Long noncoding RNA repertoire and targeting by nuclear exosome, cytoplasmic exonuclease, and RNAi in fission yeast.

Long noncoding RNAs (lncRNAs), which are longer than 200 nucleotides but often unstable, contribute a substantial and diverse portion to pervasive noncoding transcriptomes. Most lncRNAs are poorly annotated and understood, although several play important roles in gene regulation and diseases. Here we systematically uncover and analyze lncRNAs in Schizosaccharomyces pombe. Based on RNA-seq data from twelve RNA-processing mutants and nine physiological conditions, we identify 5775 novel lncRNAs, nearly 4× the previously annotated lncRNAs. The expression of most lncRNAs becomes strongly induced under the genetic and physiological perturbations, most notably during late meiosis. Most lncRNAs are cryptic and suppressed by three RNA-processing pathways: the nuclear exosome, cytoplasmic exonuclease, and RNAi. Double-mutant analyses reveal substantial coordination and redundancy among these pathways. We classify lncRNAs by their dominant pathway into cryptic unstable transcripts (CUTs), Xrn1-sensitive unstable transcripts (XUTs), and Dicer-sensitive unstable transcripts (DUTs). XUTs and DUTs are enriched for antisense lncRNAs, while CUTs are often bidirectional and actively translated. The cytoplasmic exonuclease, along with RNAi, dampens the expression of thousands of lncRNAs and mRNAs that become induced during meiosis. Antisense lncRNA expression mostly negatively correlates with sense mRNA expression in the physiological, but not the genetic conditions. Intergenic and bidirectional lncRNAs emerge from nucleosome-depleted regions, upstream of positioned nucleosomes. Our results highlight both similarities and differences to lncRNA regulation in budding yeast. This broad survey of the lncRNA repertoire and characteristics in S. pombe, and the interwoven regulatory pathways that target lncRNAs, provides a rich framework for their further functional analyses.

Widespread exon skipping triggers degradation by nuclear RNA surveillance in fission yeast.

Exon skipping is considered a principal mechanism by which eukaryotic cells expand their transcriptome and proteome repertoires, creating different splice variants with distinct cellular functions. Here we analyze RNA-seq data from 116 transcriptomes in fission yeast (Schizosaccharomyces pombe), covering multiple physiological conditions as well as transcriptional and RNA processing mutants. We applied brute-force algorithms to detect all possible exon-skipping events, which were widespread but rare compared to normal splicing events. Exon-skipping events increased in cells deficient for the nuclear exosome or the 5'-3' exonuclease Dhp1, and also at late stages of meiotic differentiation when nuclear-exosome transcripts decreased. The pervasive exon-skipping transcripts were stochastic, did not increase in specific physiological conditions, and were mostly present at less than one copy per cell, even in the absence of nuclear RNA surveillance and during late meiosis. These exon-skipping transcripts are therefore unlikely to be functional and may reflect splicing errors that are actively removed by nuclear RNA surveillance. The average splicing rate by exon skipping was ∼ 0.24% in wild type and ∼ 1.75% in nuclear exonuclease mutants. We also detected approximately 250 circular RNAs derived from single or multiple exons. These circular RNAs were rare and stochastic, although a few became stabilized during quiescence and in splicing mutants. Using an exhaustive search algorithm, we also uncovered thousands of previously unknown splice sites, indicating pervasive splicing; yet most of these splicing variants were cryptic and increased in nuclear degradation mutants. This study highlights widespread but low frequency alternative or aberrant splicing events that are targeted by nuclear RNA surveillance.

Exploring long non-coding RNAs through sequencing.

Long non-coding RNAs (lncRNAs) are emerging as an important class of regulatory transcripts that are implicated in a variety of biological functions. RNA-sequencing, along with other next-generation sequencing-based approaches, enables their study on a genome-wide scale, at maximal resolution, and across multiple conditions. This review discusses how sequencing-based studies are providing global insights into lncRNA transcription, post-transcriptional processing, expression regulation and sites of function. The next few years will deepen our insight into the overall contribution of lncRNAs to genome function and to the information flow from genotype to phenotype.

Cost-effort analysis of Baited Remote Underwater Video (BRUV) and environmental DNA (eDNA) in monitoring marine ecological communities.

Monitoring the diversity and distribution of species in an ecosystem is essential to assess the success of restoration strategies. Implementing biomonitoring methods, which provide a comprehensive assessment of species diversity and mitigate biases in data collection, holds significant importance in biodiversity research. Additionally, ensuring that these methods are cost-efficient and require minimal effort is crucial for effective environmental monitoring. In this study we compare the efficiency of species detection, the cost and the effort of two non-destructive sampling techniques: Baited Remote Underwater Video (BRUV) and environmental DNA (eDNA) metabarcoding to survey marine vertebrate species. Comparisons were conducted along the Sussex coast upon the introduction of the Nearshore Trawling Byelaw. This Byelaw aims to boost the recovery of the dense kelp beds and the associated biodiversity that existed in the 1980s. We show that overall BRUV surveys are more affordable than eDNA, however, eDNA detects almost three times as many species as BRUV. eDNA and BRUV surveys are comparable in terms of effort required for each method, unless eDNA analysis is carried out externally, in which case eDNA requires less effort for the lead researchers. Furthermore, we show that increased eDNA replication yields more informative results on community structure. We found that using both methods in conjunction provides a more complete view of biodiversity, with BRUV data supplementing eDNA monitoring by recording species missed by eDNA and by providing additional environmental and life history metrics. The results from this study will serve as a baseline of the marine vertebrate community in Sussex Bay allowing future biodiversity monitoring research projects to understand community structure as the ecosystem recovers following the removal of trawling fishing pressure. Although this study was regional, the findings presented herein have relevance to marine biodiversity and conservation monitoring programs around the globe.

Discomfort period of fattening pigs and sows stunned with CO2: Duration and potential influencing factors in a commercial setting.

Despite raising animal welfare concerns, stunning of pigs with CO2 prior to slaughter remains the most widely applied method in commercial settings. The aim of this study was to assess the discomfort period and its influencing factors in fattening pigs and sows in a commercial slaughterhouse. The discomfort period was defined as the first reaction to the gas or the environment from the point the animal enters the gondola, until complete relaxation of its head. Results showed that the discomfort period lasted 11 s longer in sows than in pigs, and that certain behaviors occurred distinctly later in sows as compared to pigs. Furthermore, higher humidity and temperature in the pit could prolong the duration of the discomfort period. Further research is needed to better understand the underlying physiological processes for both the differences seen between sows and fattening pigs as well as the influence of ambient parameters.

Animal Welfare and Meat Quality Assessment in Gas Stunning during Commercial Slaughter of Pigs Using Hypercapnic-Hypoxia (20% CO2 2% O2) Compared to Acute Hypercapnia (90% CO2 in Air).

This study assessed aversion, stunning effectiveness, and product quality of nitrogen and carbon dioxide (CO2) mixtures used for stunning pigs. A total of 1852 slaughter pigs divided into two similar batches was assessed during routine slaughter in a Swedish commercial abattoir using either hypercapnic-hypoxia (20% CO2 and less than 2% O2; 20C2O) or hypercapnia (90% CO2; 90C) gas mixtures. Behavioral indicators of aversion and discomfort were recorded. After exposure, the stunning quality was assessed through brainstem reflexes. After slaughter, the pH and electric conductivity of carcasses were assessed to estimate the incidence of pale, soft, and exudative (PSE) pork, and the presence of ecchymosis were inspected. Compared to 90C, pigs exposed to 20C2O showed a later (p < 0.05) onset of behaviors indicative of aversion, and a lower (p < 0.01) incidence of breathlessness. However, unconsciousness (i.e., losing posture) appeared earlier (p < 0.01) in 90C compared to 20C2O. In 90C, all (100%) pigs were adequately stunned, whereas in 20C2O a 7.4% of pigs showed signs of poor stunning, especially when oxygen concentrations were >2% (p < 0.001). The percentage of PSE carcasses was higher (p < 0.01) in 20C2O than 90C. In conclusion, compared to 90C, 20C2O reduced aversion and discomfort but showed lower stun effectiveness, especially when O2 was above 2%, and a slightly poorer pork quality.

Risk assessment of sheep welfare at small-scale slaughter in Nordic countries, comparing with large-scale slaughter.

BACKGROUND: During the pre-slaughter period, animals experience novel environment and procedures which may cause reduced welfare and suffering. Over the last decades, the slaughter industry has restructured into fewer and larger abattoirs, implying potential risks of transport stress, injuries, and impaired animal welfare. Since recently, however, there is growing interest in small-scale slaughter to supply locally or regionally produced meat. Risk managers at all levels thus need to assess animal welfare risks also at small-scale operations. This study aimed to assess risks of poor animal welfare at small-scale lamb slaughter (≤5000 sheep/year and ≤70 sheep/day) in Norway, Iceland, Sweden and Finland, and to compare these risks to large-scale industrial slaughter. Assessment was done applying an individual expert opinion approach during a 2-day workshop. Nine experts in lamb slaughter procedures, behaviour, physiology, health, scoring schemes and/or risk assessment provided estimates of exposure, likelihood of negative consequences following exposure, and intensity and duration of negative consequences for 71 hazards. The methods applied mainly adhered to the risk assessment guidelines of the European Food Safety Authority. The list of hazards was modified from an earlier study and distributed to the experts before the assessment. No other literature was reviewed specifically for the purpose of the assessment. RESULTS: The highest risks to animal welfare identified in both small- and large-scale slaughter were related to inadequate conditions during overnight lairage at the slaughter plant. For most hazards, risk estimates were lower in small-scale slaughter. The reverse was true for splitting of groups and separation of one sheep from the group. CONCLUSIONS: Small-scale slaughter has a potential for improved sheep welfare in comparison with large-scale industrial slaughter. Keeping the animals overnight at the slaughterhouse and prolonged fasting before slaughter should be avoided. Solutions include continuing education and training of stockpersons and, especially in large-scale slaughter, application of existing techniques for efficient transport logistics that minimise stress.

The genomic and phenotypic diversity of Schizosaccharomyces pombe.

Natural variation within species reveals aspects of genome evolution and function. The fission yeast Schizosaccharomyces pombe is an important model for eukaryotic biology, but researchers typically use one standard laboratory strain. To extend the usefulness of this model, we surveyed the genomic and phenotypic variation in 161 natural isolates. We sequenced the genomes of all strains, finding moderate genetic diversity (π = 3 × 10(-3) substitutions/site) and weak global population structure. We estimate that dispersal of S. pombe began during human antiquity (∼340 BCE), and ancestors of these strains reached the Americas at ∼1623 CE. We quantified 74 traits, finding substantial heritable phenotypic diversity. We conducted 223 genome-wide association studies, with 89 traits showing at least one association. The most significant variant for each trait explained 22% of the phenotypic variance on average, with indels having larger effects than SNPs. This analysis represents a rich resource to examine genotype-phenotype relationships in a tractable model.

The RNA exosome promotes transcription termination of backtracked RNA polymerase II.

The exosome is an RNA-decay complex that constantly monitors transcription and contributes to post-transcriptional turnover of faulty mRNAs. Yet how nuclear RNA surveillance by the exosome is coordinated with transcription is still unknown. Here we show that the RNA exosome of Schizosaccharomyces pombe can target the transcription machinery by terminating transcription events associated with paused and backtracked RNA polymerase II (RNAPII); this is contrary to the notion that the exosome acts exclusively on RNAs that have been released by RNAPII. Our data support a mechanism by which RNAPII backtracking provides a free RNA 3' end for the core exosome, which results in transcription termination with concomitant degradation of the associated transcript. These findings uncover a mechanism of cotranscriptional RNA surveillance whereby termination of transcription by the exosome prevents formation of aberrant readthrough RNAs and transcriptional interference at neighboring genes.

N-termini of fungal CSL transcription factors are disordered, enriched in regulatory motifs and inhibit DNA binding in fission yeast.

BACKGROUND: CSL (CBF1/RBP-Jκ/Suppressor of Hairless/LAG-1) transcription factors are the effector components of the Notch receptor signalling pathway, which is critical for metazoan development. The metazoan CSL proteins (class M) can also function in a Notch-independent manner. Recently, two novel classes of CSL proteins, designated F1 and F2, have been identified in fungi. The role of the fungal CSL proteins is unclear, because the Notch pathway is not present in fungi. In fission yeast, the Cbf11 and Cbf12 CSL paralogs play antagonistic roles in cell adhesion and the coordination of cell and nuclear division. Unusually long N-terminal extensions are typical for fungal and invertebrate CSL family members. In this study, we investigate the functional significance of these extended N-termini of CSL proteins. METHODOLOGY/PRINCIPAL FINDINGS: We identify 15 novel CSL family members from 7 fungal species and conduct bioinformatic analyses of a combined dataset containing 34 fungal and 11 metazoan CSL protein sequences. We show that the long, non-conserved N-terminal tails of fungal CSL proteins are likely disordered and enriched in phosphorylation sites and PEST motifs. In a case study of Cbf12 (class F2), we provide experimental evidence that the protein is proteolytically processed and that the N-terminus inhibits the Cbf12-dependent DNA binding activity in an electrophoretic mobility shift assay. CONCLUSIONS/SIGNIFICANCE: This study provides insight into the characteristics of the long N-terminal tails of fungal CSL proteins that may be crucial for controlling DNA-binding and CSL function. We propose that the regulation of DNA binding by Cbf12 via its N-terminal region represents an important means by which fission yeast strikes a balance between the class F1 and class F2 paralog activities. This mode of regulation might be shared with other CSL-positive fungi, some of which are relevant to human disease and biotechnology.