially expressed genes due to EV supplementation). Of note, expression of genes associated with regulation of epithelial cell differentiation and cytokine signaling in the epididymis were regulated by the presence of EVs. Together, our findings comprise the first report of paracrine control of segmental gene regulation by epididymal EVs in any species. These results contribute to a better understanding of epididymis biology and could lead to techniques to enhance or suppress male fertility.

Nutrient dose-responsive transcriptome changes driven by Michaelis-Menten kinetics underlie plant growth rates.

An increase in nutrient dose leads to proportional increases in crop biomass and agricultural yield. However, the molecular underpinnings of this nutrient dose-response are largely unknown. To investigate, we assayed changes in the Arabidopsis root transcriptome to different doses of nitrogen (N)-a key plant nutrient-as a function of time. By these means, we found that rate changes of genome-wide transcript levels in response to N-dose could be explained by a simple kinetic principle: the Michaelis-Menten (MM) model. Fitting the MM model allowed us to estimate the maximum rate of transcript change (Vmax), as well as the N-dose at which one-half of Vmax was achieved (Km) for 1,153 N-dose-responsive genes. Since transcription factors (TFs) can act in part as the catalytic agents that determine the rates of transcript change, we investigated their role in regulating N-dose-responsive MM-modeled genes. We found that altering the abundance of TGA1, an early N-responsive TF, perturbed the maximum rates of N-dose transcriptomic responses (Vmax), Km, as well as the rate of N-dose-responsive plant growth. We experimentally validated that MM-modeled N-dose-responsive genes included both direct and indirect TGA1 targets, using a root cell TF assay to detect TF binding and/or TF regulation genome-wide. Taken together, our results support a molecular mechanism of transcriptional control that allows an increase in N-dose to lead to a proportional change in the rate of genome-wide expression and plant growth.

Antagonistic actions of two human Pan3 isoforms on global mRNA turnover.

Deadenylation is a fundamental process that regulates eukaryotic gene expression. Mammalian deadenylation exhibits biphasic kinetics, with the Pan2-Pan3 and Ccr4-Caf1 deadenylase complexes mediating the first and second phase, respectively; however, the significance of the biphasic nature of deadenylation in mRNA turnover remains unclear. In this study, we discovered that two distinct isoforms of human Pan3 display opposing properties necessary for coordinating the two phases of deadenylation. The shorter isoform (Pan3S) interacts more strongly with PABP than the longer isoform (Pan3L) does. Pan2 deadenylase activity is enhanced by Pan3S but suppressed by Pan3L. Knocking down individual Pan3 isoforms has opposing effects on the global poly(A) tail length profile, P-body formation, and different mRNA decay pathways. Transcriptome-wide analysis of Pan3 knockdown effects on mRNA turnover shows that depleting either Pan3 isoform causes profound and extensive changes in mRNA stability globally. These results reveal a new fundamental step governing mammalian mRNA metabolism. We propose that the first phase of deadenylation, coordinated through the interplay among the two Pan3 isoforms, Pan2, and PABP, represents a cytoplasmic mRNA maturation step important for proper mRNA turnover.

Characterization of siRNAs clusters in Arabidopsis thaliana galls induced by the root-knot nematode Meloidogyne incognita.

BACKGROUND: Root-knot nematodes (RKN), genus Meloidogyne, are plant parasitic worms that have the ability to transform root vascular cylinder cells into hypertrophied, multinucleate and metabolically over-active feeding cells. Redifferentiation into feeding cells is the result of a massive transcriptional reprogramming of root cells targeted by RKN. Since RKN are able to induce similar feeding cells in roots of thousands of plant species, these worms are thought to manipulate essential and conserved plant molecular pathways. RESULTS: Small non-coding RNAs of uninfected roots and infected root galls induced by M. incognita from Arabidopsis thaliana were sequenced by high throughput sequencing. SiRNA populations were analysed by using the Shortstack algorithm. We identified siRNA clusters that are differentially expressed in infected roots and evidenced an over-representation of the 23-24 nt siRNAs in infected tissue. This size corresponds to heterochromatic siRNAs (hc-siRNAs) which are known to regulate expression of transposons and genes at the transcriptional level, mainly by inducing DNA methylation. CONCLUSIONS: Correlation of siRNA clusters expression profile with transcriptomic data identified several protein coding genes that are candidates to be regulated by siRNAs at the transcriptional level by RNA directed DNA methylation (RdDM) pathway either directly or indirectly via silencing of neighbouring transposable elements.

Conserved DNA methylation combined with differential frontal cortex and cerebellar expression distinguishes C9orf72-associated and sporadic ALS, and implicates SERPINA1 in disease.

We previously found C9orf72-associated (c9ALS) and sporadic amyotrophic lateral sclerosis (sALS) brain transcriptomes comprise thousands of defects, among which, some are likely key contributors to ALS pathogenesis. We have now generated complementary methylome data and combine these two data sets to perform a comprehensive "multi-omic" analysis to clarify the molecular mechanisms initiating RNA misregulation in ALS. We found that c9ALS and sALS patients have generally distinct but overlapping methylome profiles, and that the c9ALS- and sALS-affected genes and pathways have similar biological functions, indicating conserved pathobiology in disease. Our results strongly implicate SERPINA1 in both C9orf72 repeat expansion carriers and non-carriers, where expression levels are greatly increased in both patient groups across the frontal cortex and cerebellum. SERPINA1 expression is particularly pronounced in C9orf72 repeat expansion carriers for both brain regions, where SERPINA1 levels are strictly down regulated across most human tissues, including the brain, except liver and blood, and are not measurable in E18 mouse brain. The altered biological networks we identified contain critical molecular players known to contribute to ALS pathology, which also interact with SERPINA1. Our comprehensive combined methylation and transcription study identifies new genes and highlights that direct genetic and epigenetic changes contribute to c9ALS and sALS pathogenesis.

Chemically modified guide RNAs enhance CRISPR-Cas13 knockdown in human cells.

RNA-targeting CRISPR-Cas13 proteins have recently emerged as a powerful platform to modulate gene expression outcomes. However, protein and CRISPR RNA (crRNA) delivery in human cells can be challenging with rapid crRNA degradation yielding transient knockdown. Here we compare several chemical RNA modifications at different positions to identify synthetic crRNAs that improve RNA targeting efficiency and half-life in human cells. We show that co-delivery of modified crRNAs and recombinant Cas13 enzyme in ribonucleoprotein (RNP) complexes can alter gene expression in primary CD4+ and CD8+ T cells. This system represents a robust and efficient method to modulate transcripts without genetic manipulation.

Transcriptome and Translatome Regulation of Pathogenesis in Alzheimer's Disease Model Mice.

BACKGROUND: Defining cellular mechanisms that drive Alzheimer's disease (AD) pathogenesis and progression will be aided by studies defining how gene expression patterns change during pre-symptomatic AD and ensuing periods of declining cognition. Previous studies have emphasized changes in transcriptome, but not translatome regulation, leaving the ultimate results of gene expression alterations relatively unexplored in the context of AD. OBJECTIVE: To identify genes whose expression might be regulated at the transcriptome and translatome levels in AD, we analyzed gene expression in cerebral cortex of two AD model mouse strains, CVN (APPSwDI;NOS2 -/- ) and Tg2576 (APPSw), and their companion wild type (WT) strains at 6 months of age by tandem RNA-Seq and Ribo-Seq (ribosome profiling). METHODS: Identical starting pools of bulk RNA were used for RNA-Seq and Ribo-Seq. Differential gene expression analysis was performed at the transcriptome, translatome, and translational efficiency levels. Regulated genes were functionally evaluated by gene ontology tools. RESULTS: Compared to WT mice, AD model mice had similar levels of transcriptome regulation, but differences in translatome regulation. A microglial signature associated with early stages of Aß accumulation was upregulated at both levels in CVN mice. Although the two mice strains did not share many regulated genes, they showed common regulated pathways related to AßPP metabolism associated with neurotoxicity and neuroprotection. CONCLUSION: This work represents the first genome-wide study of brain translatome regulation in animal models of AD and provides evidence of a tight and early translatome regulation of gene expression controlling the balance between neuroprotective and neurodegenerative processes in brain.

Programmable technologies to manipulate gene expression at the RNA level.

RNA has long been an enticing therapeutic target, but is now garnering increased attention, largely driven by clinical successes of RNA interference-based drugs. While gene knockdown by well-established RNA interference- and other oligonucleotide-based strategies continues to advance in the clinic, the repertoire of targetable effectors capable of altering gene expression at the RNA level is also rapidly expanding. In this review, we focus on several recently developed bifunctional molecular technologies that both interact with and act upon a target RNA. These new approaches for programmable RNA knockdown, editing, splicing, translation, and chemical modifications stand to provide impactful new modalities for therapeutic development in the coming decades.

Targeting the Transcriptome Through Globally Acting Components.

Transcription is a step in gene expression that defines the identity of cells and its dysregulation is associated with diseases. With advancing technologies revealing molecular underpinnings of the cell with ever-higher precision, our ability to view the transcriptomes may have surpassed our knowledge of the principles behind their organization. The human RNA polymerase II (Pol II) machinery comprises thousands of components that, in conjunction with epigenetic and other mechanisms, drive specialized programs of development, differentiation, and responses to the environment. Parts of these programs are repurposed in oncogenic transformation. Targeting of cancers is commonly done by inhibiting general or broadly acting components of the cellular machinery. The critical unanswered question is how globally acting or general factors exert cell type specific effects on transcription. One solution, which is discussed here, may be among the events that take place at genes during early Pol II transcription elongation. This essay turns the spotlight on the well-known phenomenon of promoter-proximal Pol II pausing as a step that separates signals that establish pausing genome-wide from those that release the paused Pol II into the gene. Concepts generated in this rapidly developing field will enhance our understanding of basic principles behind transcriptome organization and hopefully translate into better therapies at the bedside.

CRISPR/Cas13 as a Tool for RNA Interference.

Almost all biological processes involve RNA, making it crucial to develop tools for manipulation of the transcriptome. The bacterial CRISPR/Cas13 system was recently rewired to facilitate RNA manipulation in eukaryotes, including plants. We discuss here the opportunities and limitations of using CRISPR/Cas13 in plants for various types of RNA manipulation.

RNA virus interference via CRISPR/Cas13a system in plants.

BACKGROUND: CRISPR/Cas systems confer immunity against invading nucleic acids and phages in bacteria and archaea. CRISPR/Cas13a (known previously as C2c2) is a class 2 type VI-A ribonuclease capable of targeting and cleaving single-stranded RNA (ssRNA) molecules of the phage genome. Here, we employ CRISPR/Cas13a to engineer interference with an RNA virus, Turnip Mosaic Virus (TuMV), in plants. RESULTS: CRISPR/Cas13a produces interference against green fluorescent protein (GFP)-expressing TuMV in transient assays and stable overexpression lines of Nicotiana benthamiana. CRISPR RNA (crRNAs) targeting the HC-Pro and GFP sequences exhibit better interference than those targeting other regions such as coat protein (CP) sequence. Cas13a can also process pre-crRNAs into functional crRNAs. CONCLUSIONS: Our data indicate that CRISPR/Cas13a can be used for engineering interference against RNA viruses, providing a potential novel mechanism for RNA-guided immunity against RNA viruses and for other RNA manipulations in plants.

Engineering RNA Virus Interference via the CRISPR/Cas13 Machinery in Arabidopsis.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems are key immune mechanisms helping prokaryotic species fend off RNA and DNA viruses. CRISPR/Cas9 has broad applications in basic research and biotechnology and has been widely used across eukaryotic species for genome engineering and functional analysis of genes. The recently developed CRISPR/Cas13 systems target RNA rather than DNA and thus offer new potential for transcriptome engineering and combatting RNA viruses. Here, we used CRISPR/LshCas13a to stably engineer Arabidopsis thaliana for interference against the RNA genome of Turnip mosaic virus (TuMV). Our data demonstrate that CRISPR RNAs (crRNAs) guiding Cas13a to the sequences encoding helper component proteinase silencing suppressor (HC-Pro) or GFP target 2 (GFP-T2) provide better interference compared to crRNAs targeting other regions of the TuMV RNA genome. This work demonstrates the exciting potential of CRISPR/Cas13 to be used as an antiviral strategy to obstruct RNA viruses, and encourages the search for more robust and effective Cas13 variants or CRISPR systems that can target RNA.

Origin of exon skipping-rich transcriptomes in animals driven by evolution of gene architecture.

BACKGROUND: Alternative splicing, particularly through intron retention and exon skipping, is a major layer of pre-translational regulation in eukaryotes. While intron retention is believed to be the most prevalent mode across non-animal eukaryotes, animals have unusually high rates of exon skipping. However, when and how this high prevalence of exon skipping evolved is unknown. Since exon skipping can greatly expand proteomes, answering these questions sheds light on the evolution of higher organismal complexity in metazoans. RESULTS: We used RNA-seq data to quantify exon skipping and intron retention frequencies across 65 eukaryotic species, with particular focus on early branching animals and unicellular holozoans. We found that only bilaterians have significantly increased their exon skipping frequencies compared to all other eukaryotic groups. Unlike in other eukaryotes, however, exon skipping in nearly all animals, including non-bilaterians, is strongly enriched for frame-preserving sequences, suggesting that exon skipping involvement in proteome expansion predated the increase in frequency. We also identified architectural features consistently associated with higher exon skipping rates within all studied eukaryotic genomes. Remarkably, these architectures became more prevalent during animal evolution, indicating co-evolution between genome architectures and exon skipping frequencies. CONCLUSION: We suggest that the increase of exon skipping rates in animals followed a two-step process. First, exon skipping in early animals became enriched for frame-preserving events. Second, bilaterian ancestors dramatically increased their exon skipping frequencies, likely driven by the interplay between a shift in their genome architectures towards more exon definition and recruitment of frame-preserving exon skipping events to functionally diversify their cell-specific proteomes.

Transcriptomic Analysis in Strawberry Fruits Reveals Active Auxin Biosynthesis and Signaling in the Ripe Receptacle.

The role of auxin in ripening strawberry (Fragaria ×ananassa) fruits has been restricted to the early stages of development where the growth of the receptacle is dependent on the delivery of auxin from the achenes. At later stages, during enlargement of the receptacle, other hormones have been demonstrated to participate to different degrees, from the general involvement of gibberellins and abscisic acid to the more specific of ethylene. Here we report the involvement of auxin at the late stages of receptacle ripening. The auxin content of the receptacle remains constant during ripening. Analysis of the transcriptome of ripening strawberry fruit revealed the changing expression pattern of the genes of auxin synthesis, perception, signaling and transport along with achene and receptacle development from the green to red stage. Specific members of the corresponding gene families show active transcription in the ripe receptacle. For the synthesis of auxin, two genes encoding tryptophan aminotransferases, FaTAA1 and FaTAR2, were expressed in the red receptacle, with FaTAR2 expression peaking at this stage. Transient silencing of this gene in ripening receptacle was accompanied by a diminished responsiveness to auxin. The auxin activity in the ripening receptacle is supported by the DR5-directed expression of a GUS reporter gene in the ripening receptacle of DR5-GUS transgenic strawberry plants. Clustering by co-expression of members of the FaAux/IAA and FaARF families identified five members whose transcriptional activity was increased with the onset of receptacle ripening. Among these, FaAux/IAA11 and FaARF6a appeared, by their expression level and fold-change, as the most likely candidates for their involvement in the auxin activity in the ripening receptacle. The association of the corresponding ARF6 gene in Arabidopsis to cell elongation constitutes a suggestive hypothesis for FaARF6a involvement in the same cellular process in the growing and ripening receptacle.

Corrigendum: Transcriptomic Analysis in Strawberry Fruits Reveals Active Auxin Biosynthesis and Signaling in the Ripe Receptacle.

[This corrects the article on p. 889 in vol. 8, PMID: 28611805.].